The hexameric Paf1 (RNA polymerase Ⅱ associated factor 1) complex is a crucial transcription regulator in eukaryotes.Paf1-regulated expression of specific genes in plants is closely related to diverse biological processes including growth,development,and stress responses.In order to get information on the responses of Paf1 to abiotic stresses in common wheat,homologous sequence searches were performed to identify all of the genes encoding each of the Paf1 subunits in the wheat genome.mCherry fusions of the wheat Paf1 subunit proteins were expressed in protoplasts and tobacco leaves for determination of protein subcellular localization by fluorescence microscopy.qRT-PCR assays were conducted to profile the expression of wheat Paf1 subunit genes in response to different abiotic stresses.The results showed that,in wheat,five of the Paf1 subunits,TaVIP3,TaVIP4,TaVIP5,TaVIP6,and TaPHP,were each encoded by one set of homeologous genes while the sixth subunit TaVIP2 was encoded by two sets.Plant VIP2 sequences had an N-terminal proline-rich region with variable length,and wheat TaVIP2 sequences had an additional glutamine-rich region.Protein subcellular localization assays revealed the nuclear localization of TaVIP2,TaVIP4,TaVIP5,and TaVIP6 proteins and the nuclear and cytoplasmic localization of TaVIP3 and TaPHP proteins.Gene expression analyses revealed similar tissue-dependent constitutive expression variations and similar stress-induced expression patterns of wheat Paf1 subunit genes.These genes coordinately responded to the stress of high temperature by expression upregulation and to the stresses of salt and drought by expression downregulation.Collectively,our results suggested the involvement of expression regulation of Paf1 subunit genes in the responses of wheat to abiotic stresses.

To investigate the transcriptome differences between resistant and susceptible varieties of cauliflower after inoculation with Xanthomonas campestris pv.campestris (Xcc),and to identify genes associated with cauliflower resistance to black rot disease,the susceptible variety Y1-2 and the resistant variety EC-247 of cauliflower were selected as the research subjects.Total RNA was extracted from cauliflower leaves at 0,1,3,and 5 days post-inoculation with Xcc,respectively.High-throughput parametric transcriptome sequencing was then conducted utilizing the Illumina RNA-Seq platform,followed by Real-time Quantitative PCR for validation of selected differentially expressed genes(DEGs).DEGs associated with disease resistance were screened and analyzed.The findings revealed that 6 355 genes exhibited significant differential expression between resistant and susceptible cultivars across the four time points.KEGG enrichment analysis focused on plant disease resistance pathways,identifying 47 genes involved in plant-pathogen interactions and 61 genes related to plant hormone signaling.Cluster analysis of these gene expression levels disclosed specific genes,including one CDPK,four CMLs,one PTK,one CaM,one RLK,and one SGT1 in the plant-pathogen interaction pathway,and three auxin-responsive protein genes,a TIFY gene,an indole-3-acetic acid-amido synthetase gene,two brassinazole-resistant protein genes,and a Shaggy-associated protein kinase zeta gene in the plant hormone signaling pathway.Notably,the expression of these genes was significantly higher in resistant varieties compared to susceptible ones,indicating their active response to pathogen infection at various time points.The results indicated that these differential genes might be related to disease resistance in cauliflower,which provided important genetic resources and scientific basis for molecular breeding of disease resistance in cauliflower.

Preliminary transcriptomic analysis identified ZmRAV1 as a candidate gene involved in maize's response to drought stress. To further investigate its function, this study cloned the ZmRAV1 gene, conducted bioinformatics analysis of its coding sequence, and overexpressed this gene in Arabidopsis thaliana. The function of ZmRAV1 was validated by assessing the phenotypes and physiological and biochemical indices of the transgenic Arabidopsis lines under drought conditions. The results showed that the ZmRAV1 gene had a total length of 1 176 bp and encoded 389 amino acids.It had the highest proportion of irregular coils in its secondary structure and was a hydrophilic protein that did not contain signal peptides and was non transmembrane.Subcellular localization indicated that the protein was located in the nucleus.ZmRAV1 exhibited high conservation across different species.Phylogenetic analysis indicated that ZmRAV1 shares the closest evolutionary relationship with its homolog in Miscanthus sinensis, showing a high degree of homology. After drought stress treatment,the root length of Arabidopsis thaliana lines overexpressing ZmRAV1 during germination was significantly higher than that of wild-type (WT)lines.In the seedling stage,WT showed withering or even death after drought stress,while the survival rate was lower than that of overexpressing lines.Moreover,the POD and SOD activities of ZmRAV1 overexpressing lines were higher than those of WT after drought treatment,indicating that overexpression of ZmRAV1 gene could enhance Arabidopsis thaliana's resistance to drought stress.

To explore the function of key genes in photosynthesis, a functional knockout mutant (zmC₄nadp-me) of ZmC₄NADP-ME, the gene encoding the rate-limiting enzyme of the dark reaction of photosynthesis in maize, was obtained. Evolutionary tree analysis showed that ZmC₄NADP-ME and its homologous genes exist in multiple copies in most plants, with diverse expression patterns. Phenotypic analysis revealed that the entire zmC₄nadp-me plant was yellow-green, and its seedling-stage leaves dried up and died rapidly under light. Chlorophyll fluorescence analysis indicated that Y(Ⅱ) and electron transport rate ETR(Ⅱ) of photosystem Ⅱ (PSⅡ) in zmC₄nadp-me decreased significantly, with little change in Y(NPQ), while the Y(NO) increased notably. Measurement of the absorption capacity (P700) of photosystem Ⅰ (PSⅠ) found that both the electron transport rate (ETR(Ⅰ)) and the actual photoelectron efficiency (Y(Ⅰ)) of zmC₄nadp-me dropped substantially, and the gap widened with increasing light intensity. Under specific light intensities, Y(ND) and Y(NA) of zmC₄nadp-me were greater than those of the wild type (WT). In conclusion, ZmC₄NADP-ME is essential for plant growth and development. Disruption of this gene severely stresses PSⅡ, and the plant can't alleviate this stress by increasing Y(NPQ). Meanwhile, at low light intensities, the inhibition of PSⅠ may originate from the electron donor side of PSⅠ, and as the light intensity increases, the inhibition from the electron acceptor side of PSⅠ becomes a key factor.

Cabbage fusarium wilt is a serious soil-borne fungal disease caused by Fusarium oxysporum f.sp.conglutinans (FOC),which affects the yield and quality of cabbage.In order to clarify the biological function of the transcription factor SNT2 in this pathogen,the SNT2 gene knockout mutant ΔSNT2 in F.oxysporum was successfully obtained by homologous recombination and protoplast transformation,and its phenotype and pathogenicity were analyzed.The results showed that SNT2 in F.oxysporum encoded 1 529 amino acids,had a SANT domain that binds to DNA,and the protein belonged to a hydrophilic protein.Compared with the wild type strain,the mycelial growth rate of the ΔSNT2 mutant decreased and the septation increased significantly,and the conidial production decreased significantly.Based on the results of exogenous stress,ΔSNT2 was insensitive to osmotic stress of 1 mol/L sorbitol,but its tolerance to oxygen stress,salt stress and cell wall stress of 0.1% H₂O₂,2 mol/L NaCl and 0.05% Congo red was reduced.At the same time,the pathogenicity test showed that the disease index of ΔSNT2 mutant was significantly lower than that of wild type,and the deletion of SNT2 resulted in a significant decrease in the pathogenicity of F.oxysporum.In conclusion,the transcription factor SNT2 plays an important role in maintaining the integrity of the cell wall during the interaction between the pathogen and the host,and participates in the regulation of the growth and development of F.oxysporum and the expression of pathogenicity.

In order to accelerate the breeding of pink-tomato varieties,a rapid breeding strategy was used to generate pink-fruited tomato material by CRISPR/Cas9-mediated gene editing of SlMYB12.Two adjacent target sites within the first exon of SlMYB12 were selected to construct the CRISPR/Cas9 binary vector, and then the vector was introduced into red-fruited inbred line R18-10C through Agrobacterium-mediated transformation. The homozygous mutants without exogenous Cas9 were screened using specific primers and their agronomic traits and fruit nutritional quality were analyzed. Sequencing results showed that three homozygous mutants of different mutation types were obtained and all of which were frame shift mutations caused by base deletion. Compared with wild-type red fruit tomatoes, SlMYB12-edited plants grew and developed normally,and there were no significant difference in plant height,single-fruit weight,total yield per plant,fruit total soluble solid content and lycopene level,but the mature fruit showed pink and the flavonoid naringenin chalcone(NarCh)content of tomato peels was significantly reduced in MYB12-edited plants.In summary,the procedure for the generation of pink-fruited tomato plants through CRISPR/Cas9-mediated targeted mutagenesis of SlMYB12 was set up and new pink-fruited tomato germplasms with stable inheritance were obtained.

PHR1 is a crucial factor in balancing plant disease resistance and low phosphorus stress resistance.To investigate the nature and function of the StPHR1 gene in potato and to explore the role of StPHR1 in the process of potato resistance to Alternaria solani infection,the CDS sequence of the StPHR1 gene was cloned by PCR technology using potatoes as the research material,and the structural,physicochemical properties,and phylogenetic relationships of StPHR1 were analyzed and predicted using bioinformatics software,then,the expression level of StPHR1 during the infection of potatoes by A.solani and under different hormone treatments was analyzed using qRT-PCR technology,and subcellular localization analysis of the protein was conducted using laser confocal microscopy technology.The results showed that the CDS of the StPHR1 gene was 1 353 bp,encoding 450 amino acids.The protein had a molecular formula of C₂₁₄₇H₃₃₉₉N₅₉₅O₇₁₁S₁₈,a molecular weight of 49.51 ku,and a theoretical isoelectric point of 5.07,encoding a hydrophilic,unstable protein with no signal peptide and no transmembrane structure.Its secondary structure consisted mainly of random coil and α-helix.Phylogenetic tree analysis revealed that the StPHR1 protein was most closely related to Arabidopsis thaliana; conservative domain analysis revealed that the StPHR1 protein,like other PHR1s,possesses both MYB-CC and MYB conserved structural domains at its C-terminus.Relative expression analysis found that StPHR1 was significantly induced by A.solani and salicylic acid,and it was hypothesized that StPHR1 played an important role in A.solani infection of potato and in the response to salicylic acid; and the subcellular localization showed that the StPHR1 protein was localized in the nucleus.It is hypothesized that StPHR1 may regulate potato resistance to A.solani through its MYB transcription factor activity and response to salicylic acid.

