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.

The effect of different concentrations salt stress on seedling growth and expression of related genes were explored in this study. Firstly, salt tolerant variety Yura Hama Daikon and susceptible variety Wujinhong were selected according to germination under salt stress conditions among 11 cultivars.And then the seedling height(SH) and the leaf scorch index(LSI) of Yura Hama Daikon and Wujinhong were investigated under salt stress treatment.The results showed that SH decreased and LSI increased under salt stress in both varieties.Compared with the salt-sensitive variety Wujinhong,the salt-tolerant variety Yura Hama Daikon had a smaller decrease in SH and lower LSI.Under 200 mmol/L salt stress,SH and LSI of salt-tolerant Yura Hama Daikon were 46.18% and 20.56,respectively,while those of salt-sensitive Wujinhong were 75.25% and 56.11.The transcription of RsCAT and RsSOD genes was studied in salt-tolerant and susceptible varieties under different salt concentrations by qPCR.The expression of RsCAT gene was first increased and then decreased under low salt concentration treatment,and reached the maximum at 7 day.When treated with high salt concentration,the transcription of RsCAT in Wujinhong was the highest at 48 h,while the expression level in salt-tolerant varieties increased gradually and maintained for a longer time,reaching the highest at 7 day.After high salt concentration treatment,the transcription of RsSOD gene reached the highest expression level at 24 hours,and then maintained a higher level in salt-tolerant variety.In salt-sensitive varieties,the maximum expression level of RsSOD appeared at 14 day.The above results will lay foundation for revealing the mechanism of salt stress in radish and provide technical support for radish salt-tolerant breeding.

Calmodulin is an important Ca²⁺ receptor protein in plants,which plays an important role in calcium signaling pathway and stress resistance.It is of great significance to study the mechanism of CaM protein in tomato under low temperature stress for cold tolerance breeding of tomato.Tomato calmodulin genes SlCaM3, SlCaM4 and SlCaM5 were cloned from tomato varieties Heinz1706,LA3969,Jifen 2,Jifen 3 and Nongbofenba 15,and their sequences and protein sequences were analyzed by bioinformatics;the cis acting elements in the promoter regions of SlCaM3,SlCaM4 and SlCaM5 were analyzed by plantcare;Quantitative Real-time PCR was used to analyze the expression patterns of SlCaM3,SlCaM4 and SlCaM5 under 15,5 ℃ temperature stress in different tomato varieties,and the specific expression of SlCaM3,SlCaM4 and SlCaM5 in different tissues was analyzed in combination with RNA-seq data.The results showed that the coding sequence of SlCaM3,SlCaM4 and SlCaM5 were 450 bp,and their similarity was 93.63%;the encoded amino acid sequences were identical,belonging to acidic stable hydrophilic protein with typical conservative domain of cam protein.The analysis of cis acting elements showed that the promoter regions of the three genes contained not only the necessary core elements,but also a variety of biotic and abiotic stress response elements,and showed a complementary pattern.The analysis of the expression patterns of different degrees of low temperature stress showed that the expression patterns of SlCaM3,SlCaM4 and SlCaM5 in five tomato materials showed a trend of first decreasing and then increasing at 15 ℃,and the expression of SlCaM5 increased more significantly.At 5 ℃,SlCaM3 and SlCaM4 did not change significantly,but the expression level of SlCaM5 gene increased significantly in the later stage of treatment.The high expression of SlCaM5 gene in the process of tomato resistance to low temperature indicated that it maintained the translation level of cam protein under low temperature and ensured that the function of CaM protein was not affected.The analysis of SlCaM3,SlCaM4 and SlCaM5 specific expression in different tissues of Heinz1706 showed that SlCaM3 and SlCaM4 were highly expressed in meristem,while the expression of SlCaM5 different tissues was not significantly different.

