Special Issue

Biotechnology
This special topic selects papers related to biotechnology published in Acta Agriculurae Boreali-Sinica , involving papers on crop genetics and breeding, planting resources, biotechnology,etc.Click on the relevant paper to open the web page and download the full text. In order to quote and share for readers, each article contains a complete citation format in Chinese and English (including international DOI number) and a proprietary  QR code. Long press the  QR code of the article to open the web page of the article and realize mobile sharing at the same time. Thank you for downloading, quoting, forwarding and sharing.
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  • ZOU Xiaoyue, LIU Jia, LI Zhiyong, MA Jifang, WANG Yongfang, QUAN Jianzhang, LIU Lei, BAI Hui, DONG Zhiping
    Abstract (94) PDF (61) RichHTML (15)

    In order to understand the function of SibHLH19 in foxtail millet,the CDS sequence and promoter sequence of SibHLH19 gene were separately cloned with the leaf cDNA and genomic DNA from resistance material Shilixiang as template by PCR.Promoter cis-acting elements and biological characteristics were analyzed using bioinformatics online tools.Then the expression patterns of SibHLH19 in different tissues and during the process to rust resistance were surveyed by qRT-PCR,respectively.Lastly the prokaryotic expression characteristics for the gene were detected by SDS-PAGE,laying a theoretical foundation for further research on SibHLH19 gene function and disease resistance mechanism.The results showed that the CDS sequence of the SibHLH19 transcription factor was 843 bp in length,encoding a total of 280 amino acids,the predicted protein molecular weight was 29.97 ku.The theoretical isoelectric point was 5.85,and the encoded protein chemical formula was C1296H2071N397O400S11,containing a bHLH conserved domain,belonging unstable hydrophilic protein.The largest element of the protein's secondary structure was random coils,and the smallest element was a β-turn.Evolutionary analysis showed that SibHLH19 had the higher homology to the amino acid sequences of Panicum miliaceum (RLM85279.1),Panicum hallii (PUZ71581.1)and Panicum virgatum (XP_039835205.1),and had the lowest homology with Triticum aestivum(KAF7059972.1)and Aegilops tauschii subsp.strangulata (XP_040244423.1).The analysis of the promoter cis-acting elements showed that there were multiple response elements such as hormones and stresses in the promoter region of the SibHLH19 gene.Tissue expression analysis showed that the gene was mainly expressed at the seedling stage with the highest expression in the aboveground part,and was almost no expression at the booting stage.Within 24 hours of the response to the biotic stress of rust disease in foxtail millet,the SibHLH19 gene expression was up-regulated at 8 and 16 h in the disease resistance response,while its expression was only slightly up-regulated at 16 h and down-regulated at the rest of the time points in the susceptible response.It was speculated that SibHLH19 played a positive regulatory role in the resistance response to rust disease in foxtail millet.The constructed prokaryotic expression vector pET30a-SibHLH19 could express the SibHLH19 fusion protein with an apparent molecular weight of about 44 ku after being induced by 0.1 mmol/L IPTG.

  • XIAO Shikui, LI Fang, ZHANG Wenting, LÜ Shufang, SHI Guoan, WU Jiang, FAN Bingyou
    Abstract (53) PDF (15) RichHTML (5)

    In order to explore the function of ACS gene in herbaceous peony,a full-length cDNA sequence of PlACS cDNA in Paeonia lactiflora was obtained,RACE technique and bioinformatic methods were used to analyze the protein sequence which it encoded.The CDS of PlACS was subcloned,the prokaryotic expression vector of PlACS was constructed based on pET32a vector,and then the highly efficient prokaryotic expression system was established.The results showed that the total length of PlACS cDNA(GenBank accession JX512359)was 1 752 bp,which encoded 492 amino acids.Seven conserved regions and active sites K278 were detected in PlACS protein.Phylogenetic tree analysis showed that PlACS was highest homological with ACS of P.suffruticosa.PlACS protein was determined structurally to be 40.04% α-helix,16.26% β-extended strand,6.91% β-turn and 36.79% random coil.Protein 3D structure homology modeling predicted that PlACS existed as homodimers.The optimal expression condition of PlACS protein was that when the cell density of genetic engineering strain A600 reached 0.2,IPTG with a final concentration of 0.1 mmol/L was added,and the recombinant protein was expressed for two hours at 37 ℃.It was of great significance to acquire PlACS recombinant protein with biological activity by denaturation & renaturation and identify its enzymatic activity in vitro.

  • HOU Ruize, HOU Yuxiang, XU Xiaoyong, LI Xuan, LI Meilan
    Abstract (53) PDF (18) RichHTML (12)

    CYP79B2 is a related gene that regulates auxin synthesis in Arabidopsis.By cloning the homologous gene BrcCYP79B2-1 of CYP79B2 in B.rapa ssp. cninensis,analyzing its expression in different tissues and periods,and studying its regulatory mechanism on vernal flowering in B.rapa ssp. cninensis,to lay a theoretical foundation for the subsequent functional verification of the auxin-encoding gene in B.rapa ssp. cninensis.The homologous gene of CYP79B2 was cloned from B.rapa ssp. cninensis by qRT-PCR and named as BrcCYP79B2-1.The structure,physicochemical properties and relationship of its protein were analyzed by bioinformatics method,and its protein was analyzed by qRT-PCR method.Expression levels in different tissues and growth stages in B.rapa ssp. cninensis.And its expression in different tissues and growth stages of B.rapa ssp. cninensis was analyzed by qRT-PCR method.The results showed that the full-length coding sequence of BrcCYP79B2-1 gene was 1 623 bp,encoding 540 amino acids.The physicochemical analysis of the protein showed that the molecular mass of the protein was 60.849 73 ku,and the theoretical isoelectric point was 8.71.Compared with the amino acid sequences of other species,it was found that BrcCYP79B2-1 was highly conserved in cruciferous plants,and had the highest homology with turnip,up to 99.52%;the expression levels of different organs of B.rapa ssp. cninensis were analyzed by qRT-PCR, and it was found that the expression level of BrcCYP79B2-1 was the highest in roots, followed by leaves, and the lowest in flower buds.The expression of BrcCYP79B2-1 in seedlings after 0,10,15 and 16 days of low temperature treatment showed that the expresion of BrcCYP79B2-1 reached its peak on the 10th day of low temperature treatment, that is, the vegetative growth period, while the expression of BrcCYP79B2-1 decreased during flower bud differentiation, which indicated that the expression of BrcCYP79B2-1 was related to low temperature vernalization and could affect flower bud differentiation.

  • ZHAO Jie, LIU Hongquan, ZHAO Yun, YANG Kai, FU Yongbin, GU Yuzhang, SUN Lijing, HU Mengyun, LI Hui, ZHANG Yingjun
    Abstract (37) PDF (20) RichHTML (3)

    In order to explore the relationship between the vernalization genes and winter-spring characteristics of wheat varieties in the Northern China Winter Wheat Region,271 wheat cultivars from the Northern China Winter Wheat Region were used as materials to detect the composition and distribution of four vernalization genes Vrn-A1,Vrn-B1,Vrn-D1 and Vrn-B3 by molecular markers.The heading date of these cultivars in the field was observed in Sanya,Hainan Province,and their winter-spring characteristics were investigated with the data recorded.The results showed that in this wheat region:among the four dominant alleles,Vrn-D1 (27.7%)had the highest distribution frequency in the tested cultivars and the distribution frequency decreased gradually from the South to the North;the distribution frequency of dominant Vrn-B1 alleles (3.0%)was very low in wheat materials in this wheat region;none of the test materials contained the dominant alleles Vrn-A1 and Vrn-B3;the most common vernalization gene allele combination was vrn-A1/vrn-B1/vrn-D1/vrn-B3.Further analysis of winterness-springness type of the test cultivars showed that some cultivars containing the dominant allele Vrn-D1 must be vernalized to blossom,while the wheat cultivars containing the dominant allele Vrn-B1 had a weak demand for vernalization.Among the four dominant vernalization genes,only the dominant alleles Vrn-D1 and Vrn-B1 were distributed in the tested cultivars in the Northern China Winter Wheat Region.The effect of Vrn-B1 on the vernalization development characteristics was stronger than that of Vrn-D1.

  • JIN Yifeng, GAO Yansong, WANG Qi, WANG Mengmeng, ZHAO Di, XIONG Yi, CHEN Yang
    Abstract (66) PDF (44) RichHTML (15)

    Protein kinases are important factors in plant defense system,protein kinase SnRK2 is a serine/threonine protein kinase,can play an important role in the plant stress signal transduction pathway through phosphorylation.We analyzed the expression pattern of SnRK2.4 under abiotic stresses,aiming to reveal its role in the regulation of adversity.The SnRK2.4 gene of high-quality cold-season turfgrass Poa pratensis L. was cloned using RT-PCR,the SnRK2.4 gene contained an ORF of 1 092 bp encoding a 363-amino acid protein,and its molecular characteristics were analyzed by bioinformatics.In addition,the expression pattern of this gene in different tissue parts under different abiotic stresses was observed using qPCR.The results showed that the Poa pratensis L.SnRK2.4 gene belonged to the SRK/SAPK superfamily,contained typical STKc_SnRK2 domain,tyrosine kinase phosphorylation site,casein kinase Ⅱ phosphorylation site and serine/threonine-protein kinase activity site,which had the highest homology with Brachypodium distachyon.The qPCR results demonstrated that the Poa pratensis L.SnRK2.4 gene was tissue-specific,highly expressed in panicles,and had no significant differential expression in roots,stems and leaves.Moreover,the Poa pratensis L.SnRK2.4 gene could respond positively to abiotic stresses such as drought,salt,low nitrogen,low phosphorus,ABA and BR.

  • WANG Ya, WANG Yuetao, SHEN Guanwang, WANG Fuhua, WANG Shengxuan, BAI Tao, YIN Haiqing
    Abstract (32) PDF (15) RichHTML (0)

    In order to improve the blast resistance of Shuijing 3,an excellent food-flavor rice variety,CRISPR/Cas9 gene editing technology combined with gene chip technology were used to pyramid the R gene Pigm and the non-R gene bsr-d1 into Shuijing 3.Firstly,Bsr-d1 was selected as the target gene to construct a recombinant expression vector using the CRISPR/Cas9 gene editing system,and transformed into the excellent food-flavor rice Shuijing 3 by Agrobacterium-mediated method.The homozygous bsr-d1 mutant lines without T-DNA elements,including five mutation types as T insertion,G insertion,GA deletion,CGCA deletion and CGCAGA deletion,were screened out.The japonica line Jinyu 1 containing a broad-spectrum blast resistance gene Pigm was used as the gene donor parent to cross with the homozygous bsr-d1 mutant lines without transgenic components.The Pigm gene was introduced into bsr-d1 mutant lines by cross,backcross and self-cross combing molecular breeding chip to simultaneously perform Pigm gene and background-assisted selection.The improved lines SJ3-G1,SJ3-G2,SJ3-G3,SJ3-G4,SJ3-G5,which were homozygous for the disease resistance genes(carrying both bsr-d1 and Pigm genes)and whose background recovery rates were all above 96%,were finally obtained.The improved strains of Shuijing 3 displayed enhanced leaf blast resistance compared with the wild type in inoculated identification test using Magnaporthe grisea strain GUY11.After inoculation with M.oryzae,the POD activities in the improved strains of Shuijing 3 were significantly lower than that of the wild-type control,while the H2O2 contents were significantly higher than that of the wild-type control.The improved Shuijing 3 lines with blast resistance carrying both bsr-d1 and Pigm genes are obtained by CRISPR/Cas9 gene editing technology combined with gene chip technology.