The naked seeds of zucchinii possess significant natural advantages in processing.To investigate the genetic mechanisms underlying the hull-less trait in zucchini,we utilized the hulled 17pu10 (P₁) and the hull-less 17pu08 (P₂) as parental strains,and constructed populations of F₁ (P₁×P₂),F₂,and BC₁.The phenotypes of the zucchini seeds in the progeny population were assessed.The results showed that the number ratio of hulled seeds and hull-less seeds in the progeny population was in line with 3∶1 separation ratio.This indicated that the hull-less trait was regulated by a single gene and that the hull-less allele was recessive.Genetic mapping within this interval revealed that the gene for hull-less in zucchini was located between the markers InDel3157329 and InDel3724121,with genetic distances of 1.4,2.6 cM,respectively,while the physical distance was 0.6 Mb.The annotation and function analysis of 24 genes in the interval showed that 4 genes were directly or indirectly involved in the biosynthesis of cell wall,cellulose and lignin.Further analysis of the expression differences of 4 genes showed that only Cp4.1LG12g04350 and Cp4.1LG12g04370 had significant differences in expression levels during seed development.It was inferred that Cp4.1LG12g04350 or Cp4.1LG12g04370 was the candidate gene controlling the hull-less trait.In addition,InDel markers linked to hull-less genes were developed,which could be used as markers to identify zucchini hull-less traits,so as to accelerate the breeding of high-quality zucchini seed hull-less varieties.

Cucumber powdery mildew is one of the main diseases that adversely impacts cucumber production,posing a significant challenge to its sustainable cultivation.Identifying genes related to cucumber resistance to powdery mildew can help understand the genetic principles and molecular mechanisms of cucumber resistance to powdery mildew,and provide diverse gene resources for disease resistant breeding.This study constructed F₁ and F₂ populations of QK×QG using cucumber resistant inbred line QK and susceptible inbred line QG as parents.Using the extreme trait mixed pool resequencing (BSA-seq) method,the genomic regions harboring the resistance genes of cucumber powdery mildew were preliminarily located.By integrating transcriptome data with gene annotation information,the association interval of the disease phenotype was narrowed,sequence variations were identified,and key genes were screened.The results showed that the resistance of powdery mildew may be controlled by recessive genes,and the population of F₂ showed a continuous normal distribution from resistant to susceptible.The BSA-seq analysis,combining the SNP-Index method and QTG(quantitative trait genomics)-seq method analysis highlighted the 19—21 Mb region of chromosome 5,where there were 77 annotated genes with SNP differences between samples,including 33 non-synonymous mutations.The transcriptome sequencing (RNA-seq) results showed that there were 309 upregulated genes and 697 downregulated genes in the susceptible material.The expression levels of 13 genes within the candidate segment of chromosome 5 showed significant differences after infection.Through a comprehensive analysis of differentially expressed genes and BSA,the candidate genes in this segment were narrowed to 3,and only SNP mutations were detected in the LOC101207011 gene.The candidate gene LOC101207011 was characterized by a mutation resulting in an amino acid change from Valine at position 656 to Leucine.This gene emerges as the primary focus of our investigation due to its potential role in conferring resistance to powdery mildew.

In order to reveal the expression patterns of PsbS protein in Nicotiana tabacum,the full-length NtPsbS gene sequence was cloned from cDNA of the tobacco cultivar K326,and the multiple protein sequences of NtPsbS gene and PsbS genes of seven crops including rice,tomato and soybean were compared by DNAMAN software.The phylogenetic analysis was carried out by MEGA 11 software,and the phylogenetic tree was established by neighbor-joining method.Tissue expression of NtPsbS gene in tobacco at different growth stages was detected by qRT-PCR.The plant expression vector pS1300-PsbS-GFP was constructed to find subcellular localization of the NtPsbS mature protein.Finally,the mRNA levels of NtPsbS under abiotic stresses were analyzed in K326.The results showed that the total length of tobacco NtPsbS gene was 825 bp,encoding 274 amino acids.The homology of tobacco NtPsbS protein and tomato SlPsbS protein was the highest up to 91%.The NtPsbS gene exhibited the highest mRNA levels in the leaves among the tissues,including leaves,roots,stems,seeds and other parts of K326.The mature NtPsbS protein was located in chloroplasts.For the abiotic stress,it was found that the expression level of NtPsbS was significantly increased under salt stress,cold stress and Abscisic acid(ABA)treatments.In summary,the expression level of NtPsbS was the highest in tobacco leaves at different growth stages,and it was higher when under the treatments of salt stress,cold stress and ABA,indicating that this gene may be involved in the salt and cold stress resistance and ABA metabolic pathway in tobacco,which could provide basis and reference for the functional analysis of the NtPsbS gene in future.

To reveal the sequence characteristics,subcellular localization,and expression pattern of PatASIL2,belonging to the Trihelix transcription factor family,the PatASIL2 gene was first cloned using the cDNA of Pogostemon cablin (patchouli)as a template,and the bioinformatics analysis was further performed.The PatASIL2-EGFP expressed vector was constructed and transformed into Arabidopsis protoplasm to investigate the subcellular localization of PatASIL2.The real-time quantitative reverse transcription PCR(qRT-PCR)was also performed to detect the expression profiles of PatASIL2 gene in different tissues of patchouli and under treatments of methyl jasmonate(MeJA),salt stress,drought stress,and cold stress.The results indicated that PatASIL2 gene contained an open reading frame of 1 035 bp and encoded 344 amino acids.The PatASIL2 protein was an unstable hydrophilic protein without transmembrane domains and signal peptides and had 41 serine phosphorylation sites and one Myb_DNA-bind_4 conserved domain.The phylogenetic analysis showed that PatASIL2 was classified into the SIP1 subfamily of Trihelix transcription factor family,and clustered closely with Sesamum indicum SiASIL2.The subcellular localization result indicated that PatASIL2 was a nuclear-localized protein.The qRT-PCR results indicated that PatASIL2 expressed in the young leaf,mature leaf,old leaf,stem,and root in patchouli,particularly with the highest expression in the old leaf.The PatASIL2 expression was significantly upregulated by MeJA at 12—24 h and salt stress at 3—24 h.The expression of PatASIL2 was significantly upregulated at 24 h after drought treatment,and at 12 h after cold treatment.

Light-harvesting chlorophyll a/b binding proteins are important in plant photosynthesis and abiotic stress response.To study the characteristics of GhLhcb2A1 and its expression patterns and functions in low temperature and drought response in upland cotton,full-length CDS of GhLhcb2A1 gene was cloned from the leaf cDNA of Jimian 262 by PCR.Bioinformatic analysis was conducted to learn the basic characteristics of the gene.The expression patterns and functions in low temperature and drought response were evaluated by qRT-PCR and virus-induced gene silencing.It was shown that the length of GhLhcb2A1 CDS was 798 bp,encoding 265 amino acids.GhLhcb2A1 was highly expressed in leaves and was significantly up-regulated in leaves and roots under low temperature and drought treatment.Compared with the control,its expression maximized at 3 h under low temperature and drought in leaves with 17.42 and 30.03 folds increase respectively,whereas maximized at 6 h under low temperature and 12 h under drought in roots with 11.65 and 65.04 folds respectively.Subcellular localization assay verified that GhLhcb2A1 was expressed in the chloroplasts of cells.Compared with the control plants,GhLhcb2A1 silenced plants showed a more severe phenotype of water loss and dryness under both low temperatures and drought.The accumulated malondialdehyde content in the leaves of the silenced plants was significantly higher than that of the control,while the proline content and superoxide dismutase activity were significantly lower than those of the controls,suggesting that GhLhcb2A1 silenced plant reduced the resistance to low temperature and drought.The above results implied that this gene played a positive role in regulating low temperature and drought response.

The aim was to study the biological function of FolSid1 gene in Fusarium oxysporum f.sp.lini and its protein localization in Fusarium by cloning the gene.The gene sequence of FolSid1 was cloned by homologous comparison with F.oxysporum, and based on the principle of homologous recombination, a gene deletion box containing hydromycin resistance gene(hph)was constructed by Split Marker strategy,and the gene deletion mutant(ΔFolSid1)was obtained by PEG-mediated transfer into protoplasts of the wild type.pZESH1,a green fluorescent expression vector containing FolSid1 gene was constructed,and the subcellular localization of FolSid1-EGFP fusion protein was performed.The results showed that the sequence of FolSid1 gene consisted of 5 392 bp,which contained 3 introns.Compared with the wild type and the external insertion mutant, the knockout mutant ΔFolSid1 conidia showed a significant decrease in yield, although they did not differ in morphology and size; morphological observations revealed that the growth rate of colonies from the knockout mutant was significantly slower. The experiments of subcellular localization showed that FolSid1 protein was located in the cell membrane of mycelia cell.FolSid1 gene regulated the vegetative growth of mycelium,conidiogenesis and pathogenicity of Fusarium oxysporum f.sp.lini.

