云南红梨PyMT基因的克隆、原核表达和功能分析

Cloning,Prokaryotic Expression and Function Analysis of PyMT Gene from Yunnan Red Pear(Pyrus pyrifolia)

金属硫蛋白(metallothioneins,MTs)是一种在生物中普遍存在的多功能蛋白.根据云红梨1号光特异性差减文库中获得的PyMT(Pyrus pyrifolia metallothionein)基因序列,PCR扩增4种红梨PyMT的基因组序列并进行序列分析;通过RT-PCR方法从云红梨1号果皮中扩增出PyMT基因,并将其命名为PyMT1(Pyrus pyrifo-lia metallothionein 1).构建原核表达载体pGEX-4T-1-PyMT1,在E.coli BL21中进行表达;通过在液体培养基中添加不同浓度的重金属离子,检测转化菌和对照菌的的生长曲线,分析PyMT1的功能.序列分析表明,PyMT1是典型的Metallothion 2.PyMT1氨基酸序列系统发育分析表明,PyMT1与MdMT亲缘关系最近.SDS-PAGE结果表明所表达蛋白与预期的大小一致;生长曲线结果表明工程菌对重金属离子Zn、Cu、Cd具有一定的耐受性.这些结果为进一步纯化、鉴定目的蛋白和研究其结构和功能奠定了基础.

Metallothionein is a kind of ubiquitous and multifunctional protein in organism.PyMT（Pyrus pyrifolia metallothionein） genomic sequences were amplified according to the EST sequence of the red pear ＇Yunhong-1＇s light-specific supression substration hybridization library from four cultivars of Yunan red pear and these sequences were analyzed.The PyMT cDNA sequence of ＇Yunhong-1＇ was amplified by RT-PCR and was designated PyMT1（Pyrus pyrifolia metallothionein 1）.Prokaryotic expression vector pGEX-4T-1-PyMT1 was constructed and was transformed into E.coli BL21 for expression.The growth curves of E.coli.transformed with pGEX-4T-1-PyMT1 and the control were obtained by growing them in liquid medium with different concentration of various metal ions.Sequence analysis showed that PyMT1 belongs to the typical Metallothion 2.Results of phylogenic analysis showed that PyMT1 was at the same evolutionary branch with MdMT.The SDS-PAGE results display that the expressed proteins are consistent with the anticipated size.Results of the growth curve show that PyMT1 has a certain tolerance to heavy mental ions such as Zn,Cu and Cd in vivo.The results will provide a foundation for further purification,structure and function research of PyMT1.