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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) are a class of cysteine-rich low-molecular-weight proteins that play an important role in plant tolerance to heavy metals. In this study, we cloned the FtMT2 gene (GenBank No.KY643823) from leaves of Fagopyrum tataricum and analyzed the tolerance to Cu 2+ in FtMT2-overexpressing Escherichia coli. Using genome walking technology The promoter sequence of FtMT2 was obtained and a dual luciferase reporter vector with different truncated promoters was constructed. The response of FtMT2 promoter to Cu ~ (2+) was studied by transient transformation of protoplasts of Triterpenoid mesophyll cells. The results showed that the ORF length of FtMT2 was 237 bp. The encoded amino acid sequence of FtMT2 was characterized by typical MT2 and had high homology with MT2 of dicotyledonous plants. A series of promotor truncated truncated promoters with different 5 ’flanking sequences exhibited different modes of enhanced response to Cu 2+. Response intensity analysis showed that there was dose effect in the predicted metal response element. In addition, E. coli over-expressing FtMT2 increased resistance to Cu 2+. The above results indicate that FtMT2 plays an important role in the response of tartary buckwheat to Cu 2+ and the tolerance of heavy metals. The cloning of FtMT2 and the functional analysis of its promoter have reference value for establishing detection technology of Cu2 + pollution, and provide the theoretical basis for analyzing the intrinsic mechanism of heavy metal resistance in buckwheat.