Soybean P34 protein mainly exists in soybean seeds,and its upstream promoter was likely to regulate the high expression of downstream genes in seeds.In order to further study the tissue expression pattern of soybean P34 protein gene and the regulatory activity of soybean P34 protein gene promoter,qRT-PCR was used to detect the expression of soybean P34 protein gene in soybean tissues.The 5'upstream sequence of soybean P34 protein gene(GmP34P)was cloned.The transcription initiation sites and cis-elements were analyzed by bioinformatics.The expression vector was constructed and the tobacco was transformed by Agrobacterium-mediated leaf disk method to detect GUS expression in transgenic tobacco.The results showed that the expression of P34 protein gene in soybean seeds was significantly higher than that in roots,stems,leaves and flowers.The length of GmP34P sequence obtained by cloning was 1 380 bp.Predictive analysis showed that the transcriptional start site of this sequence was base A at position 1 342,and the sequence contained a variety of cis-acting elements related to high seed expression,such as RY element,Skn-1 motif,2S seed protbanapa,etc.The plant expression vector pCAM-GmP34P containing GUS gene driven by GmP34P promoter was obtained.The positive transgenic plants were screened by hygromycin,PCR and RT-PCR.The results showed that GUS gene expression was extremely significant in transgenic tobacco seeds compared with other tissues by qRT-PCR with positive pCAM-GmP34P transgenic tobacco plants.GUS histochemical staining showed that the GmP34P promoter could regulate the high expression of downstream GUS gene in seeds.

WRKY is a unique class of transcription factors in plants,which plays an important role in plant abiotic stress response,seed dormancy and germination,growth and development,etc.In order to reveal the function and underlying molecular mechanism of GmWRKY44 gene in soybean WRKY transcription factor family,bioinformatics analysis and biology function verification of soybean Williams 82 GmWRKY44 were performed.GmWRKY44 gene was 1 077 bp in length and encoded 358 amino acids;the results of structural prediction and evolutionary analysis showed that,the secondary structure was composed of 23.46% α-helix,4.75% β-fold,58.94% irregular coil and 12.85% extended chain,and the tertiary structure was unified with the secondary structure;It contained a conserved WRKY domain, the zinc finger structure was of the C₂H₂ type, and it belonged to the WRKY IIc subfamily; GmWRKY44 is a homologous gene of Arabidopsis thaliana AtWRKY71 with a similarity of 35.56%, and the two genes had similar gene structures. RT-qPCR analysis showed that GmWRKY44 responded to salt stress and its expression level first decreased and then increased.Under salt stress,the germination rate and root length of wild-type(Col-0)and GmWRKY44 overexpressing Arabidopsis lines were inhibited to a certain extent,but GmWRKY44 overexpressing lines were significantly better than Col-0.In addition,under salt stress,the growth inhibition of GmWRKY44 overexpressing lines was lower than that of Col-0.Physiological index analysis revealed that under salt stress,the overexpression lines of GmWRKY44 exhibited significantly higher activities of superoxide dismutase(SOD),peroxidase(POD),and catalase(CAT)than Col-0,while the content of malondialdehyde(MDA)was significantly lower than Col-0.These data indicated that overexpression of GmWRKY44 could improve salt tolerance in transgenic Arabidopsis.

In order to explore new approaches for high-oil peanut breeding and establish a new method for directly developing high-oil peanut germplasm,this study employed in vitro mutagenesis breeding technology to create new high-oil peanut germplasm.Jihua 9 embryo leaflet was used as mutagenic test materials,Jihua 9 and Jihua 54 were used as control test materials,and bleomycin was used as mutagenic agent.The ovules were sterilized and placed in gradient mutagenesis medium and screened for semi-lethal concentrations of bleomycin.After somatic embryos germinated into seedlings,sterile peanut seedlings were used as rootstocks,and transplanted to the field.Bioinformatics analysis of two known regulated peanut fat synthesis genes WRI1 and experimental feasibility validation by the correlation of WRI1 gene expression in grain and crude fat content of mutagenic plants were conducted.The results were best when the bleomycin was 3 mg/L.The crude fat content of IM13-3 was higher than that of Jihua 9(CK₁,test variety control)and Jihua 54(CK₂,high oil variety control).Two WRI1 genes,WRI1X2 and WRI1X1,encoding 366 and 357 amino acids,respectively,were both unstable hydrophilic proteins. WRI1 gene expression and crude fat content were significantly positively associated in grain.Bleomycin was first used as a peanut vitro mutagenesis agent,and IM13-3 was obtained with a crude fat content of 56.64%.It further proves the authenticity of Jihua 9 high oil mutant and the feasibility of peanut in vitro mutagenesis method. The gene expression level of the high-oil mutant WRI1 was determined and was significantly different from the control varieties. Demonstrate the feasibility of breeding methods for in vitro mutagenesis of peanut.

To investigate the function of lysophosphatidicacid acyltransferase 2(LPAT2)in Brassica napus,from which one copy(A07)of BnaLPAT2 was cloned by PCR.we constructed the overexpression vectors p35S∷BnaLPAT2-A07 and the seed-specific expression vector pNapin∷BnaLPAT2-A07,and by utilizing Agrobacterium-mediated genetic transformation method,obtained a total of 15 and 11 transgenic Brassica napus cv.Zhongshuang 6 respectively by PCR positive detection.Real-time Quantitative PCR(qRT-PCR)showed that the transcript levels of BnaLPAT2-A07 in most tissues of T₃ overexpressed rapeseed were higher than that of CK.However,in the seed-specific expression tissues of T₃ transgenic rapeseed,the BnaLPAT2-A07 genes were strongly expressed in the development and maturation stages of silique.Soxhlet extraction results showed that the oil content in the transgenic seeds driven by the 35S or Napin promoter accumulated 1.39 and 2.36 percentage point more oil than control seeds,respectively.The fatty acid components of transgenic rape were detected by gas chromatography.Compared with CK,the content of linolenic acid was increased by 3.13 and 1.47 percentage point,respectively.Taken together,the BnaLPAT2-A07 could promote seed oil synthesis,however,the specific selection function of BnaLPAT2-A07 for linolenic acid needs to be further verified.

The identification and expression pattern analysis of CmPIPs gene family in melon could provide theoretical basis and support for further exploring the function of CmPIPs gene family and the genetic improvement of melon.TBtools,MEME,MEGA X and Plant-CARE tools were used to analyze the bioinformatics of CmPIPs,and the expression level of CmPIP2;7 in the pericarp of melon at different stages after pollination,and the expression level of each member of CmPIPs in different tissues and different concentrations of plant hormone treated young leaves were visualized in the software GraphPad Prism 10.The results showed that CmPIP2;7 and CsPIP2;8 had nearest kinship;the 12 members of the CmPIPs family were mainly distributed on chromosomes 1,3,4,5,9,10 and 11;except that CmPIP2;8 had 3 CDS regions,the other members had 4 CDS regions.The promoter regions of each member of CmPIPs had multiple cis-acting elements,hormone responsive elements,such as auxin,gibberellin,and abscisic acid.The expression level of CmPIP2;7 was significantly up-regulated during rapid development and maturity of melon fruit.Members of various families of CmPIPs were expressed in different tissues of melon.After treatment with 40.0 μmol/L auxin,the expression level of CmPIP2;4 was significantly up-regulated,while the expression level of CmPIP1;1,CmPIP 2;1,CmPIP2;2 and CmPIP2;3 were extremely significantly down-regulated,and when the concentrations of abscisic acid were 0.4,4.0 and 40.0 μmol/L,the expression level of CmPIP1;1,CmPIP2;1,CmPIP2;3,CmPIP2;9 were significantly down-regulated.After treatment with 44.640 μmol/L methyl jasmonate,the expression level of CmPIP2;1 and CmPIP2;5 were significantly down-regulated,while the expression level of CmPIP2;2,CmPIP2;3,CmPIP2;7 and CmPIP2;9 were significantly up-regulated.When the concentration of ethylene glycol was 4.0 mmol/L,the expression level of each member of CmPIPs was significantly up-regulated.The gene structure,sequence characteristics,evolutionary relationship and collinearity of CmPIPs gene family members were clarified,and their expression pattern were analyzed.

As one of the subfamily members of Ca²⁺-ATPase,ACA exerts a vital role in sustaining the intracellular Ca²⁺ concentration equilibrium and modulating plant growth and development in response to abiotic stress.To acquire an in-depth comprehension of the function of the lettuce ACA gene family,bioinformatics methodologies were utilized to identify and dissect the members of the lettuce ACA gene family.The outcomes manifested that 17 ACA genes were identified in lettuce,denominated as LsACA1 to LsACA17;the LsACA genes were unevenly distributed across 8 chromosomes;the subcellular localization prediction results disclosed that all LsACA proteins were localized in the plasma membrane;the number of introns exhibited significant variation(0—32)among the members of the LsACA gene family,a total of 15 conserved domains of LsACA proteins were identified,with amino acids ranging from 21 to 50;the proportion of the secondary structure was alpha helix>random coil>extended strand>beta turn;based on phylogenetic analysis,LsACA proteins were categorized into 5 subfamilies,namely Group Ⅰ to Group Ⅴ;according to collinearity analysis,it was found that there were fragment duplications in 6 pairs of genes,the Ka/Ks of their collinear gene pairs were all less than 1,signifying that purifying selection was the preponderant force in evolution.The expression patterns of LsACA gene family members under different calcium ion concentrations were analyzed by qRT-PCR.The results showed that:compared with the control,the expression levels of 12 LsACA genes in the calcium-sensitive variety Baoshilü under low calcium treatment were extremely significantly downregulated,while the expression levels of 9 LsACA genes in the calcium-insensitive variety Yeluo were extremely significantly upregulated and the expression level of 1 LsACA gene was significantly upregulated.The ACA gene family members of lettuce were identified and analyzed,and the characteristics of the LsACA gene family members were revealed.