  • QU Dong, YAN Fei, LIU Xinrui, KANG Xue, ZENG Haitao, ZHANG Yu
    Abstract (44) PDF (8) RichHTML (3)

    In order to explore the mechanism of AcWRKY70 transcription factor in response to kiwifruit canker stress in different resistant varieties,AcWRKY70 gene was cloned from the leaf cDNA of resistant variety Xuxiang and highly susceptible variety Hongyang kiwifruit.The sequence structure characteristics of Hongyang AcWRKY70 gene,subcellular localization and evolutionary relationship of Hongyang AcWRKY70 protein were analyzed. The expression patterns of AcWRKY70 gene,including the tissue expression and different expression in resistant kiwifruit varietie and susceptible variety under Pseudomonas syringae pv. Actinidiae(Psa),salicylic acid(SA)and methyl jasmonate(MeJA)treatment were analyzed by RT-qPCR. Results showed that the full length of the Hongyang AcWRKY70 gene was 906 bp(GenBank accession number was MW881147). AcWRKY70 gene contained 885 bp of open reading frame(ORF)and encoded 294 amino acids. AcWRKY70 protein had typical WRKYGQK domain and the zinc finger structure was C2-HC. It belonged to class Ⅲ group of WRKY family and islocated in the nucleus. Phylogenetic tree analysis showed that it had the closest genetic relationship with tea plant. Both Hongyang and Xuxiang AcWRKY70 genes had the highest expression level in leaves. Under Psa treatment, the highest expression of AcWRKY70 gene reached at 12 h in Xuxiang, the expression of Hongyang reached the maximum at 48 h.Under the co-treatment of salicylic acid and Psa(SA+Psa),Methyl jasmonate and Psa(MeJA+Psa),the highest expression of AcWRKY70 gene reached at 12 h in Xuxiang. Under SA+Psa and MeJA+Psa treatment,the expression of Hongyang reached the maximum at 72,24 h,respectively. AcWRKY70 gene played a certain role in resistance stress of kiwifruit,and the response mechanism to pathogens in different resistance kiwifruit varieties may vary considerably.

  • SU Fang, HAN Diangang, YE Lingling, ZHANG Chong, YIN Shanglian, LUO Qianmin, DONG Xianlan, LI Yaoyao, LI Lingfeng, AI Jun, XIN Jige
    Abstract (30) PDF (7) RichHTML (3)

    In order to obtain COVID-19 nucleocapsid protein with similar function and activity to natural protein and apply it to practical detection. Firstly,according to Bac-to-bac insect expression system and synthetic COVID-19 nucleocapsid protein(N protein)sequence,BamH Ⅰ and Xba Ⅰ on pFastBacTMHTB vector were added to upstream and downstream primers respectively. The N gene was amplified by PCR technology,and T-Vector pMD19(simple)vector and pFastBacTMHTB vector were connected successively and recombinant plasmids pMD19-T(simple)-COV19-N and pFastBacTMHTB-COV19-N,and finally construct recombinant bacmid DH10Bac-pFastBacTMHTB-COV19-N in DH10Bac cells was expressed in insect cell Sf9. The recombinant protein was obtained and analyzed by SDS-PAGE and WB. The recombinant plasmid pMD19-T(simple)-COV19-N was identified by PCR and double enzyme digestion. The recombinant bacmid DH10Bac-pFastBacTMHTB-COV19-N was constructed in DH10Bac cells was identified by PCR and the expected two bands were 2 430,3 690 bp,respectively,which proved that the recombinant bacmid was successfully obtained. The recombinant bacmid was transfected into Sf9 insect cells. At the same time,the recombinant GFP protein control group was established. After 120 h of transfection,the recombinant N protein and recombinant GFP protein were collected and samples were prepared;SDS-PAGE and WB analysis were carried out respectively. HRP-His labeled antibody was used to verify that the transfection was successful,and both recombinant N protein and recombinant GFP protein were successfully expressed in Sf9 cells. The experimental results were consistent with the expectation,and the size of recombinant N protein band was about 46 ku. The eukaryotic expression vector of respiratory coronavirus N gene was successfully constructed and successfully expressed in insect cells,which provides an experimental basis for the establishment of ELISA detection methods and other related research.

  • ZHANG Dongmei, FENG Yayan, XIU Zhijun, YANG Chunfang, DU Meie, LI Dezhou, ZHANG Xiaoyu
    Abstract (27) PDF (9) RichHTML (3)

    In order to study the molecular mechanism of sodium silicate enhancing risistance of potato to Rhizoctonia solani,a gene StWRKY11 with high expression level in potato transcriptome induced by sodium silicate was cloned and its bioinformatics was analysed.Total RNA was extracted from potato,amplified by RT-PCR and cloned.Through bioinformatics related software,the structure prediction and prediction analysis were carried out.The results showed that StWRKY11 gene with a open reading frame of 1 005 bp was cloned from potato Atlantic,encoding 334 amino acids.The molecular formula of the expressed protein was C3013H5023N1005O1260S199,the molecular weight was 81.867 94 ku,the theoretical isoelectric point(pI)was 5.09,and the total number of atoms was 10 500.The expressed protein contained a typical WRKYGQK conserved domain,and the zinc finger structure was CX5CX23HXH,belonging to the second Ⅱ d subfamily.The secondary structural elements were α-helix,extended chain,β-folding and random coiling,among which the proportion of random curl was the highest,up to 61.68%.There were 29 phosphorylation sites in total,which might be located in the nucleus.There were cis-regulatory elements upstream of the promoter that might related to resistance stress response and cis-regulatory elements related to growth and development and hormone response.The gene was closely related to potato StWRKY5 gene,and the amino acid homology of the coding protein reached 95%.

  • SHI Yumei, CHEN Shaokang, XING Kai, ZHAO Yanhui, YUAN Jiani, SHENG Xihui, QI Xiaolong, NI Hemin, GUO Yong, WANG Chuduan
    Abstract (35) PDF (14) RichHTML (3)

    This study aims to use high-throughput sequencing technology to perform mRNA sequencing and differential analysis of longissimus dorsi muscle tissue samples from Songliao black pigs and Landrace pigs,and screen out key genes that affect pig muscle growth,meat quality and fat deposition,so as to provide pork quality research provides new reference information.The longissimus dorsi muscle tissue samples of 6 Songliao black pigs and 6 Landrace pigs were collected,their RNA was extracted,and the mRNA was sequenced by Illumina HiSeq 2500 high-throughput sequencing technology,and the obtained reads were compared,annotated and differentiated.For expression analysis,NOISeq was used to screen out differentially expressed genes and perform enrichment analysis of related biological functions.The results showed that 664 differentially expressed genes were screened from the two pig species,of which 364 genes were highly expressed in Songliao black pigs and 300 genes were highly expressed in Landrace pigs.Through the biological function analysis of differentially expressed genes,LPIN1,FADS1,FADS2,PLIN2,PPARGC1A,PRKAG2 and ACSL1 were screened to participate in the regulation of lipid metabolism and muscle development.The related pathways were fatty acid metabolism,PPAR signaling pathway,AMPK signaling pathway,insulin signaling pathway and adipocytokine signaling pathway and so on.

  • ZHENG Yusi, WANG Pei, GUO Ying, BAI Lirong, YU Dahui, ZHAO Sen
    Abstract (40) PDF (11) RichHTML (10)

    To explore the immune function and expression pattern of C-type lectin family members in Pinctada fucata,a cDNA sequence of C-type lectin gene PfCLEC17A in P.fucata was cloned through RACE technique.The molecular characteristics of PfCLEC17A were determined by bioinformatics analysis and its mRNA expression level was detected in different tissues including gill,mantle,adductor muscle,gonad,blood,foot and hepatopancreas,as well as expression level in hepatopancreas after Vibrio alginolyticus challenged using the Real-time fluorescent quantitative PCR.The results showed that a new C-type lectin gene PfCLEC17A was successfully cloned from P.fucata,the full cDNA was 699 bp in length with an open reading frame(ORF)of 534 bp,which encoded 178 amino acids.The deduced amino acid sequence was predicated containing a CTL domain.Phylogenetic tree analysis showed that the PfCLEC17A of P.fucata and other shellfish proteins were clustered together in a branch.The Real-time fluorescent quantitative PCR results showed that PfCLEC17A was expressed in all tissues detected in P.fucata,and the highest was found in hepatopancreas,which was significantly higher than the other tissues.The high expression level in hepatopancreas,one of the most important immune tissues in mollusc,suggested that PfCLEC17A gene might be involved in the body's immune reaction.The expression level of PfCLEC17A increased significantly and reached the peak at 1 h after V.alginolyticus challenge,indicating that the gene expression was induced by microbial stimulation at early stage.Therefore,based on the above findings,it could be concluded that the PfCLEC17A gene participates in the immune activation response of P.fucata and functions in immune system.

  • ZHAO Changjiang, DU Mengxiang, SONG Juqi, XU Shangyuan, HE Lin, XU Jingyu, YANG Kejun, LI Zuotong
    Abstract (424) PDF (127) RichHTML (213)

    NRL(NPH3/RPT2-Like)is a type of light-responsive protein unique to plants and plays a vital role in the phototropic signal pathway. To reveal the NRL gene maize genome's characteristics and expression,we analyzed them using bioinformatics methods combined with qRT-PCR technology. The property,structure,evolution of their encoded proteins,and growth period tissue expression and stress expression were analyzed. 31 ZmNRL genes identified were located in nine maize chromosomes,encoding protein amino acids 464-749 aa,which predicted to have chloroplast,nuclear and cytoplasmic locations. According to protein conservation,ZmNRL family was divided into four categories. Their gene structure also presented certain conservation,the most contained four exons. Analysis of the cis-elements of gene promoters revealed a large number of abscisic acids,jasmonic acid,light response,and anti-oxidation elements,among which G-box and Sp1 were two types of light-related elements. The expression of ZmNRL family genes in tissues during the growth period showed a temporal and spatial specificity,and the majority expression level was not high. Only ZmNRL2,ZmNRL4,ZmNRL24,and ZmNRL29 highly expressed. Furthermore,the characteristic modules were produced based on the data of the tissue co-expression genes. And the GO enrichment analysis of a particular leaf growth module containing six ZmNRL genes,mainly associated with the plastid organization biological processes and rRNA binding molecular functions. The expression of ZmNRL5,ZmNRL7,ZmNRL12,and ZmNRL19 genes were analyzed by qRT-PCR under salt,drought,high temperature,and Rhizoctonia solani inoculation treatments. The results showed that ZmNRL12 was significantly up-regulated in maize seedlings treated with high temperature,while ZmNRL5,ZmNRL7 and ZmNRL19 genes were down-regulated in drought,salt and pathogen treatments. In summary,31 ZmNRL genes were identified in the maize genome. They not only had apparent specific tissue expression but also participated in biotic and abiotic stress responses.

