Cloning And Functional Analysis Of Brassionsterind Biosynthesis Enzyme Gene DWF4 In Medicago Sativa L.

Alfalfa（Medicago sativa L.）is known as the"queen of forage"because of its high feed output and good nutrition.In China,alfalfa is mainly planted in poor and saline alkali land without irrigation conditions.Its output can not meet the needs of China and needs to be imported in large quantities.Therefore,it is of great significance to study how to improve the yield and resistance of alfalfa in Chia.Brassionsterind（BR）,a steroid hormone,which plays an important role in regulating plant growth and stress resistance.DWF4 gene encodes C-22 hydroxylate in BR synthesis,which is the key to brassinolide biosynthesis and endogenous brassinolide level feedback regulation.The function of DWF4gene in Arabidopsis thaliana has been studied,but the function in alfalfa is not clear.1.In this study,we isolated the gene MsDWF4,which encodes 489 amino acids and the length of MsDWF4 was 1,470 bp.MsDWF4 is an unstable transmembrane hydrophilic protein,which belongs to P450 superfamily and contains 67 kinase phosphorylation sites.The analysis result of phylogenetic tree showed that the relationship between MsDWF4 of alfalfa and DWF4 of Tribulus terrestris（Medicago truncatula）was the closest,but that between MsDWF4 and DWF4 of Gramineae was the furthest.2.Tissue-specific expression analysis showed that MsDWF4 had the highest expression in root tip,followed by flower and leaf.It suggested that BR biosynthesis might be more active in root.The qRT-PCR was used to analyze the expression of MsDWF4 under various adversities and hormones.Salt(150 mmol.L^-1 NaCl),high temperature（35℃）,low temperature（4℃）and drought（15%PEG）could induce the expression of MsDWF4 in different degrees,which indicated that MsDWF4 probably participated in the response of these stresses.Under hormone treatment,1μmol·L^-1 BR,0.1μmol·L^-1JA,1 mmoL·L^-1 MeJA and 1 mmo L·L^-1 SA inhibited the expression of MsDWF4 in different degrees,while 1μmol·L^-1 IAA and 1μmol·L^-1 ABA significantly increased the expression of MsDWF4.Under 1mmoL·L^-1 GA₃ treatment,the expression of MsDWF4 in the shoot part was first induced and then inhibited,while the expression in the root was always inhibited.It is suggested that MsDWF4 may be regulated by BR,JA,MeJA,SA,IAA,ABA and GA3 hormones in plant growth and development.3.The results of phenotype observation and salt tolerance analysis of transgenic alfalfa:The overexpression vector of MsDWF4 was constructed and transformed into Medicago sativa cv.Zhongmu No.1.The plant growth height,branch number and aboveground biomass of the transgenic alfalfa were0.4,1.14 and 2.15 times higher than those of the control group.Under the treatment of 200 mmol·L^-1NaCl,the expression level of MsDWF4 and the activities of antioxidant enzymes（cat,pod and SOD）in the transgenic alfalfa was higher than that in the control group,indicating that overexpression of MsDWF4 gene could significantly improve the growth,development and salt tolerance of alfalfa.4.The results of phenotype observation and salt tolerance analysis:the overexpression vector of MsDWF4 was constructed and transformed into Col Arabidopsis thaliana by inflorescence infection method.Compared with the control group,the root length,leaf number,petiole length,narrow and curly leaf,bolting in advance and hypocotyl length in dark condition of transgenic Arabidopsis increased by0.51 times,0.08 times and 0.23 times respectively.Under 100 mmol·L^-1 NaCl treatment,the germination rate and the root length of transgenic Arabidopsis was significantly higher than those of the control group.It is suggested that MsDWF4 not only participates in the regulation of flowering time and root elongation,but also in the regulation of seed germination under salt stress.