In order to identify the pathogenic fungi which causing tomato bacterial wilt in Fengnan District of Hebei Province.This study proved the disease as Bipolaris sorokiniana by pathogen isolation,pathogenicity test,morphological observation,and rDNA-ITS gene sequence analyses was caused by Fusarium oxysporum f.sp.radicis-lycopersici(Forl).The result showed that the mortality and morbidity of stem based inoculation were higher than root soaking inoculation.So,the disease with stem based inoculation developed faster.The study provided theoretical basis for the breeding of disease resistance and the screening of the fungicides.

It is necessary for us to clone some endogenous promoters with high transcription activity in tomato leaves and construct CRISPR/Cas9 system, which could provide an effective technology for the research of molecular design breeding in tomato. Six different truncated tomato U3 promoters had cloned by two rounds of PCR amplification from tomato variety Zhongshu 4. Six GUS fusion expression vectors were constructed and transformed into tomato leaves by the agroinfiltration. After right sequencing of the transfer PCR products, the necessary element for transcriptional function of six kinds of SlU3 promoter was analyzed using DNAMAN software. The different truncated SlU3-1P,SlU3-3P and SlU3-4P promoters from tomato variety Zhongshu 4 were performed to clone by two rounds of PCR amplification, altogether there were six different promoters. Lengths were 489,318,450,248,457,248 bp respectively. Six GUS fusion expression vectors were constructed and transformed into tomato leaves.The promoter sequence alignment analysis revealed that the tomato U3 promoter, like the Arabidopsis U3 promoter, also contained two more conserved elements, the USE and TATA boxes, and the positions between the two elements were relatively fixed.Transfected tomato leaves by agroinfiltration transformation method. The results showed that the six truncated U3 promoters could drive GUS expression in tomato leaves and tomato leaves could be stained blue and the shorter promoters also had stronger transcription activity as original promoters.Six different tomato U3 promoters had cloned with high-level transcription in tomato leaves, which could provide ideal endogenetic promoters for the construction of CRISPR/Cas9 system for tomato functional genomics research.

The aim of this study was to uncover the specific SNPs of fusarium wilt resistance gene FOC1 of Brassica oleracea, which will lay the foundation for the development of specific molecular markers in the future.Based on the identification of fusarium wilt resistance in 11 cabbage inbred lines,the variations of gene sequences and amino acid sequences of FOC1 and its alleles in 11 cabbage materials were analyzed.Results showed that there were 92 SNPs and 2 Indels between these sequences,including 18 specific SNPs related to fusarium wilt resistance,of which 86.11% were transition mutations and 13.89% were transversion mutations.The nucleotide substitutions of T/C (47.22%) and G/A (38.89%) showed higher mutation rates,while C/G and A/C accounting for about 13.89%.In addition,the four specific SNPs identified between the susceptible and resistant cabbage materials led to the variations of three amino acids.

Kales,which are widely cultivated as ornamental plants in wintertime or cold areas,play important roles for urban landscaping.Cold stress which can reduce the ornamental value is one of the most important characters for the improvement and breeding of kales.In order to elucidate the physiological characteristics during low temperature and reveal cold resistance mechanism,several varieties and strains which were bred by our group were used as research materials.The relative electrical conductivity (REC) under different low temperature treatment,semi-lethal temperature (LT₅₀) of different accessions,as well as the osmoregulation substances contents and antioxidant enzymes activities under different temperature treatment were analyzed.It was showed that the REC of kale leaves increased rapidly when temperature under -5℃,and reached maximum values at about -10℃.The TL₅₀ values of kale accessions were -10.3——13.7℃.The TL₅₀s of red types were lower than white types,as well as round and leatherleaf types were lower than wrinkle types.These results were consistent with field cold resistance performances of the varieties.Changing characteristics of the detected osmoregulation substances and antioxidant enzyme activities were different under different temperature treatments.These physiological indexes reached maximum values at -5℃ or -8℃ and decreased at lower temperature,except soluble sugar which reached maximum value at -10℃.The principal component analysis revealed that LT₅₀ value,soluble sugar and free proline contents were main components causing cold resistance variations among different varieties.These results will provide theoretical basis for the establishment of kale cold resistance evaluation system,and lay foundation for the development of new varieties with excellent cold resistance.