  • YAN Liuyan, LI Jianfeng, ZHANG Shiwen, ZHANG Bo, WANG Yongfang, ZHANG Xiaomei, ZU Chaofan, WANG Zhenshan, SANG Luman, HE Zhanxiang, JIA Xiaoping, DONG Zhiping
    Abstract (203) PDF (70) RichHTML (93)

    The SiPRR73 gene was cloned from Yangu 11 using RT-PCR technology,and through analyzing tissue-specific expression,responsive features of SiPRR73 to different photoperiods,photo-thermal combinational treatments and five abiotic stress treatments,the regulation mode of photoperiod and temperature on SiPRR73,and the responsive pattern of SiPRR73 to abiotic stresses in foxtail millet were explored. The results showed that totally 2 928 bp cDNA sequence of SiPRR73 was obtained from Yangu 11,which included 2 283 bp CDS region,encoding 760 amino acids. The SiPRR73 proteins of C4 crops including Panicum miliaceum,Panicum hallii,Sorghum bicolor and Zea mays showed relatively close relationship with SiPRR73. The second parietal leaf was the highest expression tissue of SiPRR73,but the expression level at root,stem and panicle tissues was relatively lower. The expression level of SiPRR73 was higher at light period than that at dark period under both short-day and long-day conditions,and during the whole vegetative growth phase,SiPRR73 showed higher expression level under long-day compared to short-day,which indicated that the expression of SiPRR73 was induced by light and controlled by photoperiod. The temperature determined expression peak number of SiPRR73 and the photoperiod determined occurrence time of expression peaks,so temperature and photoperiod participated in regulating of SiPRR73 expression mutually. PEG and low temperature stresses induced SiPRR73 expression totally,NaCl induced SiPRR73 expression at early stress stage,but inhibited it at later stress stage. Fe stress inhibited SiPRR73 expression at early stage,but induced it at later stage. ABA stress caused the close responsive feature of SiPRR73 to NaCl. This study indicated that SiPRR73 showed light-dependent expression feature,and photoperiod and temperature regulated SiPRR73 by interaction pattern,suggesting that SiPRR73 participated in adaptability regulation process to different photo-thermal conditions and might play a certain role in coping with drought,low temperature,ABA,NaCl and Fe stresses in foxtail millet.

  • ZHANG Bin
    Abstract (421) PDF (190) RichHTML (48)

    To investigate the function of soybean GmPP2C89 gene in plant abiotic stress response and adaptation. The expression patterns of GmPP2C89 under NaCl,PEG and mannitol treatments were detected by transcriptome data and Real-time quantitative PCR. Then,the cis-acting elements on the promoter of GmPP2C89 in response to abiotic stress were analyzed,and promoters of different lengths were cloned according to the distribution of cis-elements to construct fusion GUS vectors to obtain the corresponding transgenic Arabidopsis. The response of the promoters to NaCl,PEG and mannitol was analyzed by GUS staining. Transgenic Arabidopsis overexpressing the GmPP2C89 was constructed,and the root length,leaf MDA content and electrolytic leakage,and the expression of salt stress-related genes(SOD,POD,CAT,RD26,RD29A,and RD29B)were measured under normal and NaCl treatment conditions. The results showed that NaCl,PEG and mannitol treatments all led to a significant increase in the expression level of soybean GmPP2C89;the promoter region contained many cis-acting elements such as ABRE,DRE,G-box,MBS,MYB,MYC and TC-rich repeats which were involved in abiotic stress response,and this promoter was more responsive to NaCl treatment. In addition,under the salt treatment,the root length of transgenic Arabidopsis GmPP2C89-OX was significantly greater than that of WT,while the MDA content and electrolytic leakage were significantly lower than those of WT,and the salt tolerance was significantly enhanced;the expression of antioxidant enzyme genes(SOD and POD)and ABA pathway key gene RD29B in GmPP2C89-OX was significantly higher than that in WT. These results indicated that soybean GmPP2C89 was induced by NaCl,PEG and mannitol,and GmPP2C89 overexpression could enhance the salt tolerance of transgenic Arabidopsis by activating antioxidant and ABA pathways.

  • FENG Liting, ZHANG Jianfeng, CHI Shengqi
    Abstract (28) PDF (11) RichHTML (20)

    A multiplex RT-PCR detection system was established and studied to target four sweet potato RNA viruses,Sweet potato feathery mottle virus (SPFMV),Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus G (SPVG)and Sweet potato virus C (SPVC),which caused serious damage in sweet potato production. Firstly,the specific primers for four viruses were designed by Premier 5.0 software and synthesized according to the conserved sequences of coat protein(CP)gene of these viruses from GenBank. The cDNA synthesized was used as a template,and 4 couples of selected primers were used simultaneously to amplify the target fragments by PCR. A best multiplex RT-PCR reaction system was tested by optimizing the annealing temperature,extension time,dNTPs amount,template amount,primer concentration and so on and an optimal quadruple RT-PCR system that could simultaneously detect these four sweet potato RNA viruses was established. The results showed that an accurate and sensitive multiplex RT-PCR method for detecting the four sweet potato viruses mentioned above with respective target band had been successfully established by challenge tests in which there would be no crossover among the virus primers. The multiplex RT-PCR method established in this study could detect four kinds of sweet potato virus at the same time. It was not only accurate and sensitive,but also reduced the cost of detection and improved the efficiency of virus detection.

  • JI Xiang, SONG Zhicheng, WEI Xiaoling, YANG Yu, SUI Jiongming, GUO Baotai
    Abstract (30) PDF (19) RichHTML (22)

    The purpose was to prepare polyclonal antibody against the recombinant double CP of Potato leafroll virus (PLRV)and Potato virus S (PVS),and apply the polyclonal antibody to indirect ELISA and DAS-ELISA detections of PLRV and PVS.Prokaryotic expression vector pET22b-LRCP/SCP of the fused double CP gene of PLRV and PVS was constructed.After replacement of lysozyme treatment by ultrasonic disruption,inclusion body protein was extracted from the recombinant strain BL21(pET22b-LRCP/SCP),the target protein(recombinant double CP)was purified by nickel ion affinity chromatography and high-purity target protein of 51.2 ku was obtained.The high-purity recombinant double CP was used as antigen to immunize rabbits to prepare an antiserum with a titer of 1∶128 k.Specific reactions were respectively observed between the purified polyclonal antibody (IgG) against the recombinant double CP and the positive standard of PLRV or PVS and no cross-reaction was found between the purified IgG and other four potato major viruses (PVX, PVY, PVA and PVM). The purified IgG against the recombinant CP with the diluted concentration of 1∶3 200 still positively reacted with PLRV or PVS in indirect ELISA detection.The purified IgG and the alkaline phosphatase-conjugated IgG both with the diluted contraction of 1∶100 also positively reacted with PLRV or PVS positive standard in DAS-ELISA detection.The results showed that one type of the prepared IgG against the recombinant double CP could detect two viruses of PLRV and PVS by DAS-ELISA or indirect ELISA.

  • LIANG Peng, ZHANG Wen, FENG Dengzhen, QIANG Hao, RONG Xuan, MENG Ke
    Abstract (58) PDF (46) RichHTML (21)

    The aim of this study was to analyze the variation in meat quality and expression of intramuscular genes between Tan sheep,Dorper sheep and Small-tailed han sheep,and to preliminarily reveal the molecular mechanisms underlying the differences in their meat quality traits and also provide a theoretical basis for the selection and improvement of other sheep breeds. The meat quality of 8-month-old Dorper sheep,Tan sheep and Small-tailed han sheep was analyzed and compared,and the Illumina HiSeqTM 4000 platform was used to sequence and analyze the transcriptome of the longest dorsal muscle tissue to explore the regulatory genes associated with the differences in their meat quality traits. The results showed that a total of 820 differentially expressed genes were screened,including 99 between Dorper sheep and Tan sheep,436 between Dorper sheep and Small-tailed han sheep and 552 between Tan sheep and Small-tailed sheep. The results of GO functional enrichment analysis of the differential genes showed that each comparison group was significantly enriched in 224,517 and 657 GO entries;and the KEGG pathway enrichment analysis showed that DEGs were enriched in cAMP,MAPK,AMPK,purine metabolism,glycerophospholipid metabolism,amino acid and fatty acid metabolism and glycolysis/gluconeogenesis signalling pathways,further screening for candidate genes related to meat quality regulation and flavor substance metabolism such as FOS,PLA2G4E,LPIN1,AMPD1,AMPD3, NT5C1A,GPI,PFKM and PKM. Real-time fluorescence quantitative PCR validation of several randomly selected differential genes showed consistent expression trends with the transcriptome sequencing results,indicating the reliability of the sequencing results. These differential genes obtained in this study can be used as basic information for the breeding of new breeds(lines)of Ningxia high-quality meat sheep.

  • FU Yanfeng, ZHAO Weimin, LI Hui, LI Bixia, CHENG Jinhua, XU Xiaobo, REN Shouwen
    Abstract (45) PDF (12) RichHTML (23)

    The objective of the study was to screen the candidate genes and SNP markers for the total number born(TNB)and number born alive(NBA)in pigs,so as to prepare for the next step of improving the reproduction traits,and breeding new high-proliferation of Canadian line of large white pigs. Illumina porcine 50K chip was used to do a genome-wide SNP scan on 186 Canadian line of large white sows in one pig farm,followed by quality control,beagle fill and SNP genotyping. Combined with the phenotypic traits and population structure analysis results of these experimental pigs,genome-wide association study(GWAS)was carried out. The results of population structure analysis showed that there was no population stratification in the pig samples used in the experiment. A total of 36 867 effective SNP markers were obtained on 18 pairs(36)of autosomes for GWAS analysis of experimental pigs. The results of GWAS showed that NBA in all parities of these Canadian large white pigs was significantly associated with two SNPs:seq-rs323899658 and seq-rs329781338,respectively. The SNP of seq-rs329781338 was annotated with three genes:SSBP1,WEE2 and KIAA1147,respectively. NBA was significantly associated with 7 SNPs,namely:seq-rs81238474,seq-rs321377412,seq-rs340736313,seq-rs80782154,seq-rs81244816,seq-rs81467772 and seq-rs329781338. Five out of seven SNPs had gene annotation,and a total of 22 genes were annotated:EphA1, TAS2R60, FAM131B, CLCN1, CASP2, TMEM139, TRBV21OR9-2, PRSS2, TRBV19, TRBV24-1, U6, SSBP1, WEE2, KIAA1147, NKX2-8, ALKBH3, HSD17B12, U6, HOMER2, WHAMM, FSD2 and SCARNA15. The results of GO function and KEGG pathway analysis showed that the most significant biological process of GO function of these genes was the regulation of multi biological process,the molecular function was non transmembrane/transmembrane protein tyrosine kinase activity,and the KEGG pathway was mismatch repair.Combined with GWAS and gene function annotation results,WEE2 and EphA1 genes could be used as key candidate genes to increase the TNB and NBA of Canadian large white pigs,and provide some reference for the precise cultivation of high-yield pigs in the future.

