Function Analysis Of A Super Compact Gene Scp-1 In Cucumber And Construction Of The Yeast Two-hybrid CDNA Library

The plant height is an important trait that affect the cucumber production.To date,the research about mechanism of cucumber height regulation is rarely reported.Brassinosteroids(BRs)is one of the main hormones regulate plant height.The study of BRsdwarf mutants could help to reveal the molecular mechanism of cucumber height.Our research team previously studied about a super compact mutant C257,previous gene mapping had delimit the scp-1at 6.6 cM,and there were fourteen non-synonymous mutation SNPs located in eight gene in the candidate region combined with MutMap.In this study,we cloned the candidate gene and analyzed the function of candidate gene based on the previous work.In addition,the hypocotyl of cucumber is the first internode of the cucumber plant,the hypocotyl and the above internodes are contribute to the height of the cucumber plant.Meanwhile the length of the hypocotyl is closely related to the strong seedlings and the field planting.In this study,we constructed a cucumber yeast two-hybrid cDNA library,and screened the interacting proteins with the candidate gene CsLH1 of long hypocotyl cucumber mutant.The main results were as follows:1.According to the phenotype and skotomorphogenesis conditionof the mutant,wespeculatedit's a BR related mutant.The mutant phenotype could be rescued with exogenous application of brassinolide revealed it's a BR deficit mutant.We cloned the candidate gene CsCYP85 A of scp-1from C257 and CCMC,the result of sequence alignment showed one SNP(G in WT to A in C257)was substitution in coding region of CsCYP85 A,which resulted in premature translation termination of CsCYP85 A in mutant.We developed a CsCYP85A-dCAPS marker according to this SNP.We conducted genetic linkage analysis using this dCAPSin F2 populations cross between C257 and North American cucumber Gy14,indicated the CsCYP85A-dCAPS marker was co-segregate with scp-1;we also genotyped 412 cucumber lines in the natural populations with growth habit,suggested this SNP is unique.These results indicated the CsCYP85 A is the most possible candidate gene.According to the bioinformatics analysis,there are three copies of the CsCYP85 A gene in cucumber genome,namedCsCYP85A1,CsCYP85A2 and CsCYP85A3,respectively.CsCYP85A1 was detectable in male flower,female flower,stem,leaf and root in C257 and WT,while CsCYP85A2 and CsCYP85A3 were barely detectable.The expression of CsCYP85A1 in the wild type was feedback regulated by BL application,while in C257 its expression was not affected.2.A yeast two-hybrid cDNA library of North China cucumber CCMC leaves and hypocotyl in seeding stage were constructed.The total capacity of the cDNA libraries was 9.45×105 cfu,titer was 9.72×10~7 cfu/ml,the size of most inserts were 250-2000 bp in the library.The recombination rate of library was 80%.3.Constucted the bait vector fused with candidate geneCsLH1 of long hypocotyl cucumber mutant.Throughyeast two hybrid system,CsLH1 interacted with CsHY5 were identified.Screening of proteins interacted with CsLH1 through cNDA library,we initially got a protein: GATA transcription factor 24-like.