In order to study the influence of silicon on the expression of pepper CaMADS-box gene under abiotic stress,such as the high temperature and salt stress,we used pepper 101 as experimental materials,the physical and chemical properties of encoding protein was analyzed,phylogenetic tree was constructed,subcellular localization was predicted through bioinformatics software on the base of the cloning of pepper CaMADS-box gene.The results showed that the cloned gene encoding protein CaMADS-box was hydrophilic protein,containing MADS domain structure,belonged to MADS gene families.And its subcellular localization was in the nucleus,the molecular evolutionary tree showed that close to Nicotiana,the similarity was 67%.Fluorescence quantitative analysis showed that CaMADS-box gene expression after high temperature stress and salt stress were presented first rise after the fall of the model, the difference was to peak at 48 h, under high temperature stress, salt stress peak at 24 h, silicon handle could induce gene expression CaMADS-box, under high temperature stress and salt stress were expressed at 12 h to reach peak, which suggested that CaMADS-box was a silicon quick response genes, speculated that the silicon handle in alleviating pepper abiotic stress such as high temperature and salt stress plays an important role.

The effect of soil amendments on soil microbial communities,quality and yield of tomato in protected house were investigated for providing a scientific basis for reasonable use of soil amendments.A field experiment was initiated in a planting tomato greenhouse with different kinds of soil amendments(lime nitrogen,fosthiazate,abamectin and substrate).Changes of bacteria,fungi,quality,yield were monitored.Results showed that four different types of soil amendments could significantly increased the number of soil bacteria,fungi,actinomycetes and antagonists,while the numbers of bacteria and fungi in the later growing period of tomato compared with the middle growing period were decreased;the contents of lycopene and Vc reached the highest under substrate treatment for 8.75 mg/100g,78.60 mg/kg,respectively;the soluble sugar content ranged from 2.85% FW to 3.29% FW;compared with other treatments,the sugar acid ratio of CK,substrate and abamectin were 7.57,6.91 and 7.70,respectively,taste the best;under substrate treatment,the yield of tomato was the highest for 139 059.30 kg/ha,which was increased by 21.18% compared with CK.Considering the taste quality and yield,substrate could be used as a preliminary reference for tomatoes production.

Neutral/alkaline invertases,as important substances in plant sucrose metabolism,are mainly involved in processes such as plant growth and development,and response to adversity stress.To investigate the response pattern of AsNI to stress,this study cloned two garlic neutral/alkaline invertase genes,using Ledu purple skin garlic as the test material and they were subjected to bioinformatics and expression characterization.The results showed that the open reading frames of AsNI1 and AsNI2 were 522,1 203 bp,encoded 173,400 amino acids.AsNI1 and AsNI2 were both hydrophilic proteins predicted to be localized in the cytoplasm with a Glyco_hydro_100 structural domain.However,the amino acid sequence similarity between the two was only 25.75%,and AsNI2 contained one glycosylation site,while no glycosylation site was detected in AsNI1,and the two were distantly related.The analysis of the protein interaction network showed that AsNI2 and AsNI1 might participate in different biochemical processes.The promoter sequence analysis revealed that the promoter regions of AsNI1 and AsNI2 contained multiple cis-acting elements related to stress response,with the AsNI2 promoter having a significantly larger number of drought and low temperature stress response elements than AsNI1.The prediction of promoter transcription factor binding sites showed that they contained different kinds and numbers of binding sites,indicated that AsNI1 and AsNI2 could perform different gene functions.The qRT-PCR assay revealed that the expression of AsNI was significantly tissue-specific,with the highest expression of AsNI1 and AsNI2 in the roots and bulbs,respectively.Meanwhile,adversity stress was able to induce AsNI expression,and the response of AsNI2 was stronger than that of AsNI1 under both low temperature and drought treatments.Among them,low temperature stress mainly induced the expression of AsNI2 in leaves,and drought stress mainly induced the expression of AsNI2 in roots.The sequence characteristics and expression pattern of AsNI were analyzed to verify the stress resistance function of AsNI.