  • ZHANG Zhiwei, FAN Junchen, KANG Liru, JIA Ruifang, TIAN Zaimin, ZHAO Jun
    Abstract (80) PDF (24) RichHTML (34)

    Small GTPases Rabs is a kind of monomer GTP binding protein,which can act as a molecular switch involve in variety of physiological and biochemical reactions in plant cells. In order to explore the relationship between potato small G protein StRab5b and anthocyanin synthesis,the StRab5b gene was overexpressed in potato by Agrobacterium mediated genetic transformation,try to study the effects of StRab5b gene on anthocyanin content and PAL activity. The result showed that overexpression of StRab5b gene significantly increased the anthocyanin content in potato leaves,stems and tubers. Compared with the control,the anthocyanin content in leaves of transgenic potatoes StRab5b-L2,StRab5b-L3 and StRab5b-L5 increased by 42%,24% and 54%,respectively;in stems was increased by 59%,37% and 32%,respectively;in tubers was increased by 118%,82% and 105%,respectively. At the same time,StRab5b gene also promoted the PAL activity,a key enzyme of plant anthocyanin synthesis. Compared with the control,the PAL activity in leaves of transgenic potatoes StRab5b-L2,StRab5b-L3 and StRab5b-L5 increased by 88%,63% and 66%,respectively;in stems increased by 48%,38% and 31%,respectively;in tubers increased by 98%,78% and 64%,respectively. In summary,small GTPases StRab5b increased the content of anthocyanin in potato by regulating PAL activity.

  • LING Chen, ZHAO Hong, YAN Jun, MA Yingxue, FENG Yanfang, ZHANG Jingli, LI Bo, DENG Chao, XU Zhenjiang
    Abstract (68) PDF (16) RichHTML (34)

    In order to establish a rapid DNA molecule identification system of lettuce varieties,8 lettuce varieties of different types with significant morphological differences were chosen and used for preliminary primer-screening,and 81 pairs of primary primers were selected from 245 pairs of primers by polyacrylamide gel electrophoresis. Labeled with fluorescence at 5' end of upstream,the 81 pairs of primary primers were re-screened with 90 lettuce varieties by capillary electrophoresis,and 22 pairs of core primers were finally screened out. The corresponding reference samples for each of the core primers were also selected according to the size of amplified fragments and a SSR fingerprint database of 233 varieties was constructed based on these core primers. The results showed that 22 pairs of core primers detected a total of 269 alleles in 233 varieties. For each pair of primers,the detected alleles ranged from 4 to 22,with an average of 12.23,and the PIC value ranged from 0.37 to 0.89,with an average of 0.73. Cluster analysis showed that 233 varieties were divided into two major groups of stem lettuce and leaf lettuce. Among the leaf groups,crisphead lettuce cluster into a group,half-hitch lettuce tended to cluster into a group,and looseleaf lettuce was difficult to cluster into a group. 15 varieties in 6 groups with no difference in SSR markers were compared in a field trail. 3 pairs of varieties with no significant morphological differences were determined as the same and other varieties from different groups were determined as similar varieties because of a few morphological differences. Thus,the result of DNA clustering was basically in agreement with that of the field morphological classification. In addition,24 varieties in 8 groups with the same name can be distinguished by the molecular markers. In conclusion,a fingerprint database of 233 lettuce varieties was constructed based on the 22 pairs of core primers and a variety identification system for stem lettuce and leaf lettuce was established,which could be used for rapid identification of lettuce varieties and germplasm resources,genetic diversity analysis and similar variety selection in DUS testing.

  • DENG Jie, WANG Hanlin, LI Youyu, WANG Zie, CHEN Xiaohua, LIU Diqiu
    Abstract (51) PDF (12) RichHTML (25)

    Polygalacturonase-inhibiting proteins(PGIPs)play an important role during response to pathogen infection.A PGIP gene and its promoter were isolated from Lilium regale Wilson,which has strong resistance to Fusarium oxysporum.The open reading frame of LrPGIP was 1 008 bp in length,which encoded a protein with a length of 335 amino acid residues and contained 7 leucine-rich repeat domains.LrPGIP had high homology with PGIP of Elaeis guineensis,Cocos nucifera and Nicotiana sylvestris,which was 91%,94% and 86%,respectively.The LrPGIP showed closer relation with Phoenix dactylifera,Ensete ventricosum,E.guineensis and C.nucifera.LrPGIP was expressed in roots,stems,leaves,flowers and scales.It had the highest expression level in roots and the lowest expression level in scales.The expression level of LrPGIP was induced by F.oxysporum,and the highest expression level was at 72 h after F.oxysporum infection.The plant signal molecules including salicylic acid(SA),jasmonic acid(JA),ethephon(ETH)and hydrogen peroxide induced the expression of LrPGIP.Among them,the expression level of LrPGIP induced by ETH was the highest,which was followed by JA.The length of LrPGIP promoter fragment was 706 bp and had some cis-acting elements,such as hormone and stress responding elements.The expression cassette of the β-glucuronidase(GUS)driven by the LrPGIP promoter was transferred into tobacco for expression,and the GUS activity was obviously induced by the infection of F.oxysporum and Alternaria alternata as well as the stress of HgCl2 and NaCl.It also responded to defense-related plant hormones including JA,SA and ETH.The up-regulation of GUS activity by HgCl2 treatment was the greatest,followed by A.alternata and ETH.These data showed that the LrPGIP promoter activity was induced by plant hormones,biotic and abiotic stresses.The above results indicated that LrPGIP might be an important disease resistance gene in L.regale against F.oxysporum.

  • ZHANG Xiaohan, CHEN Yanliang, MA Xin, ZHANG Shanshan, WEI Shanjun
    Abstract (46) PDF (19) RichHTML (17)

    In order to explore the molecular mechanism of Zoysia grass in resistance to abiotic stresses,in the present study we reported the function of a cold responding gene from Zoysia matrella designated as ZmCOR410. The sequence information of the gene was obtained on the base of transcriptome and genomic data. The expression profile of ZmCOR410 in response to low temperature was detected by qRT-PCR,and the functions of the gene under abiotic stresses were estimated in Arabidopsis and yeast by genetic transformation.The results showed that the CDS of the gene was 927 bp in length,encoding an acid dehydrin that contains 308 amino acids. In the polygenetic tree of COR410 homologues from gramineous grasses,ZmCOR410 had a close relationship with the homologues of Cleistogenes songorica and Eragrostis curvula,two species that were high tolerant to drought stress. In the genomic DNA,there were four copies of core sequence of DRE-cis element in the 1 700 bp region upstream of the CDS of ZmCOR410,and its mRNA was accumulated in leaves exposed to cold. Compared to WT plants,Arabidopsis plants over expressing ZmCOR410 showed reduced injury in leaves after a freezing-temperature exposure,and showed higher survival rates under drought and high temperature stresses. Yeast cells harboring ZmCOR410 were also more tolerant to high temperature stress than the control cells. The results indicated that the product of ZmCOR410 could enhance cells resistance to freezing cold,high temperature and drought stresses,which would help Zoysia matrella going through adverse environments.

  • WANG Ya'nan, LIANG Yanyan, YAN Wenpei, ZHANG Yinping, LI Qiang, WU Xiuju
    Abstract (48) PDF (17) RichHTML (17)

    In order to further mine the enzyme gene related with methyl-eugenol synthesis and to elucidate the secondary metabolism pathway of Asarum heterotropoides. According to the transcriptome database information of Asarum heterotropoides,AhOMT1 gene was screened and cloned,and the corresponding bioinformatics analysis and prokaryotic expression analysis were carried out. The results showed that AhOMT1 contained an open reading frame of 1 089 bp,encoding 362 amino acids,with a theoretical molecular weight of 40.23 ku and an isoelectric point of 5.34. AhOMT1 protein was a hydrophilic protein without transmembrane structure and signal peptide sequence. AhOMT1 gene had dimerization domain and methyltransferase domain. AhOMT1 was close with the O-methyltransferase catalyzing flavonoids and eugenols. The prokaryotic expression vector pET-32b-AhOMT1 was successfully constructed and the recombinant protein of about 60 ku was successfully induced. The optimal induction condition was 0.2 mmol/L IPTG and induced at 16 ℃ for 16 h. Most of the recombinant proteins existed in the form of soluble proteins. AhOMT1 protein was separated and purified with Ni2+ resin,and the maximum amount of protein was obtained by eluting with 200 mmol/L imidazole. AhOMT1 gene of Asarum heterotropoides was cloned for the first time,prokaryotic expression vector was constructed,and the best induction conditions of the protein were screened.

  • LI Hui, KANG Zepei, QIU Caisheng, DAI Zhigang, QIU Huajiao
    Abstract (538) PDF (36) RichHTML (37)

    To provide a solid foundation for studying the biological function of WRKY family members in kenaf in response to salt stress,all members of WRKY family were identified and their expression patterns were analyzed.Physical and chemical properties,phylogeny and conserved functional domains of WRKY gene family members were analyzed by bioinformatics method.The expression characteristics of WRKY gene family members under salt stress were analyzed by RT-PCR.The results showed that a total of 33 WRKY family members were identified,which were unevenly distributed on 12 chromosomes.There were certain differences in the physical and chemical properties of each member,such as amino acid number,molecular weight and theoretical isoelectric point.The conserved sequence WRKYGQK of each member did not change.Phylogenetic tree analysis showed that 33 WRKY family members were divided into 3 groups,GroupⅠ,GroupⅡ,Group Ⅲ,of which Group Ⅲ contained 5 subgroups.The real-time fluorescence quantitative RT-PCR results showed that there were 26 WRKY family members induced by salt stress,of which 23 had positive regulation and 3 had negative regulation.A total of 33 WRKY family members of kenaf were identified,of which 26 WRKY family members were involved in the salt stress response of kenaf.

  • ZHANG Jinyu, XU Xinjuan, CHAO Maoni, ZHANG Xiaohong, WU Xiangyuan, GAO Jitao, HUANG Zhongwen
    Abstract (132) PDF (25) RichHTML (121)

    Zinc finger proteins are transcription factors widely studied in eukaryotes,and play important roles in plant growth and development,and responses to stresses.In order to deeply understand the gene function of zinc finger protein in soybean,the full-length CDS sequence of GmZAT12 gene was cloned from Shangdou 1201,and the characteristics of coded protein by this gene was predicted by bioinformatics analysis.The subcellular localization of GmZAT12 protein was detected by the tobacco epidermal injection system, and the expression pattern of GmZAT12 gene in different tissues of soybean and abiotic stress was analyzed by Real-time PCR (qRT-PCR) technology. The results showed that GmZAT12 CDS contained 516 bp,encoding 171 amino acids and the molecular weight of the protein was 19.264 28 ku with a theoretical isoelectric point(pI)of 9.02.Its main components were random coils and α-helices and it contained 20 phosphorylation sites, mainly serine phosphorylation sites.Sequence analysis indicated that GmZAT12 possessed two conserved C2H2 zinc finger domains.The result of subcellular location indicated that GmZAT12 protein was localized in the nucleus.The results of qRT-PCR showed that CmZAT12 gene expressed mainly in roots,leaves and seeds of soybean,while low expression in flower and stem, and was induced by high temperature,low temperature,NaCl and ABA.The fact implied that this gene might be involved in abiotic stress signaling pathways.

