金属硫蛋白（metallothioneins,MTs）是一类富含半胱氨酸的低分子量蛋白,在植物重金属耐受性中发挥重要的作用。本研究从苦荞（Fagopyrum tataricum）叶片中克隆了FtMT2基因（GenBank No.KY643823）并分析过表达FtMT2的大肠杆菌（Escherichia coli）对Cu^2＋的耐受性;利用基因组步移技术获得FtMT2的启动子序列并构建不同截短启动子的双荧光素酶报告载体,采用瞬时转化苦荞叶肉细胞原生质体研究FtMT2启动子对Cu^2＋的响应。结果表明,FtMT2的ORF长度为237 bp,其编码的氨基酸序列具有典型的MT2的特征,且与双子叶植物的MT2有较高的同源性。5′侧翼序列缺失的系列启动子截短体对Cu^2＋表现为不同的增强响应模式,响应强度分析显示,截短体包含的预测金属响应元件存在剂量效应。此外,FtMT2过表达的大肠杆菌对Cu^2＋的耐受性增加。以上结果表明,FtMT2在苦荞对Cu^2＋的响应及重金属的耐受性中发挥重要作用。FtMT2的克隆及其启动子的功能分析对于建立Cu^2＋污染的检测技术具有参考价值,同时为解析苦荞重金属抗性的内在机制提供了理论依据。
Metallothioneins （MTs）, which are a kind of widely distributed and low molecular weight cysteine- rich proteins, were found to play an important role in heavy metal tolerance. In this study, a cDNA fragment corresponding to the type II metallothionein （MT2） coding region （GenBank No. KY643823） was isolated from tartary buckwheat （Fagopyrum tataricum） leaf. FtMT2 encoded a deduced peptide of 78 aa residues with a calculated molecular mass of 7.87 kD and contained the cysteine-rich domains with the presence of C-C, C- X-C, and C-X-X-C （X was other amino acids other than cysteine） motifs at amino-terminus and C-X-C motifs at carboxy-terminus. Sequence and homology analysis showed that the FtMT2 protein sequence shared high homology with other plant type 2 MT-like proteins. In order to clarify the regulatory mechanism of FtMT2 in response to Cu^2＋ stress, 2.l kb upstream sequence of FtMT2 was isolated by genome walking. The 2.1 kb fragment and its 5＇ flanking deletions were tested for promoter activity using the dual luciferase reporter system and transient transformation in mesophyll protoplasts of F. tararicum. The promoter activities of all the fragments were up-regulated, and the full length promoter showed strongest inducibility in response to taw treatment. The role of FtMT2 protein in protecting against Cu^2＋ toxicity was demonstrated in pGEX4T- 1- FtMT2-beared Escherichia coli, which could obviously increase the host E. coli cell＇s tolerance. Functional characterization of upstream region of FtMT2 provides the regulatory mechanism for establishing the method for copper contamination monitoring and offers an underlying basis for further revealing the molecular mechanisms of heavy metal resistance of Tartary buckwheat.
Journal of Agricultural Biotechnology