Chalcone synthase(CHS)is an important structural gene that regulates the early biosynthesis of flavonoid pathways in plants,and plays a role in plant growth and development and stress response.Previously,the key genes StCHS4 and StCHS5 for anthocyanin biosynthesis were identified in the potato CHS family by expression analysis.To further explore the function of potato StCHS4 and StCHS5 in the biosynthesis of flavonoids and anthocyanins,the characterization of StCHS4 and StCHS5 proteins was analyzed by online website.The 35S∷StCHS4-GFP and 35S∷StCHS5-GFP recombinant vectors were constructed by homologous recombination method based on the pRI101 binary vector,and then were transformed into Agrobacterium GV3101 strain.The subcellular localization of StCHS4 and StCHS5 proteins was determined by transient transformation of Nicotiana benthamiana.N.tabacum was used as the experimental material for transient overexpression and stable genetic transformation to analyze the content of total flavonoids and anthocyanins after overexpression of StCHS4 and StCHS5 genes.The results showed that the secondary structures of StCHS4 and StCHS5 proteins were mainly α-helix and random coil.StCHS4 was an unstable hydrophilic protein,and StCHS5 was a stable hydrophilic protein.The sequence alignment revealed that StCHS4 and StCHS5 had a close relationship with the CHSs of Capsicum annuum and Solanum lycopersicum,respectively.The results of subcellular localization revealed that StCHS4 and StCHS5 proteins were localized in the cytoplasm and cell membrane.In transient overexpression of tobacco,StCHS4 and StCHS5 genes significantly enhanced anthocyanin accumulation at 3—5 days after injection.Three transgenic N.tabacum lines of StCHS4 and StCHS5 gene were obtained,respectively.Compared with the wild type,the expression of StCHS4 and StCHS5 in transgenic plants was significantly higher,and the contents of total flavonoids and total anthocyanins were higher than those in the wild type.The total flavonoid content in StCHS4-OE3 and StCHS5-OE1 transgenic plants was significantly increased.The anthocyanin content in StCHS5-OE1 and StCHS5-OE2 plants increased by 89%,131%,respectively.The above results demonstrated that StCHS4 and StCHS5 were the key CHS genes in the flavonoid pathway of Solanum tuberosum,and the overexpression of StCHS4 and StCHS5 contributed to the biosynthesis of anthocyanins and flavonoids.

DNA damage significantly hinders impact plant growth and development.NBS1 plays an important role in the cellular response to DNA damage,to analyze the functional difference between NBS1 and its alternative splicing isoform,NBS1-3.Specific primers were designed to clone NBS1 and NBS1-3 from the complementary DNA of Arabidopsis thaliana,and the sequences of the NBS1,NBS1-3 and the three-dimensional structure of the proteins were analyzed.Transgenic Arabidopsis thaliana lines overexpressing of NBS1 and NBS1-3 were generated respectively,and nbs1 homozygous mutant plants were identified,detecting the expression levels of NBS1 in overexpressed lines NBS1 and NBS1-3.To further elucidate the functional distinction between NBS1 and NBS1-3,wild type,mutant and overexpressed plants were treated with 0.6 mmol/L Methyl Methanesulfonate(MMS)and observed damage area.The results of quantitative showed that the expression levels of NBS1 in overexpressed lines NBS1 and NBS1-3 were higher than wild type.PI staining of root tips showed that after 0.6 mmol/L MMS treatment,the mutant showed the highest relative damage area,while the NBS1-3 overexpressed lines exhibited the least damage,followed by NBS1 overexpressed lines and wild type.These results suggest a potentially more important role of NBS1-3 in DNA damage repair compared to NBS1.

To understand the member characteristics of the WRKY transcription factor family in hydrangea and their roles in response to leaf spot disease,this study utilized the bioinformatics method to characterize WRKY family members in Hydrangea macrophylla Endless Summer,and systematically analyzed the protein physicochemical features,gene structure,systematic evolution,collinearity and expression patterns under the infection with Corynespora cassiicola of WRKY family members.The results showed that there were 84 non-redundant HmWRKY members in H.macrophylla genome.All of the HmWRKYs were hydrophilic proteins and unevenly distributed on 18 chromosomes of H.macrophylla,encoding 112—1 046 amino acids.In addition,HmWRKY members were divided into 3 subgroups(Group Ⅰ—Group Ⅲ),which contained a conserved DNA binding domain composed of WRKYGQK and C2H2.The sequence length of HmWRKY varies greatly from 512 bp to 40 338 bp,and 8 collinear gene pairs with Ka/Ks ratios less than 1 were detected,indicating that the HmWRKY family experienced purification selection in evolution.18 HmWRKY members showed significantly differential expression after infection with C.cassiicola,of which 9 up-regulated and 9 down-regulated.The results indicated that these HmWRKY genes might play important roles in response to leaf spot disease of hydrangea.

4-coumaric acid:coenzyme A ligase(4CL),as the main biosynthetic enzyme of flavonoids and lignin,is closely related to the formation of plant flavonoids.In order to explore the relationship between the 4CL gene family of Glycyrrhiza uralensis and the synthesis and accumulation of isoglycyrrhizin,this study determined the content of isoglycyrrhizin in G. uralensis after drought stress,and the expression characteristics and bioinformatics analysis of the 4CL gene family of G. uralensis Fisch.to understand the relationship between the accumulation characteristics of isoglycyrrhizin and the 4CL gene of G.uralensis.The results showed that isoliquiritigenin was mainly accumulated in the roots of G.uralensis,and drought stress could significantly increase the content of isoliquiritigenin in the roots of G.uralensis.The content of isoliquiritigenin after 2 h of polyethylene glycol(PEG)stress was 3.91 times that of the control group(0 h).Drought stress could induce the up regulation of Gu4CL gene in the underground part.The expression levels of Gu4CL2, Gu4CL4 and Gu4CL5 were higher after PEG stress induction,while Gu4CL2 was most significantly up-regulated after PEG stress.The expression of Gu4CL2 was similar to that of isoliquiritigenin.Bioinformatics analysis of Gu4CL gene showed that 11 genes had two conserved polypeptide motifs Box Ⅰ(SSGTTGLPKGV)and Box Ⅱ(GEICIRG),and 11 genes were distributed on 11 Scaffold fragments.Promoter cis-acting element analysis showed that Gu4CL2, Gu4CL4 and Gu4CL5 genes contained more abscisic acid and jasmonic acid response elements than other Gu4CL genes.Therefore,drought stress may induce the synthesis of jasmonic acid and abscisic acid,regulate the expression of Gu4CL2, Gu4CL4 and Gu4CL5 genes in licorice roots,and increase the accumulation of isoliquiritigenin.This study provides a research basis for optimizing the cultivation techniques of G.uralensis,improving the adaptability and quality of cultivated land in saline-alkali land,exploring the role of Gu4CL gene family in G.uralensis,and exploring the synthesis mechanism of isoliquiritigenin.

Currently,traditional breeding methods cannot fully meet the demands of the cotton market and production. Therefore, molecular biotechnology can be employed to accelerate the development of new cotton varieties. Transcription factors have emerged as significant tools in the study of cotton gene function, genetics, and breeding during this period. Among them, the MYB transcription factor family is one of the largest transcription factor families present in many plants, and it performs multiple roles in plant growth and development. Hence, the MYB transcription factor holds high research value. While the function of MYB transcription factors has been extensively studied in model plants, research in non-model plants, particularly cotton, remains limited and is mostly concentrated in upland cotton. To further understand MYB transcription factors, this paper reviews the research progress on MYB transcription factors in various plants and cotton. It covers their classification basis, structural characteristics, evolution, roles in response to biotic and abiotic stress, cotton fiber development, and secondary metabolism. Additionally, it provides statistics on the known functions of cotton MYB transcription factors. This review aims to deepen our understanding of cotton MYB transcription factors and serve as an important reference for future studies on the functions and mechanisms of MYB transcription factors in different cotton species.

This study aims to provide theoretical guidance for the effective use of sister line materials,accelerate the promotion of the wheat Annong 1687 variety,and offer a reference for the genetic improvement of new wheat varieties.Wheat Annong 1687 and its sister lines and parents was used as materials.We combined phenotypic identification of field agronomic traits with genotypic identification of wheat materials using 55K microarrays.The data was analysed using SPSS and mapped it with the RIdeogram package.Significant differences in spike length were found between parents and some sister line combinations.Specifically,parents Annong 1106 and Xinong 822,and sister lines Annong 1687 and Xinong 822,showed very significant differences.The parental lines Annong 1106 and Xinong 822,as well as the sister lines Q6,8 and 105,133,exhibited a total of 909 differential SNP sites across 19 chromosomes.Notably,chromosome 5B contained 500 of these sites,which were concentrated in intervals of 63—87 Mb and 400—410 Mb.Cloning and sequencing of candidate genes within the intervals revealed that two genes encoding growth hormone-inducible proteins,TraesCS5B01G225000 and TraesCS5B01G058700,had missense mutations resulting in amino acid changes.Mutations in TraesCS5B01G225000 resulted in a codon change to the termination codon TGA and termination of amino acid synthesis.The bi-parental and sister line combinations had five and two missense mutations,respectively.It is hypothesized that these two genes may have an effect on the length of wheat spikes.

To study the genetic diversity of astringency in walnut germplasm resources,118 germplasm resources from 4 different astringency walnut populations were used as materials,which was studied by using SSR capillary electrophoresis fluorescence labeling technology,and the dendrogram of 4 populations was constructed.The results showed that a total of 93 allelic variants were detected by 12 pairs of primers,ranging from 2 to 18,with an average of 7.75 alleles detected by each pair of SSR primers.Polymorphism information content (PIC) ranged from 0.357 9 to 0.785 2,with an average of 0.541 1.The number of polymorphic sites (Np) was 91,the percentage of polymorphic sites (PPB) was 97.85%,the number of observed alleles (Na) was 1.978 5,the effective number of alleles (Ne) was 1.198 5,Shannon's information index (I) was 0.209 7,Nei's diversity index (H) was 0.126 3,number of total genetic diversity index (Ht) was 0.126 7 and intra-population genetic diversity index (Hs) was 0.122 2,indicating that the genetic diversity and variation of the four walnut populations were not high.The coefficient of genetic differentiation among populations was 0.035 5,indicating that the genetic variation within populations was 96.45%.The percentage of polymorphic loci in the slightly astringent population was the largest,which was 72.04%,indicating that the slightly astringent population had the richest genetic diversity among the four populations.UPGMA cluster analysis showed that the genetic identity between four populations ranged from 0.989 8 to 0.997 1,indicating that the genetic distance among four populations was close.The four populations can be divided into two groups at the coefficient of 0.993 4.The first group included unastringent,slightly astringent and more astringent populations,with the astringent populations as a separate group.By calculating the gene frequency of 93 locis in different populations,41 locis of 12 pairs of SSR primers were found to be able to distinguish the resources with different levels of astringency.SSR capillary electrophoresis fluorescence labeling technology was used to analyze the genetic diversity related to astringency in 4 walnut populations.According to the transcriptomic sequencing results,12 pairs of primers were selected for SSR analysis.A total of 93 SSR locis were amplified,and the percentage of polymorphic sites was 97.85%,indicating that the primers had high polymorphism and were suitable for genetic diversity analysis of walnut,which can be used for subsequent studies on genetic diversity and breeding of astringency in walnut.