  • JIA Zhiqiang, XU Yunyu, GAO Xue, TAO Hongzheng, CHEN Zengmin, LIU Yating, LI Yongzhong
    Abstract (16) PDF (6) RichHTML (11)

    In order to study the response mechanism of pepper CaWRKY30 transcription factor and Tomato spotted wilt orthotospovirus,it was experimental materials with pepper Xiangyan 11.The CaWRKY30 coding sequence was obtained by RNA extraction,RT-PCR,split gel and cloning.Biological information analysis results showed that CaWRKY30 full length was 1 122 bp,encoding 373 amino acids,the gene encoded protein contains 1 WRKY conservative domain and 1 C2H2 domain,belonged to a typical Ⅱ(e)subfamily member.System evolution analysis showed that the relative relationship with the potato StWRKY22 amino acid sequence was recently.It was found that CaWRKY30 was positioned in the nucleus and cell membranes in its cigarette seedlings,and leads to cell membranes.The results of Real-time fluorescence quantitative PCR analysis showed that the viral accumulation of Tomato spotted wilt orthotospovirus mechanical friction-vaccination was found that the viral accumulation was gradually increased from 1 to 14 days after inoculation,and virus accumulation reached its maximum in 14 days,after inoculation 14 days,viral accumulation gradually declined.At the same time,CaWRKY30 was induced by Tomato spotted wilt orthotospovirus,when the inoculation 1-14 days,the CaWRKY30 expression was raised,and the peak was reached in 14 days,the expression in 14 days gradually decreased.In summary,it obtained the CaWRKY30 transcription factor gene sequence,which was located in the nucleus and cell membrane,and preliminarily explained the expression trend of CaWRKY30 transcription factors under the stress of Tomato spotted wilt orthotospovirus.

  • JI Ying, SUN Feng, WU Shuhua, LI Shuo, TU Liqin, GAO Danna, CUI Xiaoyan, CHEN Xin, JI Yinghua, GUO Qingyun
    Abstract (23) PDF (14) RichHTML (11)

    To characterize the genomic structure of Cowpea mild mottle virus (CpMMV)Jiangsu isolate and clarify its taxonomic status and evolutionary characteristics,4 pairs of specific primers were designed and the genomic fragments were amplified using cDNA obtained from reverse transcription of total RNA of a soybean sample collected from Jiangsu as a temple.The amplified products were cloned and sequenced,and the obtained fragments were assembled to get the full genomic sequence.Sequence analysis revealed the genome of Jiangsu isolate were 8 194 bp in length,containing 6 ORFs and 2 untranslated regions(UTRs):5' UTR(72 nt)and 3' UTR(117 nt).Among the six proteins encoded by CpMMV,CP shared highest amino acid sequence identities with other isolates(96.5%-100.0%),while RdRp(81.1%-98.2%),TGB1(81.0%-97.0%)and TGB3(80.9%-95.6%)shared relatively low sequence identities,indicating CP might be a conservative gene and RdRp,TGB1,TGB3 might show good diversity in different isolates.The phylogenetic analysis based on the whole genomic sequence revealed that Jiangsu isolate was clustered with other CpMMV isolates in a branch and it shared highest nucleotide sequence identities with 2 Chinese isolates(98.2% with Anhui isolate,96.0% with Hainan isolate),compared with other foreign isolates such as the United States,Brazil,India,Mexico,Kenya and so on(<82.7%).These results clarified the genomic structure of Jiangsu isolate,which shared similar character with other CpMMV isolates and phylogenetic analysis revealed Jiangsu isolates clustered with other isolates from China with higher sequences identities,compared with foreign isolates.

  • GUO Fangrui, LIU Yi, YANG Kexin, CHU Meirong, ZHANG Quanfa, ZHANG Yafei, WANG Shutong, CAO Keqiang
    Abstract (25) PDF (11) RichHTML (8)

    Based on the establishment of the T-DNA insertion mutant library,in order to screen out the mutants with significantly decreased pathogenicity and clarify the insertion site information,the phenotypic and pathogenicity differences of the mutants were tested.The results laid a foundation for further elucidating the pathogenic molecular mechanism of Fusarium oxysporum.In our previous work,a T-DNA insertion library of F.oxysporum HS2 was constructed via optimized Agrobacterium tumefaciens-mediated transformation(ATMT).In the present study,totally 300 transformants generated by T-DNA insertions were randomly selected and the alien gene was detected via PCR.The colony morphology,growth rate,sporulation,mitosis stability and other biological traits were observed and measured. The pathogenicity differences of mutants were compared by begonia radicle inoculation method. The flanking sequence analysis of insertion site and Southern Blot analysis were performed on the mutants with significantly weakened pathogenicity.The results showed that 283 tested mutants had inserted the target gene with green fluorescence.After the T-DNA insertion mutant was continuously transferred for five generations,it could be stably inherited on the medium containing hygromycin B,and the colony morphology and color had no significant change.In the pathogenicity test of 283 mutants,the pathogenicity of 87.9% mutant strains was weakened.Among the attenuated mutants,HS2-520,HS2-1016 and HS2-2109 almost lost pathogenicity.Eight mutants with significantly weakened pathogenicity were selected for Southern Blot analysis.The results showed that six mutants were single-copy inserted strains and one was double-copy inserted strains.The TAIL-PCR flanking sequences of the eight mutants were amplified.After sequencing by the company,12 flanking sequences were obtained after comparison with the genomic sequence of wild strain HS2.And three of them were identified as gene sequences of F.oxysporum.Six single copy insertion mutants with significantly reduced pathogenicity were obtained,and flanking sequence information of insertion sites of two mutants were confirmed.

  • AN Qingming, WANG Xing, WU Zhenyang, MENG Jinzhu, ZHAO Yuanyuan, SONG Xingchao
    Abstract (28) PDF (13) RichHTML (14)

    Carcass muscle growth and development is an important evaluation index that affects the breeding efficiency of goats.The aim of this study was to analyze key candidate genes for muscle growth and development of Guizhou white goats with different genders by RNA-Seq,and provide new reference information for the research on the muscle growth and development of Guizhou white goat.We collected the longissimus dorsi muscle to extract RNA and determined the slaughter performance of 6 Guizhou white goats with different genders,which were two years old and were feeded in same level.Then screened differentially expressed genes,analyzed the signal pathway of related genes.Meanwhile,RT-qPCR was used to verify the screened differentially expressed genes.The results showed that the raw reads which obtained by sequencing were filtered,a total of 78.99 Gb Clean Data were obtained in six samples.Each single sample was obtained Clean reads between 83 030 104 and 95 739 024,and the comparison efficiency with the reference genome was between 93.75%-94.79%,a total of 25 089 transcripts were obtained,of which 1 077 were significant differentially expressed genes,and 194 were new transcripts.Among them,563 were up-regulated and 514 were down-regulated in longissimus doris tissue of male sheep.GO functional enrichment analysis was performed on 1 077 differentially expressed mRNAs,which 587 were significantly enriched(Q-value≥0.05) and concentrated in 35 groups of three major categories.KEGG signaling pathway analysis revealed that annotated differential genes participated in 243 signaling pathways,among which the PI3K/AKT signaling pathway were the most enriched,including 32 genes,among them,17 genes were significantly up-regulated and 1 gene was down-regulated,meanwhile,the coexpression score of COL4A1 and COL4A2 genes were the highest in this pathway,the ITGAV protein interaction with other proteins was the most abundant.6 genes that may be closely related to muscle growth and development in white goat were screened out,among which FHL3,WFIKKN2 and SOX6 genes were up-regulated in male longissimus doris tissue,QSOX2,MYH2 and LAP genes were down-regulated.RT-qPCR showed that the expression trend of the 6 candidate genes were consistent with high-throughput sequencing results,and the expression levels differences of these genes were significant,which showed the sequencing results were reliable.Totally,1 077 differentially expressed mRNAs,which 194 were new transcripts were obtained of longissimus doris tissue in Guizhou white goat with different genders by RNA-Seq technology,the screened differentially expressed genes and PI3K/AKT signaling pathway were related to the growth and development of longissimus dorsi muscle of Guizhou white goats.

  • RAN Li, LÜ Jinshi, ZHANG Hao, WANG Yong, ZHU Jiangjiang, LI Yanyan, MENG Qingyong, LIN Yaqiu
    Abstract (16) PDF (14) RichHTML (13)

    In order to clarify the role of APOC3 gene in the differentiation of intramuscular adipocytes of goats,the APOC3 gene sequence was cloned by RT-PCR,and the biological information was analyzed by online software.Quantitative Real-time PCR(qPCR)was used to detect the expression of APOC3 gene in intramuscular adipocytes of goats at different tissues and differentiation stages.APOC3 overexpression vector was constructed by double-enzyme digestion method,and the effects of APOC3 gene overexpression on lipid droplet accumulation were determined by oil red O staining.At the same time,the mRNA relative expression level of marker gene of adipogenic differentiation was detected by qPCR.The results showed that the ORF region of APOC3 was 294 bp in length and encoded 97 amino acids with a functional domain of 23-88 aa. APOC3 was expressed in 14 tissues including heart,liver and spleen,and so on,which the highest level of APOC3 was found in liver.APOC3 expression was the highest at 48 h and extremely significantly higher than that before differentiation.After APOC3 was overexpressed,lipid drop accumulation increased,and the relative expression levels of marker genes SREBP1 and CEBPβ were extremely significantly up-regulated,PPARγ was significantly up-regulated,and Pref-1 was significantly down-regulated.APOC3 might promote the differentiation of intramuscular adipocytes by up-regulating SREBP1,CEBPβ,PPARγ and down-regulating Pref-1.

  • YANG Xiaobei, CHEN Eryong, LI Chengwei
    Abstract (29) PDF (13) RichHTML (22)

    LMI1 is a key gene in the regulation of serrated leaf development.In order to study the mechanism of okra leaf development in cotton,we cloned GaLMI1-like gene and GaLMI1-like promoter by PCR amplification technology from Shixiya 1,which is an Asia cotton species of a genome.The sequence lengths were 681,1 439 bp,respectively.Domain analysis found that GaLMI1-like contained a homeobox domain.Then,we constructed a reorganization overexpression vector p6MYC-GaLMI1-like.The transgenic Arabidopsis showed incised leaf shape when GaLMI1-like was overexpressed in Arabidopsis.Moreover,we analyzed the cis-acting elements of the promoter sequence of GaLMI1-like.It was found that it had basic acting elements,such as CACA-box and TATA-box,and also had photoresponsive element,root,stem and mesophyll-specific expression elements.GUS fusion expression vector of GaLMI1-like promoter was constructed and transformed into Arabidopsis thaliana.GUS staining results showed that the promoter could drive GUS gene expression in root column,stem and leaf,and the expression was especially high in leaf.These results indicated that GaLMI1-like had the function of regulating the formation of incised leaves,and its function was realized by the strong expression of GaLMI1-like regulated by GaLMI1-like promoter in cotton leaves.