The effect of SA on physiological indexes and gene expression were studied in tomato seedlings under low potassium stress in order to provide an important theoretical basis for the relationship between SA and low potassium stress. In this study,the different concentrations of salicylic acid (SA) (0.10,0.25,0.50,1.00 mmol/L) affect antioxidant enzyme activities and gene expression in tomato seedlings under low-potassium stress were investigated.The results showed that the chlorophyll (Chl) and proline (Pro) contents,as well as catalase (CAT),ascorbate peroxidase (APX),peroxidase (POD) and superoxide dismutase (SOD) activities,were significantly increased,and malondialdehyde (MDA) concentration decreased under 0.25 mmol/L SA treatment compared with that in the control.Meanwhile,the gene expression levels of APX,CAT,POD,and SOD in tomato leaves were analyzed through quantitative Real-time PCR.Four gene expression levels of antioxidant enzymes were considerably increased after 48,96 h treatment.These results suggested that SA promoted the gene expression levels of APX,CAT,POD,and SOD to alleviate the damage of low potassium stress,and decreased the accumulation of ROS and enhanced resistance in tomato seedlings under low-potassium stress.

This experiment consists of eight varieties of tomato were collected from home and abroad,the indexes of physiological and biochemical characteristics,field resistance,yield and quality were determined after the haze.The results showed that:during the weather of low temperature and haze,solar greenhouse's own environmental conditions far could not meet the requirements of the temperature,light and humidity of tomato.Under stress conditions,the varieties with stronger resistance were Jinpeng No.8 and Fenyan No.1,their fruit drop rates were 2.5%,3.7%,dead rates of plant were 38.8% and 25.0%,the diseases incidence rates were 35.5% and 35.0% respectively,These indicators were in the lower level than other varieties.And Jinpeng No.8 had the highest yield,54 300 kg/hm²,followed by Fenyan No.1and T1701,which production were more than 50 000 kg/hm². Fruit quality,the maximum of sugar acid ratio was Jinpeng No.8,and the taste was best;The minimum of the nitrate content was Fenyan No.1.The whole fog and haze,the smallest decrease amplitude of chlorophyll was Jinpeng No.8,Fenyan No.1 wasthe second.Physiological and biochemical indexes,the varieties of accumulated more contents of soluble sugar and proline were Jinpeng No.8,Fenyan NO.1,the value of SOD and POD were also higher,The content of MDA didn't have obvious differences between species in this experiment.Therefore,under low temperature and weak light,the varieties with self-regulation,strong adaptability were Jinpeng No.8,Fenyan NO.1,which results of physiological indexes were consistent with the results of field experiments.

In order to detect and identify the viral causing agent that infecting cucurbit vegetable plants in Shandong, 867 cucurbit plant samples of suspected viral diseases from 11 vegetable planting areas in Shandong were collected during July to August, 2014.9 primer pairs specific to Cucumber mosaic virus (CMV), Cucumber green mottle mosaic virus (CGMMV), Watermelon mosaic virus (WMV), Tobacco mosaic virus (TMV), Zucchini yellow mosaic virus (ZYMV), Squash mosaic virus (SqMV), Melon yellow spot virus (MYSV), Cucurbit chlorotic yellows virus (CCYV), Prunus necrotic ringspot ilarvirus (PNRSV) and Melon necrotic spot virus (MNSV) were used respectively to perform PCR detection.The results showed that viruses carried rate was 34.8%, 10.4%, 20.0%, 41.7%, 27.0%, 7.8%, 2.6%, 1.7%, 0 and 0.9% respectively.The natural infection of these viral diseases except PNRSV in Shandong was confirmed, and the incidence of CMV and TMV were higher than others.Two or more than two viruses mixed infections were also very common in field.At the same time, those CMV positive samples from 11 regions were selected for subgroup classification.Coat protein(CP) gene and the whole RNA3 nucleotide sequences were determined.Sequence alignment and phylogenetic analyses were performed.The results showed that the CMV isolates of cucurbit vegetables in Shandong area were all members of CMV subgroup IB with the highest similarity to a South Korea isolate As(AF013291), no other strains were found.