In order to investigate the effect of cinnamic acid 4-hydroxylase(C4H)on the accumulation of flavonoids in Anoectochilus roxburghii,the total flavonoid content of different tissues of A.roxburghii was determined by using the aluminium nitrate assay.Based on homologous cloning and RACE techniques,the C4H gene(ArC4H,GenBank accession number ON455229)was isolated and cloned from A.roxburghii;the expression of the C4H gene in different tissues of A.roxburghii was analysed by Real-time Quantitative PCR(qRT-PCR).The results showed that the flavonoid contents of different tissues of A.roxburghii differed significantly(leaf>stem>root),with the total flavonoid content of leaf being more than three times that of root and stem;the phylogenetic tree analysis revealed that A.roxburghii was close to most orchids and the open reading frame of the ArC4H gene was 1 518 bp,encoding 505 amino acids,and belonged to the cytochrome P450 superfamily,with a theoretical molecular weight of 65.17 ku and an isoelectric point of 9.21,which belonged to hydrophilic proteins;ArFLS had sequence conservatism,and was expressed in different tissues.qRT-PCR analysis showed that the gene had the highest expression in stems,followed by roots,and the expression in leaves was extremely low,and the total flavonoid content also showed a similar trend.Accordingly,the gene was initially identified as ArC4H gene.

Bioinformatics and expression patterns of Dicer-like(DCL),Argonaute(AGO)and RNA-dependent RNA polymerase(RDR)gene families in the whole genome of Solanum habrochaites were analyzed,so as to provide references for further study on the functions of DCL,AGO and RDR gene families in the response of S.habrochaites to abiotic and viral infection.Using Arabidopsis thaliana DCL,AGO and RDR genes as reference sequences,the genome of S.habrochaites LA1777 was searched by local perl language and software such as Pfam and SMART,and the members of ShDCL,ShAGO and ShRDR gene families were determined.Bioinformatics analysis of DCL,AGO and RDR family genes in S.habrochaites was carried out by means of ExPASy,GSDS 2.0,MEGA,Tbtools and SWISS-MODEL.According to abiotic stress treatment,Tomato chlorosis virus(ToCV)treatment and Real-time Fluorescence Quantitative PCR technology,the expression patterns of these genes were analyzed.Seven ShDCL,15 ShAGO and 6 ShRDR genes were identified from S.habrochaites,which were distributed on chromosome 5,7 and 6 respectively.The encoded proteins were similar in structure to DCL,AGO and RDR in other plants,and all of them contained conserved domains unique to this family.Phylogenetic analysis showed that these genes were divided into 4 subgroups,and there were high structural and functional similarities between S.habrochaites and S.lycopersicum.ShDCL2a,ShDCL2c,ShDCL3,ShDCL4,ShAGO1b,ShAGO3,ShAGO4b,ShAGO5,ShAGO7,ShAGO10a,ShAGO10b,ShRDR1,ShRDR2,ShRDR3a,ShRDR6a and ShRDR6b were significantly up-regulated after various abiotic stresses and ToCV infection.It is speculated that these genes play important roles in abiotic stress and virus infection.

The folding mode of Chinese cabbage leaf ball is the main character that determines the appearance shape,taste and stress resistance of commercial organs.In order to explore the internal molecular mechanism of the formation of the folding mode of Chinese cabbage,we cloned the full length sequence of the transcription factor BrPIF5 gene from overlaping and outward-curling Chinese cabbage as experimental materials,and conducted bioinformatics analysis,constructed the plant overexpression vector,and used Agrobacterium to mediate the transformation into tobacco to obtain positive transformation plants.The expression level of BrPIF5 gene in tobacco was detected by qRT-PCR.The results showed that the protein encoded by BrPIF5 gene was a hydrophilic protein with a continuous and complete open reading frame of 634 bp,containing 210 amino acids.The protein was composed of more α-helical structure and random curl,including an AP2/ERF domain.BrPIF5 protein and the other 9 gene family members contained a conserved motif 1,and the position was different from that of other gene family members,which was located in the front of the protein sequence.Phylogenetic tree showed that BrPIF5 gene had close evolutionary relationship with SoPIF15,BhPIF1,BoPIF4,AtPIF4 and BrPIF4 family members.The tobacco strain with overexpression of BrPIF5 was obtained by Agrobacterum-mediated genetic transformation,and the leaves of the tobacco positive transformation strain showed inward curling.qRT-PCR showed that the expression level of BrPIF5 gene in the overlaping Chinese cabbage was higher than that in the outward-curling Chinese cabbage,and the gene expression level in the positive tobacco plants was higher than that in the control.It was further proved that BrPIF5 gene controlled the inward curling of Chinese cabbage leaves,thus promoting the formation of leaf ball folding type.

In order to clarify the role of the Zn(Ⅱ)2Cys6 transcription factor gene VDAG_ 04814 in the growth, development and pathogenicity of the Verticillium dahliae. It constructed a VDAG_04814 gene knockout mutant using homologous recombination mediated by polyethylene glycol. Wild-type and mutant strains were inoculated separately onto PDA media supplemented with hydrogen peroxide, sodium chloride, potassium chloride, sorbitol, sodium dodecyl sulfate, Congo red, as well as onto media overlaid with sterile cellophane, to analyze their levels of resistance to oxidative stress, salt stress, osmotic stress, stress on cell wall and plasma membrane integrity, and strain penetration ability. Their pathogenicity was assayed, and the fungal biomass in potato plants was detected. After hygromycin selection and PCR validation, the correct knockout transformants were able to amplify DNA bands of 1 500 bp upstream and downstream, respectively, as well as the full-length 4 500 bp knockout fragment sequence. The results demonstrated that the growth rate and melanin formation ability of the ΔVDAG_04814 mutants were significantly reduced. On media subjected to oxidative stress and salt stress with the addition of hydrogen peroxide, sodium chloride, and potassium chloride, ΔVDAG_04814 showed a higher inhibition rate compared to the wild-type. On osmotic stress media with sorbitol, the growth inhibition rate of ΔVDAG_04814 was significantly lower than the wild type. No growth inhibition was observed for ΔVDAG_04814 on media subjected to cell wall and membrane integrity stress with the addition of sodium dodecyl sulfate and Congo red. On media overlaid with sterile cellophane, no colonies grew for ΔVDAG_04814, whereas the wild-type strain produced normal colonies. Pathogenicity tests indicated that the wilting index of ΔVDAG_04814 was significantly reduced compared to the wild-type, with wilting index ranging from 47.22 to 55.56. It has been demonstrated that VDAG_04814 can regulate the growth, development, stress resistance, penetration ability and pathogenicity of V. dahliae towards potato. This study provides a new target for the control of potato Verticillium wilt disease.

Temperature stress is one of the main nonbiological stresses that affect the quality and yield of spinach.Investigating the molecular response mechanism of spinach to temperature stress is crucial for spinach stress tolerance breeding.To provide a theoretical basis for the research of the mechanism of spinach resistant to cold stress and heat stress,this study used the cold-tolerant inbred line D3 and the heat-tolerant inbred line M10 of spinach as experimental materials and analyzed their transcriptomes and metabolomes under cold and heat stress to explore the transcriptional and metabolic mechanisms underlying spinach tolerance.Transcriptomic analysis showed that the pathways in which the DEGs were the most enriched in D3 and M10 were essentially the same under cold stress and heat stress.Metabolomics analysis showed that under cold stress,they were coenriched in the pyrimidine metabolism and lysine degradation in KEGG pathways.Under heat stress,these were mainly enriched in the tryptophan metabolism,toluene degradation,biosynthesis of various other secondary metabolites,and glycine,serine and threonine metabolism in KEGG pathways.The joint transcriptomic and metabolomic analysis indicated that through data analysis and gene annotation.SpADH(sov2g036390),SpSHMT(sov1g001130)and SpALDH-1(sov4g007150)were identified as the candidate genes for cold stress tolerance in spinach.SpALDH-1(sov4g007150),SpALDH-2(sov1g043320)and SpNPC(sov1g040610)were identified as candidate genes for heat stress in spinach.Among them,SpALDH-1(sov4g007150),which may be a regulatory gene for spinach stress tolerance,was significantly expressed under both cold and heat stress.

In order to investigate the function of watermelon calcium-dependent protein kinase (CDPK) in grafted seedlings and abiotic stress environments, this study used RT-PCR technology to clone the ClCDPK(Cla97C01G019720) gene from watermelon grafted seedlings and performed bioinformatics analysis on it. Further designed specific primers with Kpn Ⅰ and Sal Ⅰ enzyme cleavage sites based on the ClCDPK sequence,conducted amplification and double enzyme cleavage, and connected with pCAMBIA1300 to successfully construct the expression vector pCAMBIA1300-35S-ClCDPK for the target gene.Using RT-qPCR technology, the gene expression levels of ClCDPK were measured in self rooted seedlings (ZG) and grafted seedlings (JJ) after being subjected to salt and drought stress, respectively.The results showed that the ORF of ClCDPK gene was 1 647 bp, encoding 548 amino acids. Its protein contained STKc_CAMK and FRQ1 functional domains, and was a hydrophilic protein. Subcellular localization prediction showed that the protein was located in the nucleus. Evolutionary tree analysis of ClCDPK with CDPK from six other plants revealed that it was closely related to CDPK from Cucurbitaceae melons and pumpkins, with protein sequence homology alignment exceeding 92.64%, indicating high homology.The RT-qPCR expression results showed that the expression level of ClCDPK in grafted seedlings was significantly higher than that in self rooted seedlings. With the duration of stress, the expression levels of ClCDPK in grafted and self rooted seedlings first increased and then decreased, and under the same stress treatment, the expression level of ClCDPK in grafted seedlings was higher than that in self rooted seedlings.This study indicated that ClCDPK responded positively to salt and drought stress, and the ability of grafted seedlings to resist stress was higher than that of self rooted seedlings. It is speculated that ClCDPK is one of the key factors in watermelon's response to grafting, thereby improving the salt and drought resistance of watermelon grafted seedlings.