  • CHENG Chunhong, YOU Jingfan, CAI Zhaoming, YE Chunhong, CHEN Li
    Abstract (27) PDF (6) RichHTML (9)

    During the growth and development stages,tuber mustard is always affected by abiotic stresses.As a plant stress hormone,ABA plays an important role in regulating plant response to abiotic stress.BjuEAR1-1 was a homologue of negative regulator EAR1 in ABA signaling pathway,and its mutant ear1 exhibited an ABA sensitive phenotype.Therefore,to explore the biological function of BjuEAR1-1 will provide a direction for the regulation mechanism of resistance to adverse environment stresses,and also provide an important theoretical basis for the effective use of gene resources to cultivate new varieties of BjuEAR1-1 with resistance to adverse stresses and stable yield.The BjuEAR1-1 coding sequence was cloned by PCR method and the plant overexpression vector 35S∷PTF101-GFP-BjuEAR1-1 was constructed by enzyme digestion and ligation method.The subcellular localization of BjuEAR1-1 was analyzed by transient transformation technology of tobacco leaves.The cis-element of BjuEAR1-1 gene promoter was analyzed,and the gene expression patterns of BjuEAR1-1 under different abiotic stresses and different tissues and organs were detected by fluorescence quantitative PCR.The result of subcellular localization assay showed that BjuEAR1-1 was localized in the nucleus and cytoplasm of tobacco.BjuEAR1-1 was expressed in root,stem,leaf,flower,seed pod and swollen stem of tuber mustard,with the highest expression in root.The expression level of BjuEAR1-1 was significantly induced under low temperature and ABA stresses,and there was no significant difference under salt stress. BjuEAR1-1 was localized in the nucleus and cytoplasm and regulated the response of tuber mustard to low temperature and ABA stresses.

  • ZHANG Jingjing, LI Bing, SHI Yufan, GAO Xiurui, PAN Xiuqing, SONG Xue, WU Yanrong
    Abstract (33) PDF (14) RichHTML (14)

    In order to analyze the molecular mechanism of different watermelon peel firmness,and provided a theoretical basis for discovering key genes related to watermelon peel firmness,the high firmness(901)4-1-1-M and low firmness BSH with similar growth period but significant difference in peel hardness were used as experimental materials.The peel with the maximum difference 30 days after pollination was selected for transcriptome sequencing analysis and the Illumina HiSeqTM sequencing platform was used to analyze the molecular mechanism of different watermelon peel firmness,Real-time fluorescence quantitative PCR (qRT-PCR) was used to verify the sequencing results.A total of 1 085 differentially expressed genes (DEGs) were screened by transcriptome sequencing,including 555 up-regulated genes and 530 down-regulated genes.Gene Ontology(GO)analysis showed that 1 085 DEGs were significantly enriched in cell components,molecular functions and biological processes,including cell wall,cell periphery,external encapsulation structure,extracellular region,tetrapyrrole skeleton,redox enzyme activity,transferase activity and pectin esterase activity.Kyoto Encyclopedia of Genes and Genomes (KEGG)analysis indicated that 19 DEGs,including Cla97C04G075800,Cla97C02G044950,Cla97C09G165820,Cla97C10G195660,Cla97C01G025380,etc.,were enriched in the most significant enrichment biosynthesis of phenylpropanoid,which eventually lead to the metabolites of Syringyl lignin,5-Hydroxy-guaiacy lifnin,Guaiacy lifnin and P-Hydroxy-phenyl lifnin.The correlation coefficient of DEGs expression levels by qRT-PCR and RNA-seq data was 0.791,which indicated that the transcriptome test data were reliable.This study explained the reason of watermelon peel firmness difference between(901)4-1-1-M and BSH from the transcriptional level.

  • CHEN Jing, HU Rong, LIU Yong, QIN Yi, XIONG Xinghua
    Abstract (28) PDF (10) RichHTML (24)

    To further understand the role of the promoter in the regulation of the expression of the Brassica napus FIL gene(BnaFIL),it used the DNA extracted from the leaves of Brassica napus as a template based on the genomic data of Brassica napus.For cloning,the length was 1 326 bp.Using PlantCARE online analysis software to perform bioinformatics sequence analysis on the promoter sequence,the results showed that the sequence contained some conserved DNA modules involved in the photoreaction and essential elements for core promoters such as CAAT-box and TATA-box.Expression related cis-acting regulatory elements CAT-box and photosensitive response elements.Replace the CaMV35S promoter on the pBI121 plant vector with the promoter sequence,fused the promoter with the GUS gene to obtain the pBnaFIL-GUS expression vector,and transfer the vector into Arabidopsis thaliana by the method of Agrobacterium inflorescence.Flower promoter recombinant plasmid-positive transgenic lines and late flower promoter recombinant plasmid-positive transgenic lines.After that,GUS staining analysis was performed on the transgenic Arabidopsis plants,and the expression effect of the promoter was tested.Finally,the GUS gene expression was found in different transgenic Arabidopsis plants.The results showed that there were differences in promoter expression between early-flowering materials and late-flowering materials.The promoters of early-flowering materials could drive gene expression better than those of late-flowering materials.It was inferred from this that the driving effect of the promoter regulates the early and late flowering of rape,resulting in different expression effects of the FIL gene in different materials.

  • TANG Zhongli, LU Xiaonan, ZHAO Rui, LIU Qinghua, LI Meilan, LEI Fengjin, XU Xiaoyong
    Abstract (29) PDF (11) RichHTML (21)

    Carotenoids are the most widely distributed class of pigments in nature,which endow plants flowers,fruits and other organs with bright colors.The synthesis process of carotenoids contains a variety of metabolic enzyme genes.In order to reveal the molecular mechanism of carotenoid accumulation in yellow peeled zucchini,lay a foundation for further studying the function of carotenoid synthase gene in zucchini,this study identified zucchini carotenoid anabolism family genes based on the zucchini genome database,and further screened and cloned the key enzyme genes for carotenoid synthesis and metabolism by publicly published transcriptome data and qRT-PCR verification.The results showed that 48 carotenoid metabolizing enzyme gene members were detected in zucchini genome;and two key genes PSY1 and LCYE2 were screened out and showed different expression during the different developmental stages based on the transcriptome data of Sweet REBA/Lady Godiva.qRT-PCR analysis also showed that, except for PSY1 gene 0 d and LCYE2 gene 2 d, the relative expression of PSY1 and LCYE2 in different developmental stages of yellow peeled zucchini were significantly higher than that of white peeled zucchini.The full-length CDS of PSY1 and LCYE2 genes were cloned and sequencing analyzed from white and yellow peeled zucchini,respectively,which suggested that PSY1 genes of both materials were 51 bp more than the predicted coding sequence in zucchini reference genome,and were all consistent with the published sequence of PSY gene(GenBank number:XM_023695146.1),while the sequences of LCYE2 gene was not significantly different from the reference genome.Phylogenetic tree analysis showed that zucchini PSY1 was homologous to the reported Cucurbita moschata PSY(JN176311.1)with the best similarity as high as 98.80%.

  • DAI Mengyuan, GAO Mei, LI Wenchang
    Abstract (37) PDF (28) RichHTML (22)

    Gibberellin oxidase gene(GAox)is a key enzyme in the synthesis and regulation of gibberellin,and effect on plant height through regulating active GA level.To analyze the gene of gibberellin oxidase in castor,we identified gibberellin oxidase gene(RcGAox)from the castor bean whole genome by bioinformatics,and analyzed physicochemical properties,conserved domain,phylogeny,promoter cis-acting element.The RcGAox gene expression pattern was analyzed by tissue specific expression of online database and apical tender stem transcriptome sequencing.A total of 30 gibberellin oxidase genes were identified from the castor genome,including 7 RcGA2ox,4 RcGA3ox,and 19 RcGA20ox.The molecular weight was ranged from 26.12 to 44.31 ku,and the isoelectric point was ranged from 5.06 to 7.82.Gene structure analysis showed that the number of introns was ranged from 1 to 2.Protein conserved domain analysis showed that all the genes shared conserved Motif 1,Motif 2 and Motif 4.Phylogenetic analysis showed that RcGAox genes were clustered into five subfamilies Ⅰ,Ⅱ,Ⅲ,Ⅳand C20 GA2ox,and subfamiliesⅠ,Ⅱ,Ⅲ correspond to GA2ox,GA3ox and GA20ox respectively.Promoter cis-acting element prediction showed that light-related elements had largest number and uniform distribution in predicting region,and 18 genes had 1 to 2 gibberellin-related element.There were 7,2,and 1 RcGAox genes specifically expressed in endosperm,male flowers and leaves respectively.Transcriptome sequencing showed that 5 genes were expressed in tender stems.It was supposed that RcGA2ox7,RcGA20ox1 and RcGA20ox14 might be the main gene involved in gibberellin synthesis pathway to regulate castor plant height.These results might provide theoretical basis for further studies on the RcGAox gene function in castor bean.

  • ZHANG Zongxiang, HUANG Zhengrong, WU Xuefan, LIU Nannan, LI Xiaoxiao, DONG Zhaorong, SONG He
    Abstract (506) PDF (22) RichHTML (19)

    Yield and nitrogen accumulation of maize will decline under soil acidification,but the physiological mechanism is not clear.Field experiment was conducted with four different soil acidity gradients:nautral acid(pH=7,CK),weak acid(pH=6,T1),medium acid(pH=5,T2)and strong acid(pH=4,T3),comparing yield,nitrogen accumulation,grain protein content,nitrogen metabolism-related enzyme activities,gene expression,nitrate nitrogen,ammonium nitrogen,soluble protein,free amino acid content in leaf and stem of maize.The results showed that compared with CK,the yield of T1,T2 and T3 treatments declined by 4.2%,30.7% and 52.3%,respectively.Grain number per spike decreased by 1.8%,28.1% and 42.8%;grain protein content showed a downward trend with T3 treatment significantly reduced by 14.5%.At the big flare stage,with the increase of soil acidity,nitrogen accumulation in leaves showed a downward trend,it was significantly decreased in T2 and T3 treatment by 28.1% and 56.2%,respectively.In stem,the nitrogen accumulation increased firstly and then decreased.Compared with CK,T1 treatment was significantly increased by 33.1%,and T3 treatment significantly decreased by 65.4%.At the big flare stage, the activities of nitrate reductase, glutamate dehydrogenase and glutamate synthase in leaf and stem under T3 treatment were significantly higher than those in CK, while the activities of glutamine synthase in leaf were significantly decreased. The amino acids in stem decreased first and then increased.With the increase of soil acidity,the expressions of ZmGln2 and ZmFd-GOGAT were up-regulated,which promoted the assimilation of NH 4 + released by photorespiration and NH 4 + produced by NO 3 - reduction;the down-regulated expression of ZmGln1.2-ZmGln1.4, ZmNADH-GOGAT2 in leaf and ZmNADH-GOGAT1 in stem decreased the assimilation of NH 4 + released by catabolism.By up-regulating or down-regulating the expression of relevant genes,maize could promote the production of more free amino acids and soluble proteins during nitrogen metabolism to resist acidification stress,but also reduced the nitrogen accumulation,resulting in lower yield and grain protein content.