To explore the reasons for difference in mechanism of cold resistance of winter rapeseed of Brassica rape and Brassica napus.The method of combining field and pot culture was adopted,and using 8 different cold resistance of winter rapeseed varieties as materials,before wintering period with 5 to 6 true leaves,field experiments researched botanical morphology and accumulation of dry matter,the physiological characteristics were measured by pot experiment after the treatment of 24℃→10℃→5℃→0℃→-5℃→-10℃ 48 h.The results showed that winter Brassica rape was creeping growth,growth point was under the ground,accumulation of dry matter was mainly concentrated in the underground part,such as fresh matter of underground parts and dry matter of underground parts of winter Brassica rape had increased,which average increased 236.1%,263.0% than winter Brassica napus respectively.It was showed that the photosynthetic organic product of strong cold resistant varieties was preferentially allocated to the underground part during the vegetative growth stage,to establish a large root system to provide metabolic energy for the safe winter.With the change of temperature,the physiological and biochemical indexes of different varieties was vary greatly,SOD activity of Longyou 7 increased 10.7% than CK at low temperature of -5℃,CAT and POD activity of Longyou 7 increased 24.7%,28.6% than CK respectively at 0℃,SP content of winter Brassica rape average increased 32.3% than winter Brassica napus at 0℃,SS content of winter Brassica rape average increased 71.4% than winter Brassica napus,and MDA content of winter Brassica napus average increased 52.8% than winter Brassica rape at low temperature of -10℃,this indicated that the strong cold resistant varieties could protect themselves from damage of low temperature condition,the CAT,POD,SP were protective substance of against chilling injury,but SOD,SS were protective substance of against freezing.Therefore,the winter Brassica rape had obvious advantages in the morphology and physiological level than winter Brassica napus,the morphological advantages was that to withstand cold weather of extreme,to provide metabolic energy of during the wintering period and regreen next year;At physiological levels,the activities of protective enzyme and content of osmotic regulation of winter Brassica rape increased significantly at low temperature stress to protect the structure of cell membrane,and the accumulation of MDA was decreased,to alleviate hurt of winter rapeseed leaves at low temperature,so the over wintering was ensured highly,it was important to provide a theoretical basis for the research on the cold resistance of winter rapeseed of Brassica rape and Brassica napus in the North of China.

To explore the root disease resistance genetic regularity,by root disease resistance difference material,G57 and G70,the F₂ segregation population containing 500 per plant was built.Through artificial inoculation of the parents,F₁ and F₂ segregation population phenotypic characterization results were statistically analyzed. The results showed that root disease resistance of materials was controlled by a single dominant gene. In order to further localization of resistance genes in test materials,we used 678 molecular markers of parents,F₁ and F₂ population screening verification,resistance genes was preliminarily located in molecular marker KBRH129J18 and TCR02-F.This test was based on two chain tags,chain development design closer chain of molecular markers,ultimately with resistance genes chain 5 of SSR molecular markers,respectively,Bra0345-1,Bra0235-2,Bra0235-1,Bra19317,Bra019392. Their genetic distance with resistance genes were 2.4,2.4,2.4,2.5,3.3 cM.Validated,development and design of polymorphism markers in Chinese cabbage had good applicability.