关键词: 紫花苜蓿, 油菜素内酯, MsDWF4, 非生物逆境, 耐盐性

Abstract:

【Objective】 Cloning the Brassinolide (BRS) synthetase gene MsDWF4 of alfalfa (Medicago sativa L.), analyzing the gene characteristics and gene expression pattern, performed the salt-tolerance analysis, therefore to clarify the function of MsDWF4 in abiotic stress resistance and provide a reference for revealing the molecular mechanism of MsDWF4 in regulating abiotic stress resistance in alfalfa. 【Method】 The homologous gene of DWF4 in M. sativa was cloned by using homology-based cloning according to the known DWF4 gene sequence of Arabidopsis. Sequence characteristics were analyzed by bioinformatics tools. Quantitative Real-time PCR (qRT-PCR) was applied for detecting the gene expression in different alfalfa tissues and the expression patterns under multiple abiotic stress (high temperature, cold damage, drought and high salt) and hormones treatment (auxin, brassinolide, abscisic acid and jasmonate). The MsDWF4 overexpression vector was constructed and transformed into alfalfa by Agrobacterium mediated transformation, and MsDWF4 overexpression transgenic alfalfa plants were obtained. The transgenic alfalfa lines were treated with high salt (200 mmol·L ^-1 NaCl) and the activities of antioxidant enzymes were analyzed to study whether MsDWF4 could improve the salt tolerance of alfalfa. 【Result】 The length of MsDWF4 CDS was 1 470 bp, which encoded a protein of 489 amino acids, belonging to the single oxygenases of cytochrome P450 family. The MsDWF4 had 67 kinase phosphorylation sites. Sequence and phylogenetic tree analysis show that MsDWF4 protein was most closely related to DWF4 protein of legume plant M. truncatula and had the farthest relationship with Gramineaeis. Tissue-specific expression analysis illustrated that MsDWF4 had the highest expression in the root tip, followed by flowers and leaves. High temperature, cold, PEG, NaCl, ABA and IAA all positively induced the expression of MsDWF4 gene in the alfalfa. After BR treatment, the expression of MsDWF4 was down regulated in shoot and root. The expression of MsDWF4 was inhibited by JA treatment. A 35S∷MsDWF4 overexpression vector was constructed and transformed into alfalfa by Agrobacterium mediated method. PCR identification showed that MsDWF4 gene has been transferred into alfalfa and 6 positive transgenic lines were obtained. Under salt stress, the expression of MsDWF4 gene and the activity of antioxidant enzymes of alfalfa overexpression MsDWF4 gene are higher than that of the control plants. 【Conclusion】 The cDNA sequence of alfalfa brassinolide synthetase gene MsDWF4 was obtained. It was found that the gene expressed the highest in root tip and its expression is responded to a variety of abiotic stress and exogenous hormone treatment. MsDWF4 gene enhanced the resistance to salt stress in transgenic alfalfa. The results showed that MsDWF4 may participate in many kinds of stress response processes and positively regulate the salt tolerance of alfalfa.

Key words: Medicago sativa, brassinosteroids, MsDWF4, abiotic stress, salt resistance