Serine hydroxymethyltransferase(SHMT),as an important enzyme involved in basic metabolism,plays an important role in plant cell metabolism,photorespiration and defense activities.To understand the bioinformatics function of the SHMT gene family in watermelon,explore its gene expression characteristics under abiotic stress,and provide a basis for the functional development of watermelon SHMT and the breeding of watermelon stress-resistance genes.Bioinformatics methods were used to identify SHMT family,and RT-qPCR was used to analyze the expression patterns of ClSHMTs in different tissues and abiotic stresses.The results showed that 8 ClSHMTs gene family members were identified in the whole genome of watermelon,which were unevenly distributed on 6 chromosomes and named ClSHMT1—ClSHMT8 in turn.There were some differences in the physical and chemical properties of each gene family member,such as the number of amino acids,molecular weight,isoelectric point.The protein contained 471—585 amino acids,with molecular weight of 51.87—65.00 ku and isoelectric point of 6.57—8.52,all of which were hydrophilic proteins.The subcellular localization prediction was mainly distributed on mitochondria.Gene structure and protein conserved motifs analysis showed that the ClSHMTs structure consisted of 4—15 exons and 3—14 introns,and all ClSHMTs contained conserved SHMT domains.Furtherly,phylogenetic analysis with 6 species such as cucumber and wheat showed that 50 SHMTs were divided into 3 sub-families,Group Ⅰ—Ⅲ.Promoter of ClSHMTs contained cis-acting elements related to light response,plant hormone response and stress response.The expression pattern analysis showed that 6 ClSHMTs were expressed in different tissues of watermelon,and the expression levels of ClSHMT1,ClSHMT4,ClSHMT5,ClSHMT8 in leaves were significantly higher than those in other tissues.Under low temperature,drought and salt stress,the expression abundance of ClSHMTs varies,but the expression was mainly up-regulated.In conclusion,this study systematically analyzed the SHMT gene family in watermelon,and will provide a reference for the further study of the biological functions of ClSHMTs.

In order to study the role of phototrapping pigment binding protein gene in potato photosynthesis,the phototrapping pigment binding protein gene StCP24 (LOC102586836)was bioinformatics analyzed and cloned into potato variety Dongnong 310 to obtain transgenic potato.The morphological indexes,leaf physiological indexes,fluorescence parameters,and gene relative expression of transgenic and control lines were measured under different light intensities after clonal propagation.The function of StCP24 gene was analyzed.The effect of this gene on chloroplasts was determined by ultrastructural observation.The results of bioinformatics analysis showed that StCP24 protein was a hydrophilic protein,and the promoter of StCP24 gene contained elements such as photoresponse,defense and stress response.Phylogenetic analysis showed that the amino acids encoded by StCP24 gene in potato were closely related to those encoded by StCP24 gene in cultivated tomato.Under different light intensity treatments,StCP24 gene overexpressed potato significantly increased stem diameter,leaf area,leaf weight,root length,chlorophyll a,b content,Fv/Fm,ETR,ETRmax and qP compared with wild-type potato.The overexpression of StCP24 gene can make the lamellar stacking of thylakoid granules more tightly and to a higher degree.In conclusion,the overexpression of StCP24 gene can increase the content of chlorophyll a and b in leaves,improve the electron transfer rate in photo cooperative photoreaction,and promote the growth of transgenic potato plants.

Tiller-related traits are important characteristics of wheat plant type,which determine plant structure and affect grain yield.In order to understand the inheritance and drought resistance of tiller-related traits in wheat under different water conditions,and to excavate the loci related to tiller-related traits,240 wheat varieties (lines) were selected as the subjects of this study,based on the phenotypic identification of tiller angle,effective tiller number and yield per unit area under normal irrigation (NI) and drought stress (DS) conditions,and the comprehensive evaluation of drought resistance,combined with 90K gene chip,genome-wide association study (GWAS) was to identify genetic loci for tiller-related traits and to screen for superior germplasm.The tiller angle,effective tiller number and yield per unit area showed significant difference,and the coefficient of variation ranged from 0.07 to 0.33.According to D-value,the drought resistance of Zhongyou 206 was the best.A total of 54 stable genetic loci significantly associated with tiller angle and other traits were detected, distributed on all chromosomes except 3D, 4D and 5D. Three identical stable loci were commonly detected under both treatments, located on chromosomes 2B, 4B, and 6B. Additionally, four pleiotropic loci were commonly detected in different traits, located on chromosomes 2B, 2D, and 5B.At the same time,the haplotype analysis of Ra_c491_902 (R²=5.45%—17.91%),which was significantly correlated with tiller angle on chromosome 2B,showed that there were three haplotypes:TA-Hap1,TA-Hap2 and TA-Hap3,the haplotypes (lines) containing TA-Hap1 were mainly derived from Huanghuai winter wheat regain.Five candidate genes related to tiller angle were screened by screening the stable genetic loci detected under different treatments.Gene annotation of the genes selected on Ra_c491_902 showed that the genes encoding cytochrome P450 family protein can be used as important genes such as regulating tillering angle,plant drought resistance and defense,to explore the association between genes and phenotype,and lay the foundation for the genetic improvement of tiller-related traits in wheat.

To investigate the characterization and function of LbaHY5 gene of Lycium barbarum,explore the role of LbaHY5 in the degree of stem straightening in Lycium barbarum.Using Ningqi No.1 as the test material,a preliminary analysis was conducted on the structure and function of the LbaHY5 gene using bioinformatics,subcellular localization,and qRT-PCR methods,the results indicated that the LbaHY5 gene cloned from Ningqi No.1 had a full-length open reading frame of 483 bp,encoding 160 amino acids,with a molecular weight of 17.52 ku and an isoelectric point of 9.69,classifying as a hydrophilic protein.Conserved domain and multiple sequence alignment analysis showed that LbaHY5 protein contained a type bZIP domain,which belonged to the bZIP gene family.Evolutionary tree analysis showed that the LbaHY5 protein in goji berries was highly homologous to the HY5 protein in Solanaceae plants such as tomatoes and potatoes.In addition,promoter analysis showed that the promoter region of this gene was rich in multiple functional elements,which were respectively related to processes such as light signaling,hormone signaling pathways,and abiotic stress response.The subcellular localization indicated that LbaHY5 was located on the nucleus.The transcript level of LbaHY5 gene was the highest in the flower and the lowest in the stem.During the fruit development period,the expression level of LbaHY5 gradually increased.The results speculated that LbaHY5 gene might play an important role in the growth and development of floral organs and fruits in Lycium barbarum.

In order to elucidate the sequence characteristics and expression pattern of PatGTL1,a member of Trihelix transcription factor family,and lay a foundation for the further function study of PatGTL1,the total RNA of Pogostemon cablin (patchouli)was extracted and reverse-transcribed to cDNA.The specific primers were designed according to the transcriptome sequence of PatGTL1,and the cDNA was used as a template to clone PatGTL1 gene.The bioinformatics analysis was further performed to reveal the structural,physicochemical properties,and phylogenetic relationships of PatGTL1.Besides,the pAN580-PatGTL1-EGFP vector was built and transformed into Arabidopsis protoplastsa to detect the subcellular localization of PatGTL1.The Real-time Quantitative reverse transcription PCR(qRT-PCR)was also carried out to investigate the expression patterns of PatGTL1 gene in different tissues of patchouli and under treatments of methyl jasmonate(MeJA),salt stress,drought stress,and cold stress.The results showed that the open reading frame of the PatGTL1 gene was 1 497 bp,encoding 498 amino acids.The PatGTL1 protein was an unstable hydrophilic protein that contained 42 serine phosphorylation sites and was predicted to be located in the nucleus without transmembrane domains and signal peptides.Multiple sequences alignment showed that PatGTL1 contained two GT1 conserved domains,and was classified into GT-2 subfamily of Trihelix transcription factor family.According to the phylogenetic tree,PatGTL1 was clustered closely with Sesamum indicum SiGTL1.The qRT-PCR analysis indicated that PatGTL1 expressed in the root,stem,and leaf in patchouli,particularly with the highest expression in the young leaf.The PatGTL1 expression was significantly upregulated by MeJA and showed an increasing trend at 3—24 h after treatment,but downregulated by drought stress.The highest expression of PatGTL1 was showed at 3 h after salt treatment,and at 12 h after cold treatment.These results enrich the Trihelix transcription factor family study of patchouli.

The seed yield per spike and spike axis dry weight affect yield per plant.In order to reveal the genetic basis,QTLs were mapped with populations F₂ and BC₁ (F₁×P₂) derived from the cross combination 9048×16-201 using CIM and ICIM-ADD methods.A total of 7/10 (CIM/ICIM-ADD) QTLs were detected in the F₂ population,including 3/6,3/3 and 1/1 QTLs conferring PSSY,PSADW and PBSADW respectively,with a contribution rate of 15.81%/24.09%,2.84%/8.65% and 6.49%/6.56%,respectively.And 1/3 QTLs were identified in the BC₁ population,including 0/2 and 1/1 QTLs underlying PSSY and PBSADW,with a contribution rate of 0/10.28% and 11.60%/10.22% respectively.Among all the detected QTLs,qPSSY3.1 and qPBSADW3.1 were stable QTLs,and the latter was also the main-effect QTL,with contribution rates ranging from 3.76%—7.00% and 6.49%—11.60%,respectively.They overlapped in the marker interval RCM920—RCM950,forming a QTL cluster (i.e.,QTL-cluster4).Additionally,a dozen pairs of epistatic QTLs were detected with the epistatic effect of these traits accounting for more than 19% of the total contribution rate,indicating that the epistatic effect was an important genetic component of seed yield per spike and spike axis dry weight in castor.The results not only provided a reference for the understanding of the genetic structure of seed yield per spike and spike axis dry weight but also offered available molecular markers for marker-assisted selection.