  • TANG Long, ZHAO Yuwei
    Abstract (373) PDF (154) RichHTML (57)

    Application of some hormonal signaling compounds,as brassinolides and their derivatives,could significantly improve the salt stress resistance in plants.The purpose of present work was to test whether the over-expressing of Methylsterol monooxygenase gene(SMO),a key gene coding a bio-synthesizing enzyme of sterol in plants,could promote the salt-stress tolerance of target plants.PnSMO1.1,gene encoding of methylsterol monooxygenase in the plant species of Pharbitis nil was firstly cloned and then used as target gene for following genetic transformation process,while wild-type Pharbitis nil seedlings were used as the receptor plants for constructing of the transgenic lines which over-expressed PnSMO1.1 genes.In this work,the PnSMO1.1 gene transformed Pharbitis nil lines were constructed via an ovary injection transformation method.Plantlets from individual PCR identified transgenic plant lines were used as materials to detect vegetative growth figures and some pivotal physiological indicators,for instance,contents of malondialdehyde (MDA),relative conductivity,as well as castasterone and 6-deoxo-castasterone contents in cells under stresses with gradient NaCl conditions varied from 0—200 mmol/L.The results showed that the over-expression PnSMO1.1 significantly improved the relative growth of roots and hypocotyls in transformants than in wild-type (WT) or vacant plasmid transformed control (BL) plants under 100—250 mmol/L NaCl stresses.Compared to WT and BL seedlings,significantly higher accumulation of 6-deoxo-castasterone,but lower relative conductivity (rEC) values,castasterone accumulation or MDA contents were found in transgenic lines under various NaCl stresses.Stresses such as salinity,drought and freezing temperatures,had severely suppressed the vegetative growth of plants,as well as their yields.These results highlighted that over-expression of the PnSMO1.1 gene could significantly improve the salinity-stress resistance of transgenic plants by delicately adjusting the dynamic homeostasis of brassinolides in cells,and protecting the structural integrity of plasma membrane.

  • HAN Zhenghong, DUAN Yuxuan, XU Shanbin, WANG Jingguo, LIU Hualong, YANG Luomiao, JIA Yan, XIN Wei, ZHENG Hongliang, ZOU Detang
    Abstract (135) PDF (92) RichHTML (40)

    In order to promote the breeding of long-grain japonica rice varieties,two knock-out vectors,pYLCRISPR/Cas9-GS3-RNA and pYLCRISPR/Cas9-GS3-GS9-RNA,were constructed with japonica rice varieties Dongfu 139,Longjing 31 and Dongnong 427,the genes of GS3 and GS9 were edited by Agrobacterium tumefaciens in vitro.In the end,GS3,GS9 and GS3,GS9 double-gene mutations without T-DNA elements were obtained in the T2.The results showed that the grain length and 1000-grain weight of gs3 mutant plants of three varieties were significantly increased compared with those of wild type,grain width,seed setting rate and grain number per panicle did not change significantly,grain length of gs9 mutant increased significantly,and grain width decreased significantly,while 1000-grain weight,seed setting rate and grain number per panicle did not change significantly,and grain length of gs3gs9 mutant increased more than gs3 and gs9,at the same time,grain width decreased significantly,1000-grain weight increased significantly,while seed setting rate and grain number per ear did not change significantly.To sum up,three japonica rice varieties,Dongfu 139,Longjing 31 and Dongnong 427 were improved by using CRISPR/Cas9 technique,which accelerated the breeding process of new long-grain japonica rice varieties.

  • SUN Lili, JIN Xiujuan, ZHAO Kai, Md Ashraful Islam, LU Juan, WANG Shuguang, SUN Daizhen
    Abstract (127) PDF (128) RichHTML (31)

    Cysteine protease,a kind of hydrolase in the process of protein degradation,participates in the senescence and maturation of plants and plays an important role in the growth and development of plants.In this study,a senesence-specific cysteine protease(SAG39)was screened from the previously obtained transcriptome data of wheat at senescence stage.In order to study the role of TaSAG39 gene in wheat at senescence,the gene structure and expression pattern of TaSAG39 were analyzed by bioinformatics analysis and Real-time fluorescence quantitative(qRT-PCR)technology.The results showed that the amino acid sequence encoded by the cysteine protease gene SAG39 had the closest relationship with the ancestral species of wheat,such as Aegilops tauschii,Triticum dicoccoides and Triticum durum,and it included the unique active site Cys-His-Asn and EFNIN structures of the papain sub-family.The full-length genomes of TaSAG39-5A,TaSAG39-5B and TaSAG39-5D were 1 455,1 435,1 439 bp,respectively,and the length of the coding sequence was 1 041,1 050,1 038 bp,respectively,with each containing two exons and one intron.The TaSAG39s proteins consisted of 346,349,345 amino acids.With relative molecular weight of about 37 ku and isoelectric point of 5.53—5.67,these proteins were stable negatively charged hydrophilic proteins.Its main components were random coils and α-helix,and there were signal peptides in the N segment of the protein.It had the conserved Inhibittor_I29 and Petidase_C1 domains,and contained 35—37 phosphorylation sites,mainly serine and threonine phosphorylation sites.TaSAG39 protein may interact with cysteine protease inhibitors and 14-3-3 protein.The promoter contained cis-acting elements relative to abscisic acid,methyl jasmonate,auxin,light,drought,low temperature and stress response.The results of qRT-PCR confirmed that gene TaSAG39 was induced to express by natural aging,darkness,drought,methyl jasmonate,high temperature and auxin,among which natural aging was the most significant.

  • JING Fanli, ZHANG Peipei, MIAO Yongping, CHEN Tao, LIU Yuan, YANG Delong
    Abstract (130) PDF (135) RichHTML (24)

    In order to clarify the expression characteristics and biological functions of the sucrose phosphate phosphatase gene(SPP),and further understand the regulatory mechanism of SPP involved in sucrose biosynthesis.Herein,three TaSPP homologs,TaSPP-5A,TaSPP-5B and TaSPP-5D,located on the fifth homologous group,were cloned using the cDNA of wheat variety Jinmai 47 as the template.The physical and chemical properties,gene structure,cis-acting elements,phylogenetic tree and protein conserved domains of TaSPP were analyzed by the method of bioinformatics.The expression pattern of TaSPP was analyzed by qRT-PCR.The results showed that TaSPP-5A,TaSPP-5B and TaSPP-5D contained eight exons and seven introns.TaSPP-5A and TaSPP-5D encoded 422 amino acids,while TaSPP-5B encoded 413 amino acids.Phylogenetic analysis showed that TaSPP in wheat and its related species belonged to the same evolutionary branch with highly genetic similarity.The specific expression analysis showed that TaSPP genes were expressed in roots,stems,flag leaves,leaf sheaths,flower and seeds,whereas the higher expression levels were identified in flag leaves and stems.The expressions of TaSPP gene could be induced by ABA,PEG-6000,NaCl and IAA,indicating that TaSPP genes could play an essential role in abiotic stress tolerance in wheat.

  • ZHAO Huiying, YANG Guangkai, GAO Yan, ZHANG Xiaojun, HAO Yanyan
    Abstract (97) PDF (66) RichHTML (16)

    Gene STAYGREEN(SGR)plays an important role in the process of plant chlorophyll degradation and metabolism.To explore the biological function of gene SGR2,MdSGR2 gene sequence was cloned from the peel of Granny Smith by RT-PCR.The homology,physicochemical properties,protein structure and cis-acting elements of the promoter were predicted and analyzed by bioinformatics software.The plant overexpression vector was constructed by double enzyme digestion.The results showed that the complete open reading frame(ORF)cDNA of gene MdSGR2 was 840 bp.Encoding 279 amino acids in total,it belonged to the Staygreen superfamily.Physicochemical analysis showed that the total molecular weight of the protein was 31.27 ku,and the isoelectric point pI was 8.52,indicating that the protein was an unstable hydrophilic protein.Homology analysis showed that MdSGR2 encoded amino acid sequence was of the highest homology with Pyrus ussuriensis,reaching 84.12%.The protein structure analysis showed that the secondary structure and tertiary structure of MdSGR2 was determined structurally to be 40.55% α-helix and 44.44% random coil.Promoter analysis showed that the promoter region of the gene contained cryogenic cis-acting elements,photo-responsive elements and induced cis-acting elements such as abscisic acid,salicylic acid and methyl jasmonate response elements.These results suggest that gene MdSGR2 may be regulated by many factors such as environment and hormones.The plant overexpression vector pCAMBIA2301-MdSGR2 was successfully constructed.

  • LI Hui, WEN Chunxiu, LIU Lingdi, WEN Saiqun, TANG Yinghong, JIANG Tao
    Abstract (89) PDF (26) RichHTML (23)

    In order to investigate the function of 5-phosphomevalonate kinase PMK gene in biosynthesis pathway of terpenoids in Perilla frutescens L.,we analyzed the transcriptome data of Perilla frutescens and mined the reference sequence of PMK.The PMK gene was cloned from Perilla frutescens by gene cloning technique,and was analysed using bioinformatics and Real-time PCR(qRT-PCR).The results showed that the ORF of PfPMK gene was 1 524 bp,encoding 507 amino acids.Bioinformatics analysis showed that the molecular weight of PfPMK was 54.73 ku and the isoelectric point was 5.20,which was a hydrophilic protein.The amino acids of PfPMK were higher homologous with SmPMK,SsPMK,SbPMK and PvPMK,which indicated PfPMK protein was highly conserved in the evolutionary process.The phylogenetic analysis showed that PfPMK was closely related to PMK of Salvia miltiorrhiza and Salvia splendens.WoLF-PSORT predicted that PfPMK protein might be located in the plasma membrane or endoplasmic reticulum.Real-time fluorescence quantitative PCR results showed that PfPMK gene was expressed in roots,stems and leaves of Perilla frutescens,and the expression level in roots was higher than that in leaves and stems.The fluorescence quantitative PCR analysis showed that the expression level of PfPMK gene was higher in the middle and late September.The full length of PfPMK gene was first cloned from Perilla frutescens.Bioinformatics analysis showed that PfPMK gene belonged to 5-phosphomevalonate kinase gene and participates in the biosynthesis of terpenoids in Perilla frutescens.

