Identification Of Cucumber Heat-resistant Genes And Preliminary Analysis Of Their Role In Thermal Response

Cucumber?Cucumis sativus L.?is an important vegetable cash crop in China,which has a large cultivation area and a wide planting range,and has good economic value.Cucumber is a thermophilic species,but is vulnerable to heat stress.High temperature is one of the main abiotic stress factors affecting cucumber yield and quality in summer cultivation or protected area production.When the ambient temperature exceeds 35°C,it will cause heat damage to cucumber,resulting in weak plant growth,low pollination success rate and fruit abnormality,which will seriously affect the economic value of cucumber.With the increasing global warming,frequent high temperature weather will seriously affect cucumber production.Therefore,studying the molecular mechanism of cucumber heat tolerance,locating cucumber heat resistance genes,and breeding heat resistant varieties are important.In this study,we use forward and reverse genetics methods to identify and analyze heat-resistant candidate genes of cucumber,which is helpful to study the molecular mechanism of heat tolerance in Cucumber.In this study,the phenotype and genetic analysis of heat sensitive 1?hs1?mutant were performed.The results showed that under high temperature of 35?,the leaves of hs1 mutants showed albinism,which developed from leaf margin to leaf center,and the whole leaves were dead and withered when it was serious,and the growth of mutants was obviously suppressed,while the leaves of North China cucumber'9930'kept evergreen under high temperature.Genetic analysis showed that the hs1 mutant was a recessive single gene controlled mutant.In order to identify the genetic difference between hs1 and 9930,we used hs1 and9930 as parents to hybridize,and got F₂ population.The BSA method was used to sample pools of plants with heat-sensitive and non-heat-sensitive phenotypes in the F₂generation population for genome resequencing.According to the genomic resequencing data,the correlation analysis between SNPs and phenotype was performed,and hs1 was roughly mapped.Correlation analysis showed that the hs1 gene was located at the end of chromosome 1 between the two markers InDel-8 and InDel-9,with a physical distance of about 1.26 Mb.Based on the results of rough mapping,two markers,indel-8 and indel-9,were used to analyze the genotypes of 1200 F₂generation plants,and a total of 33 exchange singles were selected.Based on the resequencing data,between the two markers InDel-8 and InDel-9,five SNP markers were developed.These markers were used to analysis 33 exchange individuals,and the hs1 gene was finally located between the two markers SNP-3 and SNP-4 on chromosome 1.The physical distance was about 232.3 kb,and there were 19 candidate genes in the interval.In order to identify more genes involved in the heat shock response of cucumber,9930 was used as the experimental material for transcription sequencing.Through transcriptome sequencing,the gene expression level of cucumber seedlings after 0,3and 6 hours of high temperature treatment at 42?was analyzed,and a large number of genes related to heat resistance were obtained,which were differentially expressed.These differentially expressed genes are mainly involved in metabolic pathways and protein synthesis pathways.Among the differentially expressed genes,there are many genes of the Hsp family,including Hsp20,Hsp70,and Hsp90,and these genes are up-regulated by heat stress,indicating that these genes may play a positive regulatory function in cucumber heat shock response.In addition,the expression levels of many transcription factors are also increased by heat induction,including AP2,MYB,WRKY,bHLH and HSF.Combined with transcriptome sequencing data,this study used a bioinformatics method to analyze the Hsf genes in cucumber,including their structural characteristics,chromosomal locations,tissue expression characteristics,phylogenetic relationships,and protein interaction predictions.As an important heat shock transcription factor,CsHsf-7 has predicted protein interactions with CsHsp20s,CsHsp70s and CsHsp90s,indicating that CsHsf-7 may improve the heat tolerance of cucumber by activating the expression of downstream CsHsps.Using the method of transient transformation,the CsHsf-7 overexpression vector driven by 35S promoter was injected into cucumber cotyledons,and 2 days after the injection,the cucumber seedlings were heat-treated at42°C for 12 hours.The results showed that the cotyledons of cucumber injected with the empty vector?35S::?were wilted obviously,and the cotyledon bent downward at a large angle.However,the heat tolerance of cucumber seedlings injected with 35S::CsHsf-7 was significantly stronger than control,and the cotyledon bending angle was small,indicating that CsHsf-7 plays an important role in the heat shock response of cucumber and can improve the heat tolerance of cucumber seedlings.qRT-PCR results showed that overexpression of CsHsf-7 can induce the expression of CsHsp genes,indicating that CsHsf-7 may be an important regulator of heat shock response in cucumber.In a word,the heat-resistant genes of cucumber were identified and analyzed using forward,reverse genetics and bioinformatics methods,which laid a foundation for the study of heat-resistant molecular mechanism of cucumber.