To investigate the role of calcium-dependent protein kinase (CDPK) in wheat growth and stress response,the TaCDPK17 gene was cloned from common wheat and its sequence structure,expression pattern,and stress resistance function were preliminarily analyzed.The results showed that the length of the TaCDPK17 gene coding region was 1 701 bp, encoding 566 amino acids and possessing typical structural features of the CDPK family, including one conserved serine/threonine kinase domain and four EF hand shaped domains. Evolutionary tree analysis of TaCDPK17 and CDPK17 from 12 other plants showed that TaCDPK17 had high homology with the CDPK17 sequence of gramineous crops,especially Aegilops tauschii and barley.The promoter region of TaCDPK17 gene contained multiple cis regulatory elements related to hormone signaling pathways,light response.Among them, there are more abscisic acid (ABA) responsive elements (ABRE) and methyl jasmonate responsive elements (CGTCA). The expression analysis based on Real-time Fluorescence Quantitative PCR showed that the expression level of TaCDPK17 increased to varying degrees after induced by 100 μmol/L ABA, 100 μmol/L methyl jasmonate, 20% PEG6000, and 250 mmol/L NaCl. Under stress conditions of 2 μmol/L ABA and 100 mmol/L NaCl, the germination rate of Arabidopsis seeds overexpressing TaCDPK17 was significantly higher than that of the wild type. Meanwhile, overexpression of TaCDPK17 alleviated the inhibitory effects of ABA or osmotic stress treatments on seedling root growth. During stomatal closure, transgenic plants overexpressing TaCDPK17 are more sensitive to ABA and exhibit a stronger stomatal closure trend compared to wild-type plants. These results indicated that TaCDPK17 plays an important role in stress response and hormone signaling in wheat.

In order to further explore the genetic rule of grain and stalk yield of sorghum,grain sorghum Xinliang 52 and Sudan grass TS 185 were hybridized as parents to obtain F₂ and F_2∶3 populations,115 pairs of polymorphic primers were used to construct the genetic linkage map of 430 F₂ progeny populations by interval mapping.A total of 86 QTLs were detected for 12 agronomic traits,including number of tillers, number of blades, stem diameter, panicle length, plant height, stem fresh weight, whole plant fresh weight, shelling rate, panicle weigh, thousand kernels weight, grain weight per panicle and grain number per panicle, with LOD value as the threshold value.The QTL of stem fresh weight was located between sam17164-sam15397 on chromosome 1.On chromosome 2,the QTL of number of tillers was located in Xcup64-Xcup26 region,leaf was located in Xtxp019-sam01138 region,and stem fresh weight was located in Xcup26-Xtxp080 region.Panicle length QTL was located between sam44791 and sam33751 on chromosome 3.The QTL of shelling rate was located at sam39622-sam43980 on chromosome 7.The QTL for whole plant fresh weight located in sam10491-sam17740 on chromosome 8 and the QTL for number of tillers located in sam710901b-sam59778 on chromosome 10 were all newly detected loci.

The adult plant resistance gene Lr12 exhibits excellent resistance in production systems.To fine map and develop reliable molecular markers for Lr12,a cross was made between the susceptible variety Thatcher and the resistant near-isogenic line RL6011 containing the Lr12 gene.The F₁ generation resulting from this cross was self-pollinated to generate F₂ individual plants and F_2∶3 families.Field evaluations were conducted using a mixture of five highly virulent leaf rust pathotypes (PHTT, THKS, THTT, PHTS, and PHKS) to inoculate F₂ individual plants and F_2∶3 families for adult plant resistance assessment and genetic analysis of resistance.Subsequently,genotyping was performed using a 16K liquid chip on 10 resistant and 10 susceptible individuals from the F₂ generation to identify SNP markers closely linked to Lr12.This enabled the determination of the chromosomal physical interval containing the resistance gene,the development of SSR molecular markers,and the construction of a genetic linkage map.The results indicate that the segregation ratio of resistance to leaf rust in 3 494 F₂ individuals derived from the RL6011(Lr12)/Thatcher cross was consistent with a 3∶1 ratio ( {\mathrm{\chi }}_{3:1}^2=0.14;P=0.71). In the assessment of 685 F_2∶3 families, the segregation ratio among resistant individuals, resistant heterozygous individuals, and susceptible individuals conformed to a 1∶2∶1 ratio ( {\mathrm{\chi }}_{1:2:1}^2=2.01;P=0.37), suggesting that Lr12 is a dominant gene and the population segregation follows Mendelian single-gene inheritance patterns. Genetic linkage map analysis localized the adult plant leaf rust resistance gene Lr12 between SSR molecular markers YK12817 and YK12928,within a genetic interval of 0.38 cM.This corresponds to a physical interval of 2.09 Mb within the physical range of 579.44 Mb to 581.53 Mb on chromosome 4BL of the Chinese Spring reference genome(IWGSC.Ref.V1.0).These findings provide a solid basis for predicting candidate genes.

The EXORDIUM(EXO) gene was identified in Arabidopsis thaliana as an brassinosteroid(BR)-responsive gene that promotes plant growth by mediating cell expansion.In order to investigate the function of EXO gene in Brassica napus and its expression pattern in different tissues at flowering stage,we used B.napus Zhongshuang 6 as the material,cloned the sequence of the coding region of the EXO gene named BnEXO,and carried out bioinformatics analysis,and used Real-time Fluorescence Quantification to determine the relative expression of BnEXO gene in B.napus in roots,stems,leaves,petals,buds,and pericarps at flowering stage.The results showed that the CDS sequence of BnEXO gene was 945 bp,BnEXO was a stable hydrophilic non-transmembrane protein,which belonged to secreted proteins and was expressed extracellularly,and the secondary structure of the protein was dominated by the random coil.The results of expression analysis in different tissues showed that the expression of BnEXO gene in different tissues was in the order of petals,pericarps,buds,stems,roots and leaves,and the highest expression was found in petals.In addition,20 B.napus EXO genes(BnaEXO),11 B.rapa EXO genes (BraEXO),and 11 B.oleracea EXO genes (BoEXO)were identified in this study based on the protein sequences of eight EXO gene family members in A.thaliana.Most proteins of gene family members were stable proteins,localized extracellularly,with amino acid lengths ranging from 271 to 411 aa,isoelectric point predictions ranging from 5.76 to 9.60,and molecular masses ranging from 28.76 to 46.21 ku.Phylogenetic analysis classified the EXO genes into five subgroups,EXOA,EXOB,EXOC,EXOD,and EXOE,with the least number of members in the EXOB subgroup.Gene structure analyses showed that most members contained only one exon and no intron,and the sequences of EXO gene family members were highly conserved.The results of cis-element analysis of the promoter region of the members in B.napus indicated that the BnaEXO genes play important roles in plant growth and development and in adversity stress.

To explore the expression patterns of potato genes and provide theoretical basis for the subsequent study of gene resistance to potato diseases.Three endogenous small G protein genes StRac5,StRac7 and StRac13 were cloned from potato,and their bioinformatics and expression patterns were analyzed.The results showed that StRac5,StRac7 and StRac13 contained similar conserved sequences to the small G proteins in Arabidopsis and rice,all belonging to ROP proteins.The protein domains of StRac5 and StRac13 were identical with AtRop1,AtRop3,AtRop5,AtRop6 of Arabidopsis and rice OsRac5,OsRac6,OsRac7,and StRac7 was identical with AtRop9.Phylogenetic tree analysis showed that StRac7 belonged to class Ⅱ and was closest to Arabidopsis AtRop9.StRac13 belonged to class Ⅲ and was the closest genetic relative to Arabidopsis AtRop7.StRac5 belonged to class IV and was the closest genetic relative to rice OsRac5 and OsRac6.The relative expression levels of StRac5,StRac7 and StRac13 genes in different tissues of potato were leaf>root>stem.Compared with the expression in stems,the expression of StRac5,StRac7 and StRac13 in leaves were increased by 1 222.4%,2 531.3% and 468.2%,respectively.After inoculation,the relative expression of StRac5,StRac7 and StRac13 genes increased first and then decreased,and the relative expression of StRac5 and StRac13 genes increased by 62.2%,40.4%,respectively,compared with 0 h after inoculation 24 h.The relative expression of StRac7 gene was increased by 827.8% at 72 h after inoculation compared with 0 h.After ABA,SA,GA and 6-BA treatment,the relative expression of StRac5,StRac7 and StRac13 genes showed a downward trend.After JA and IAA treatment,the relative expression of StRac5,StRac7 and StRac13 genes showed an up-regulated trend.Therefore,StRac5,StRac7 and StRac13 genes can respond to potato late blight infection,and their expression patterns are different in different tissues and under different hormone treatments.