  • SHI Jianlei, XIONG Zili, SU Shiwen, WANG Kelei, ZAI Wenshan
    Abstract (90) PDF (45) RichHTML (19)

    To explore bacterial wilt resistance genes,RNA sequencing was used to characterize the transcriptomes of resistant and susceptible tomato inbred lines with Ralstonia solanacearum inoculation(RsI).The results showed that a total of 75.02 Gb high-quality data were generated in 12 libraries.With the fold change(FC)≥2 and false discovery rate(FDR)<0.01 as the standard,970 and 695 differentially expressed genes(DEGs)were identified in the two tomato lines,respectively.The 1 312 DEGs accounted for 3.71% of the total.Among them,the numbers of up-regulated genes were 457 and 450,respectively,totaling 693;the numbers of down-regulated genes were 513 and 245,respectively,totaling 621.Among these DEGs,836 genotype-specific DEGs were highlighted.These DEGs were mainly divided into 47 functional groups such as metabolism,regulation,response,binding,and catalysis,and 88 metabolic pathways such as DNA replication,secondary metabolite synthesis,plant-pathogen interaction,and signal transduction by GO and KEGG annotation.Specifically,4 NBS resistance genes,6 plant-pathogen interaction genes,11 plant hormone signal transduction genes,22 defense response genes,32 protein kinases,65 transcription factors,and several other important functional genes were involved,indicating that they played important roles in response to Rs.Promoter analysis revealed that these genes possessed multiple defense and stress response elements.The output was confirmed using RT-qPCR for 50 representative genes.It was found that more than half of the genes were consistent with RNA-seq in expression.Solyc02g086980.3 and Solyc04g011670.3 might be involved in the resistance response,whereas Solyc01g073985.1,Solyc09g092580.4,Solyc09g098100.4,and Solyc10g081300.1 might be the opposite.Together,these gene expression profiles serve as fundamental information to understand the potential molecular basis in the response to Rs in tomato,and facilitate the application of related resistance genes in breeding.

  • ZHU Yiming, LIU Yanxiao, HE Jiuqing, DUAN Lingtao, ZHOU Erxun
    Abstract (66) PDF (10) RichHTML (20)

    Colletotrichum higginsianum is a hemibiotrophic fungus that causes cruciferous anthracnose, a serious damage to the flowering Chinese cabbage industry in South China. Effectors secreted by pathogens can help pathogens invade host plants and play an important role in the interaction between pathogens and plants. To investigate the role of C. higginsianum effectors in the pathogenesis of cruciferous anthracnose. Based on previous research results from the Laboratory of Tropical and Subtropical Fungi, South China Agricultural University, a potential effector ChEP085 with a 21 amino acid signal peptide at the N-terminus was screened out. To clarify the function of this effector, the cDNA of Arabidopsis thaliana Col-0 infected by C. higginsianum was used as the template, and the expression of ChEP085 gene was determined by qRT-PCR. It was found that the gene ChEP085 had the maximum expression after 24 h of infection. The gene ChEP085 was verified by Agrobacterium tumefaciens mediated Potato virus X(PVX) tobacco transient expression system. The results showed that the gene could stably induce tobacco cell necrosis. The gene ChEP085 was constructed into the enhanced green fluorescent protein vector pBinceGFP and transiently expressed on tobacco leaves for subcellular localization. It was found that the gene was localized in cytoplasm and cell membrane. After deletion of the signal peptide located at the N terminus, the transiently expressed effector ChEP085 was distributed in the nucleus, cell membrane and cytoplasm, indicating that the signal peptide affects the localization of ChEP085. Finally, the gene ChEP085 was knockout by homologous recombination technology, and it was found that the deletion of this gene had no significant effects on the colony morphology and mycelial growth rate of C. higginsianum, but it would lead to the reduction of conidiation and significantly weakened the pathogenicity of C. higginsianum, indicating that ChEP085 play an important role in the conidiation and pathogenesis of C. higginsianum.

  • WANG Jiarong, DONG Rui, ZHANG Mengyu, GAO Pu, ZHANG Peipei, LI Zaifeng, LIU Daqun
    Abstract (76) PDF (23) RichHTML (22)

    To identify leaf rust resistance genes in 40 wheat materials from the International Maize and Wheat Improvement Center (CIMMYT), combined with pedigree analysis, gene derivation and molecular marker detection, 36 known disease resistance gene vector varieties and 40 wheat materials were vaccinated with 17 physiological species of leaf rust bacteria with different toxicity.By comparing the infected types of the tested wheat lines and the known rust resistance genes,the possible known resistance genes in the tested wheat materials could be postulated.At the same time,12 markers closely linked with known resistance genes were used to detect the 40 CIMMYT lines,and the results could be verified with the gene postulation.Furthermore,40 tested lines were planted in Baoding,Hebei Province and Zhoukou,Henan Province,respectively,and inoculated with mixed Pt races for leaf rust evaluation at adult plant stage in the field.The results showed that 7 wheat lines contained Lr1,Lr10 was found in 9 lines,and each of Lr11 and Lr34 was present in 10 lines.Each of Lr14a,Lr15 and Lr26 was postulated to present in 2,4 and 3 lines,respectively.Each of Lr37 and Lr46 was identified in 22,39 wheat lines,respectively,by using molecular marker detection.In the field test 22 wheat lines showed adult plant resistance to leaf rust.

  • HAN Xiaoyong, JIANG Lu, YIN Jianmei, JIN Lin, ZHANG Peitong
    Abstract (100) PDF (32) RichHTML (20)

    In this study,the transcription level of resistance genes to the infection of Colletotrichum gloeosporioides were explored at the molecular level and provided candidate genes for further study of disease resistance mechanism.Suyu 8 is a new yam variety highly resistant to anthracnose,which was bred by tissue culture mutagenesis of the highly susceptible strain 024.In order to find out the anthracnose resistance genes,Suyu 8 and strain 024 leaves were inoculated for different time by Colletotrichum gloeosporioides strain 4-2,the differentially expressed genes were analyzed by transcriptome sequencing.Concurrently,the uninoculation leaves of Suyu 8 were used as control.The number of differentially expressed genes,inoculation for 24,48,72,96 h,were 197,132,187 and 313,respectively.After removing the common differentially expressed genes at each time point,we obtained 711 differentially expressed genes.Go enrichment showed that the differential genes were mainly related to response to biological stimulation,defense response,cell wall metabolism and oxidation-reduction process.KEGG enrichment analysis revealed that many metabolic pathways related to plant disease resistance were changed.The expression of multiple genes varied which defense-related plant hormone signal transduction pathways,such as auxin,jasmonic acid,and ethylene.Among them,five early auxin-responsive genes,the key genes of jasmonic acid and abscisic acid biosynthesis were up-regulated,while ERF036 was down-regulated,which might negatively regulate the infection of Colletotrichum gloeosporioides.Several cytochrome P450 genes,ubiquitin ligases involved secondary metabolite modification and phytosterol synthetases,defensins and lectins were up-regulated.WRKY,MYB and TIFY transcription factors positively or negatively regulate the expression of disease resistance genes.Under the regulation of transcription factors,PR protein,NBS-LRR disease resistance genes,receptor kinases were highly expressed,the expressions of CAT and SOD genes of antioxidant protective enzyme systems were activated by reactive oxygen species.In addition,the enzymes related to starch and sucrose synthesis were up-regulated,and enzymes related to starch degradation were down regulated.The expression trends of LAX4,IAA4,IAA21 were consistent with that of transcriptome sequencing.The relative expression of LAX4,IAA4 and IAA21 in resistant varieties were significantly higher than susceptible varieties,suggesting that auxin signaling pathway is beneficial to the immune response to anthracnose for yam.

  • LI Jun, YAN Zhihao, JIA Wanli, XU Mengmeng, ZHANG Jingfeng, XU Qiuliang, HAN Haoyuan, QUAN Kai
    Abstract (84) PDF (38) RichHTML (25)

    Adipose triglyceride lipase(ATGL)is the main rate limiting enzyme in the process of fat hydrolysis.In order to analyze the structure and transcriptional regulation mechanism of ATGL promoter in dairy goat,the 5' flanking sequence of ATGL was amplified by PCR and analyzed by bioinformatics.Luciferase reporter gene vectors of five deletion fragments with different lengths of promoter were constructed and transfected into mammary epithelial cells of dairy goat.The cells were treated with rosiglitazone and T0901317 respectively to detect the effects of PPARG and SREBP1 on ATGL promoter activity.The results showed that the 5' flanking sequence of ATGL gene of dairy goat was 2 721 bp,including 2 024 bp upstream from the transcription start site.The ATGL promoter contained eukaryotic promoter elements TATA-box and CpG island.It was also found that there were transcription factors-binding sites of FOXO,SREBF1,PPARG,C/EBPα and E2F1 by the online software prediction.The core region of ATGL promoter was located at -256—+1,and there were negative regulatory elements at -527—-256.When cells were treated with rosiglitazone and T0901317,it was found that both of them could significantly up-regulate ATGL promoter activity.The response regions of rosiglitazone and T0901317 were -527—-256 and -882—-527,respectively,indicating that PPARG and SREBP1 could regulate ATGL promoter activity.

  • JIA Weizhe, JIAO Bo, WANG Jiao, CHEN Wenye, YANG Fan, LIU Yongwei, DONG Fushuang, ZHAO Liqun, ZHOU Shuo
    Abstract (399) PDF (133) RichHTML (29)

    In order to explore stress resistance genes in wheat and study the molecular mechanism of calmodulin-like genes in plant stress resistance,a calmodulin-like gene TaCML8-A was cloned from wheat by electronic cloning combined with RT-PCR.Bioinformatics analysis showed that the open reading frame length of the gene was 519 bp,encoding 172 amino acid sequences,including PTZ00184 and FRQ1 superfamily,four EF-hand domains,including four calcium binding sites.The molecular weight of the encoded protein was 18.31 ku and the isoelectric point was 4.54.It belonged to acidic protein.Subcellular localization showed that TaCML8-A protein was distributed in the nucleus and cell membrane.Nucleotide sequence alignment showed that TaCML8-A had the closest genetic relationship with rice OsCML14,with a similarity of 79.17%.qRT-PCR results showed that the expression of TaCML8-A gene in shoot and root of wheat increased under salt,osmotic and cold stress,indicating that plants might respond to thoese stresses by increasing the expression of TaCML8-A.During heat shock,TaCML8-A gene was induced and inhibited in root and shoot,respectively,so as to play different functions.The calmodulin-like gene TaCML8-A was successfully cloned from wheat and analyzed for expression.It is preliminarily speculated that the gene might be involved in regulating abiotic stress of plants,so as to provide basic theoretical support for the follow-up study of its biological function.

  • JIA Liqiang, LIU Xun, DING Bo
    Abstract (154) PDF (75) RichHTML (17)

    In order to study the role of ZmbZIPs in the stress tolerance of maize,using maize inbreed line Zheng 58 for exprerimental material,expression analysis of 9 ZmbZIPs were performed under 200 mmol/L NaCl,20% PEG6000,4 ℃ low temperature and nitrate or ammonium deficiency stresses.Evolutionary tree analysis indicated that 9 ZmbZIPs were divided 3 subgroup.RT-qPCR analysis showed that 8 ZmbZIPs were detected in maize tissues,whereas ZmbZIP80 were not,indicated it could be pseudogene.Under simulated salt,drought,low temperature and nitrogen stress conditions,8 ZmbZIPs exhibited different expression pattern in response to various stresses,ZmbZIP37 and ZmbZIP53 were induced while ZmbZIP49 and ZmbZIP79 were downregulated obviously under NaCl stress,ZmbZIP37 and ZmbZIP53 of leaves significantly suppressed under nitrate deficit stress while ZmbZIP42 and ZmbZIP49 were upregulated in responsive to ammonium deficient stress.The result indicated 8 ZmbZIP played widely roles against stress conditions.Expression pattern of 9 ZmbZIPs gene differed in different tissues or under various adverse stresses.The study can offer scientific data for further reveal ZmbZIP biological function.