Cloning Of Key Enzyme Gene VvDET2 In Brassinosteroids Biosynthetic Pathway From Vitis Vinifera L.and Expression Analysis Of Related Genes Under Drought Stress

In this experiment,Cabernet Sauvignon(Vitis vinifera L.)was used as a test material to analyze the contents of campesterol(CR),campestanol(CN),castasterone(CS)and brassinolide(BL)and the gene expression levels of DET2,DWF1,CPD and BR6ox1 in brassinosteroids(BRs)synthetic pathway.The effects of EBR pretreatment and drought stress on the synthesis of BRs in grape seedlings were further analyzed.The full-length Vv DET2 gene was cloned and the bioinformatics of Vv DET2 gene was analyzed.The experiment enriched the understanding of the regulation of Vv DET2 gene during grape growth and drought stress,and provided a theoretical basis for the further study of the overall regulation model of BRs mediating grape growth and development.The main conclusions are as follows:(1)There were significant differences among the kinds and contents of BRs in different tissues and organs of grapes.The contents of synthetic downstream BRs(CS and BL)were higher in the above-ground parts of plants,such as immature seeds,leaves and tendrils.The contents of synthetic upstream intermediates(CN and CR)were higher in the mature seeds and underground of grape seedings,such as roots and stems.BRs biosynthetic genes also had different temporal and spatial specific expression patterns.The expression levels of DET2,DWF1 and BR6ox1 genes were higher in vegetative organs such as leaves and tendrils than those in other tissues.BR6ox1 was also highly expressed in roots,while the expression levels of DET2,DWF1 and CPD were lower in roots.Furthermore,the CPD gene was mainly expressed in grape seeds.(2)The c DNA sequence of the DET2 gene of Cabernet Sauvignon was cloned and its encoded amino acid sequence was deduced.The size of the gene fragment was 774 bp and it encoded 257 amino acids.The analysis found that the closest relationship with the DET2 protein of grape was Echinacea purpurea,and the most distantly related were plum and peach.The DET2 protein of grape had a conserved domain of the PEMT family.We speculated that grape DET2 protein was mainly involved in the second step reaction of plant BRs biosynthesis pathway and characteristically catalyzed the 5-alpha reduction of BRs synthesis precursor.We also analyzed that DET2 protein was a hydrophobic,non-secretory membrane protein.(3)EBR treatment could alleviate the decreasing of relative water content and the maximum fluorescence efficiency of PSII in grape leaves under drought stress.Exogenous EBR could relieve the inhibition of CR,CN and CS levels by drought stress to maintain the stability of endogenous BRs contents in grape seedings.Compared with CK,the expression levels of the four genes were all down-regulated by drought stress.EBR pretreatment effectively relieved the inhibition of DET2 gene transcription by drought stress.However,the EBR mitigation effects on DWF1 and CPD gene expression levels were not significant.