苹果MdDRB1的表达与功能分析

Expression and Function Analysis of Apple MdDRB1 Gene

以‘嘎拉’苹果(Malus×domestica‘Royal Gala’)为试材,扩增Md DRB1基因,分析其序列结构,同时原核诱导Md DRB1蛋白并观察其亚细胞定位。利用q RT-PCR检测Md DRB1在非生物胁迫下的表达量,通过遗传转化苹果苗鉴定Md DRB1在非生物胁迫中的功能。基因结构分析显示,Md DRB1具有2个内含子和3个外显子。亚细胞定位显示,Md DRB1主要定位于细胞核,少数定位在细胞质。原核诱导结果显示,Md DRB1融合蛋白以包涵体的形式存在。同时发现Md DRB1反义转基因愈伤中与抗性相关的mi RNA的表达水平上调。在PEG、ABA、盐和低温处理的材料中Md DRB1的表达水平明显上调。另外,Md DRB1过量表达明显提高了转基因苹果组培苗的抗性。推测Md DRB1在抗逆胁迫响应中有重要作用。

A gene named MdDRB1 cloned from Malus × domestica‘Royal Gala＇was analyzed. To understand sequence characteristics of the gene,prokaryotic expression and subcellular localization were carried out. In addition,real-time quantitative PCR were performed to determine the expression levels of MdDRB1 in response to various abiotic stresses. Finally,MdDRB1 was genetically transformed into apple seedlings to identify its function. The analysis of gene structure revealed that there were two introns and three exons in genomic sequence of MdDRB1. Subcellular localization showed that MdDRB1 were mainly distributed in the nucleus,a small number distributed in the cytoplasm. Then,the induced protein showed that MdDRB1 fusion protein existed in the form of inclusion body. Furthermore,the mi RNA related to resistance expression level raised in antisense transgenic apple callus. In addition,real-time quantitative PCR found that the MdDRB1 gene expression level significantly raised after treated with PEG,Na Cl,ABA and 4 ℃. Overexpression of MdDRB1 remarkably increased the tolerance of transgenic apple seedlings to abiotic stresses. These results indicated that MdDRB1 might be involved in the response of apple to abiotic stresses.