拟南芥Pro:RFE6-GUS载体的构建及其转基因植株的筛选

Construction of Arabidopsis Pro:RFE 6-GUS vector and identification of transgenic plants

重金属污染主要来自于工业和农业废水的排放,而重金属对生物往往造成不可逆的伤害,重金属镉便是其中一种。为探究镉胁迫对拟南芥AtRFE6基因表达的影响,文章以哥伦比亚背景(Columbia,Col)的拟南芥为实验材料,构建拟南芥Pro:RFE6-GUS转基因植株。以野生型拟南芥DNA为模板,扩增AtRFE6启动子片段,并对该启动子和GUS质粒进行双酶切和连接;然后将重组质粒热转入大肠杆菌DH5α进行鉴定和测序,测序正确的质粒转入农杆菌,并侵染野生型拟南芥;对筛选获得的转基因植株进行镉胁迫处理并GUS染色,结果表明拟南芥在受到镉胁迫时,AtRFE6基因的表达量增加,说明AtRFE6可能参与调节植物对镉胁迫的响应。

Heavy mental pollution mainly comes from the discharging of industrial and agricultural wastewater,and these heavy metals often cause irreversible damage to organisms,cadmium is one of them.In order to explore the effect of cadmium stress on AtRFE6 gene expression in Arabidopsis thaliana,Arabidopsis thaliana with a Columbia background(Columbia,Col)was used as experimental material to construct Arabidopsis Pro:RFE 6-GUS transgenic plants.Wild-type Arabidopsis DNA was used as a template to amplify the AtRFE6 promoter fragment,and perform double digestion and ligation of the promoter and GUS plasmid.Then,the recombinant plasmid was thermally transferred into E.coli DH5αfor identification and sequencing,and the plasmid with right sequence was transferred to agrobacterium and infected wild-type Arabidopsis.The transgenic plants were treated with cadmium stress and then dealt with GUS staining.The results showed that the expression of AtRFE6 gene increased when Arabidopsis was subjected to cadmium stress,indicating that AtRFE6 might be involved in the regulation of cadmium stress response.