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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。
The Md DRB1 gene was amplified using ’Gala’ apple (Malus × domestica’Royal Gala ’), and its sequence structure was analyzed. At the same time, prokaryotic expression of Md DRB1 protein was observed and its subcellular localization was observed. The expression of Md DRB1 under abiotic stress was detected by q RT-PCR. The function of Md DRB1 in abiotic stress was identified by genetic transformation of apple seedlings. Gene structure analysis showed that Md DRB1 has two introns and three exons. Subcellular localization showed that Md DRB1 mainly located in the nucleus and few localized in the cytoplasm. Prokaryotic induction showed that the Md DRB1 fusion protein was in the form of inclusion bodies. At the same time, it was found that the expression level of resistance-related miRNA in Md DRB1 antisense transgenic calli was up-regulated. The expression level of Md DRB1 was significantly up-regulated in PEG, ABA, salt and hypothermia treated materials. In addition, Md DRB1 overexpression significantly increased the resistance of transgenic apple plantlets. It is speculated that Md DRB1 plays an important role in stress response.