Function Verification Of Trichome Candidate Gene BolTRY-l And Screening Its Upstream Interaction Proteins In Wild Brassica Oleracea L.

In nature,Plants have evolved a variety of morphology,such as plant trichome,to resist phytophagous invasion.Trichome is a special hairy structure of epidermal cells in the aboveground part of plants,which can not only protect plants from diseases and pests,but also have certain economic and medicinal value.Trichome is divided into single cell and multicellular according to morphology.The single cell trichome is much simple in their structures,such as the trichome of Arabidopsis thaliana and cruciferous vegetables.While the multicellular trichome is very complex in their structures,such as the trichome of Solanum lycopersicum and Artemisia annua.B.oleracea L.is a Brassica plant of Cruciferae,which contains many varieties,such as var.capitata L.,var.botrytis L.,var.italica L.,var.capitata f.rubra L.,var.alboglabra L.,var.acephala DC.B.oleracea vegetables are the main vegetable types in China.In the process of growth,the yield and quality are often reduced by various diseases and inst pests.In the early days,a kind of wild B.oleracea（named C01）was found in our lab,which was covered with trichome.The trichome had significant resistance to aphids and cabbageworm.The trichome of C01 belongs to single cell non-glandular trichome.Previous studies have constructed F₂ and F₃ populations with C01 and B.alboglabra（named C41）.This studies have showed that C01 trichome are controlled by recessive unigenes.QTL mapping and QTL-seq analysis indicated that the QTL was located in 13.24-14.28 Mb of C01 chromosome.The interval contains 111genes.And a total of 98 SNP were detected.We focused on 10 genes and found that three SNP variants on Bol13124 genes were significantly associated with trichome（P<0.05）via correlation analysis.The further q RT-PCR analysis showed that the gene was highly expressed in C41 and low expression in C01,and the expression of the gene in non-trichome plants was significantly higher than that in trichome plants.Therefore,the gene was identified as a candidate gene.Blast P analysis found that the similarity of this gene to TRY amino acid sequence of the reported gene（negatively regulated trichome）in Arabidopsis was 64.8%.And so the gene was named BolTRY-l in this study.A variety of research methods and technical means were carried out in this study.First,BolTRY-l genes were cloned in C01 and C41,and their biological functions were identified by genetic transformation in A.thaliana and B.oleracea.This study focuses on genetic improvement of cultivated crops using wild germplasm resources.The results are of great significance for revealing the molecular mechanism of the trichome of B.oleracea.At the same time,it also provides theoretical basis and important genetic resources for inst resistance breeding of B.oleracea L.The main results are as follows:1.Cloning,expression analysis and subcellular localization of BolTRY-l genes in C01 and C41We cloned the CDS of the gene in C01 and C41,respectively.The sequence alignment showed that there was no difference in CDS,and the sequence length was237 bp,encoding 78 amino acids,which was consistent with the sequence XM＿013732072.1 in the reference genome TO1000 of B.oleracea and contained a MYB＿DNA binding domain.Using real-time fluorescence quantitative PCR to determine the expression levels of BolTRY-l genes in seven parts of C01 and C41（leaves at the two-leaf,four-leaf,seedling,mature and aging stages,and stem tips and stems at seedling stage）.The results showed that the expression of BolTRY-l genes in other tissues of C41 was significantly higher than that of C01（P<0.01）.By Agrobacterium tumefaciens,the subcellular localization vector was transfected into Nicotiana benthamiana epidermal cells.The fluorescence microscope showed that green fluorescence was found in the nucleus and cell membrane of tobacco epidermal cells,indicating that the BolTRY-l protein was located in the nucleus and cell membrane.2.Genetic transformation of BolTRY-l genes in Arabidopsis and B.oleraceaTransforming the overexpression vector p Bin35Sred3-BolTRY-l into wild-type Arabidopsis Col-0（WT）and Arabidopsis try mutants.Using the red light carried by the carrier to screen the transgenic plants,the positive transgenic plants were determined by PCR identification and RT-PCR identification.All the positive plants showed no trichome,indicating that BolTRY-l overexpression inhibited the formation of Arabidopsis trichome.Further,the p Bin35Sred3-BolTRY-l was transferred into C01.Combined with phenotypic screening,PCR labeling and RT-PCR identification,positive transgenic plants were identified.The results showed that positive transgenic B.oleracea showed no trichome.preliminary validation of BolTRY-l overexpression inhibited the formation of trichome in B.oleracea.3.BolTRY-l promoter cloning and cis-acting element analysis in C01 and C41We cloned promoter sequences of BolTRY-l in C01 and C41,named Pro BolTRY-l-C01 and Pro BolTRY-l-C41,respectively.Sequence alignment analysis has showed that there are significant differences between Pro BolTRY-l-C01 and Pro BolTRY-l-C41 sequences,the similarity is 49.9%.The comparison with the published reference genomes of two B.oleracea（TO1000 and Capitata）showed that the similarity between Pro BolTRY-l-C41 and TO1000 is 99.8%,The similarity between Pro BolTRY-l-C01 and Capitata is 84.3%.The main differences were in the-47 to-346bp upstream area before the transcription initiation positon.The cis-acting element prediction found four special cis-acting elements in Pro BolTRY-l-C01,including MYB recognition sites,GC-motif,sp1 and WUN-motif.4.Interaction of BolMYB34-l proteins with BolTRY-l promotersPrevious research showed that the five protein NAC40,SPL9,GL3,TTG1 and MYB34-l interacted with TRY promoters in different plants.So we cloned the five genes in C01 and C41 in this study.Sequence alignment showed that the NAC40 sequence length in the C01 and C41 were 1038 bp,encoding 345 amino acids,with a difference of SNP site.The SPL9 sequence length was 1113 bp,encoding 370 amino acids,with a difference of SNP site.The MYB34-l sequence length was 1011 bp,encoding 336 amino acids,with a difference of 2 SNP site.The TTG1 sequence length was 1014 bp,encoding 337 amino acids,with a difference of 1 SNP site.The GL3 sequence length in the C01 is 1515 bp,Code 504 amino acids,in the C41 is 1513 bp,encoding 503 amino acids,and there are 24 different SNP sites between them.A yeast one-hybrid assay and double luciferase system was performed to verify these proteins interaction with BolTRY-l promoter.The results showed that in C01,the yeast zygote of the combination of p GADT7-BolMYB34-l and p Ab Ai-BolTRY-l-C01grew normally in the plates,while the other combined colonies did not grow.Interestingly,the result in C41 was the opposite of that in C01.