Expansin (EXP) plays a crucial role in plant adaptation to environmental stress by regulating the relaxation of cell wall components and enhancing flexibility.The aim of this study was to investigate the characteristics of the EXP family members and their expression patterns under salt stress,and to explore the salt tolerance genes in Aquilegia coerulea.Genome-wide identification and analysis of the AcEXP family of Aquilegia coerulea was using bioinformatics methods and its expression pattern under salt stress was analysed using RNA-seq expression data and qRT-PCR.The results revealed that the entire genome of the Aquilegia coerulea contained 27 EXP genes,distributed across six chromosomes and one scaffold.The secondary structure of proteins was dominated by β-sheet and random coil.The majority of the proteins were hydrophobic,and all 27 EXPs were found in the cell wall.The phylogenetic tree showed that members of the AcEXP family were classified into four subfamilies:EXPA,EXPB,EXLA and EXLB.Members of the same subfamily possessed similar gene structure and protein conserved motifs.The AcEXP genes contained multiple elements that responded to phytohormones and stress.The transcriptome data was used to analyze the expression pattern of AcEXP under salt stress.Twenty-one EXPs responded to salt stress,but the expression patterns were different in roots and leaves.AcEXPA8/9/12,AcEXPB1,AcEXLB1 and AcEXLB2 were differentially expressed in leaves and roots.The qRT-PCR results further verified the expression pattern of AcEXP genes under salt stress.It comprehensively analyse the basic characteristics of the EXP gene family members of Aquilegia coerulea and expression changes under salt stress.The study preliminarily demonstrates that AcEXPA8/9/12,AcEXPB1,AcEXLB1 and AcEXLB2 are involved in the salt stress response process and responded positively.

MADS-box transcription factors are widely found in plants and play important roles in plant growth and development and secondary metabolism.To investigate the expression of MADS-box transcription factor family in different accumulation periods of capsaicin.MADS-box transcription factor family was identified by using transcriptome data from different accumulation periods of capsaicin,and their functions were preliminarily analyzed.Subcellular localization,conserved motifs,phylogenetic tree and chromosomal localization of members of the MADS-box transcription factor family of chili peppers were carried out.The results showed that a total of 95 MADS-box transcription factors were identified in the Capsicum annuum L.transcriptome data;containing 105-395 amino acids;with molecular weight ranging from 11.55-44.46 ku;with theoretical isoelectric points ranging from 5.16-10.01;mainly expressed in the nucleus,and all of them contained MADS conserved structural domains,and phylogenetic analysis showed that MADS proteins could be divided into eight subfamilies.There were 73 CaMADS family members localized to 12 chromosomes.There were 26 differentially expressed MADS-box genes,six of which were up-regulated during C1 vs C2 and down-regulated during C2 vs C3.Based on KEGG enrichment and protein interactions,it was predicted that CaMADS13 might be involved in lignin synthesis in chili peppers.CaMADS24 might be involved in the synthesis of capsaicin and lignin synthesis precursor,coumaroyl-coenzyme A.Bioinformatics analysis was used to identify the MADS-box family of transcription factors in chili peppers,which provides data reference and theoretical basis for in-depth study of the molecular mechanism of action in the secondary metabolism of capsaicin.

In order to identify the key genes controlling rind hardness and breed crack-resistant watermelon varieties.An F₂ segregating population was created using the high-firmness line 901 and the low-firmness line BSH.Both BSA-seq and RNA-seq approaches were utilized to map the genes responsible for rind hardness.The results of BSA-seq revealed an interval region of 2.14 Mb on chromosome 10,spanning from 1 620 000 to 3 760 000,where the intersection of SNPs and InDels identified 150 candidate genes.Among these,two genes showed non-synonymous mutations,and one gene exhibited a frameshift mutation.Correlation analysis between BSA-seq and RNA-seq identified 6 correlated genes,including Cla97C10G187120, Cla97C10G187020,Cla97C10G187430,Cla97C10G187510,Cla97C10G187280,and Cla97C10G186540.Through bioinformatics analysis,the candidate gene Cla97C10G187120 was identified.The result of qRT-PCR indicated that the transcriptome data was reliable.And the relative expression of the candidate gene Cla97C10G187120 was lower in the line 901 than the line BSH.This study lays a crucial foundation for understanding the molecular mechanisms underlying watermelon rind hardness.

To clone the PpLAC7 gene sequence of Jinxiu yellow peach,investigating its gene sequence information and its role in flesh browning.The cDNA sequence of PpLAC7 was cloned from Jinxiu yellow peach by homologous cloning.The promoter region elements,physicochemical properties,secondary and tertiary structure,phylogenetic tree and amino acid sequence alignment of PpLAC7 gene were performed bioinformatics by using bioinformatics software.In addition,it also investigated the subcellular localization and the expression pattern of PpLAC7 during the process of fresh browning in Jinxiu yellow peach.The results of bioinformatics analysis indicated that the promoter region included light responsiveness,MeJA-responsiveness,drought-inducibility,seed-specific regulation elements,etc.The full length of PpLAC7 gene was 1 692 bp,encoding 563 amino acids,molecular weight was 61.867 ku,total number of atoms was 8 621,GRAVY was -0.028,theoretical pI was 5.95,instability index was 36.48,aliphatic index was 87.26.The secondary structure of PpLAC7 protein mainly contained α-helix of 14.74%,extended strand of 28.77%,β-turn of 6.22% and random coil of 50.27%.Phylogenetic analysis showed that PpLAC7 had high similarity to MdLAC7.Deduced amino acid sequence indicated that PpLAC7 protein,the same as other species,also contained three typical copper ion domains.Subcellular localization analysis revealed that PpLAC7 was located in endoplasmic reticulum.The expression of PpLAC7 gene was up-regulated during the storage of Jinxiu yellow peach fruit.Combining the relationship of flesh browning index and PpLAC7 expression,it indicated that PpLAC7 may play an important role in the browning process of Jinxiu yellow peach fruit.

To evaluate the potential of SpCas9-NG in maize genome editing,the key gene for chlorophyll synthesis,ZmSCD,was selected as the target.The absence of this gene causes seedlings to exhibit albinism,providing a visual assessment of editing efficiency.Based on the PAM sequence recognition rules of SpCas9 (5'-NGG-3') and SpCas9-NG (5'-NG-3'),target sites were designed on the second and third exons of ZmSCD.These target sequences were successfully constructed into SpCas9 and SpCas9-NG knockout vectors,which were then introduced into the maize inbred line KN5585 using Agrobacterium-mediated genetic transformation.The callus tissues were cultured until leaf tissues differentiated,and the albinism rate was recorded to determine the editing efficiency of the different editors.The genomes of albino seedlings were then sequenced.Through three rounds of genetic transformation,SpCas9 produced 76,125 and 28 callus tissues,while SpCas9-NG produced 100,69 and 30 callus tissues.The results showed that the gene editing efficiency of SpCas9 across the three transformations was 14.47%,13.60%,and 10.71%,respectively,while SpCas9-NG editing efficiency was 12.00%,10.14% and 13.33%.Sequencing results of the albino seedlings revealed overlapping peaks near the target sites for both editors.The study demonstrates that SpCas9-NG had comparable editing efficiency to traditional SpCas9 in maize,showing similar gene editing capabilities.In contrast,SpCas9-NG has a broader PAM sequence adaptability,allowing for more flexible target design.This flexibility enables more precise and diverse editing within the maize genome.

Salt-alkali stress has become one of the important factors restricting agricultural production in my country.Exploring the molecular mechanism of salt tolerance of crops has important theoretical and practical value for crop breeding.The purpose of this study is to clone the ZmMPI gene in corn and transform corn plants.First,qRT-PCR was used to analyze the ZmMPI expression changes in plants treated with saline-alkali solutions.Then DNAMAN software was used to perform multiple comparison analysis of the ZmMPI protein sequence.MEGA 7.0 software was used to construct a phylogenetic tree,and a series of software were used to analyze the ZmMPI protein sequence.ZmMPI performed bioinformatics analysis.Finally,molecular cloning technology was used to successfully clone the coding sequence of the ZmMPI gene,construct a plant overexpression vector,and use Agrobacterium-mediated genetic transformation method to transform the corn inbred line B104.The overexpression transgenic plants were transformed at the genome level,transcription level and protein level.Identify and analyze changes in expression levels.The results showed that the expression level of the ZmMPI gene showed an overall trend of first increasing and then decreasing after being subjected to salt-alkali stress;the ZmMPI protein sequence comparison result showed a similarity rate of 64.15%,and the phylogenetic tree showed that ZmMPI had the highest homology with Zea mays subsp.parviglumis ABA34115.1.The protein contained a protein domain Potato_inhibit,which had an α-helix,a random coil and a β-turn.It was relatively hydrophobic and had 10 predicted Potential phosphorylation sites;the identification results of the 49 transformation events obtained showed that the ZmMPI gene in 13 over-expressed transgenic lines could be expressed normally at the genome level,and the ZmMPI gene in 10 over-expressed transgenic lines could be transcribed and translated normally.Finally,10 overexpression transgenic lines capable of normal transcription and translation were obtained,laying the foundation for further exploring the molecular mechanism of ZmMPI gene in response to salt-alkali stress.

Drought stress has a serious effect on the growth and development of maize,which leads to a decrease of maize yield.Purple acid phosphatase is a phospholipase protein involved in many physiological and biochemical functions of plants.In order to further study the role of purple acid phosphatase family genes in the process of stress resistance of maize,this paper explored the response mode of ZmPAP26b gene under drought stress,and Real-time fluorescence Quantitavive analysis was used to analyze the relative gene expression in different maize inbreeding lines under simulated drought conditions;ZmPAP26b(GenBank:NC_050104.1)was cloned from maize,and PAP genes in Zea mays,Arabidopsis thaliana,Oryza sativa L.,Triticum aestivum L.,Sorghum bicolor and Brachypodium distachyon were identified and bioinformatic analysis was performed.Meanwhile,prokaryotic overexpression strains were constructed for functional verification.The results showed that the expression of this gene decreased in drought tolerant materials and increased in drought sensitive materials under drought stress.The CDS length of this gene was 1 431 bp,encoding 476 amino acids.A total of 228 PAP genes were found in six species,divided into 4 subfamilies by phylogenetic analysis.The 19 PAP genes in maize were distributed on 9 chromosomes and had similar conserved domains.Analysis of promoter cis-acting elements showed that they contained elements responding to drought and hormones.Prokaryotic expression experiments showed that the growth of strains containing the recombinant plasmid pET28a-ZmPAP26b was inhibited compared with non-loaded strains under 10% PEG-6000 and 15%PEG-6000 simulated drought stress.In summary,it is speculated that ZmPAP26b is negatively regulated under drought stress.