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通过定点突变,在小麦高分子量(HMW)谷蛋白基因5′上游调控序列的TATAbox与基因翻译起始密码子ATG之间,改变三个碱基,产生新的BamHⅠ内切酶位点.在调控序列中,选择四个合适的内切酶,对上游序列进行缺失,分离到四个大小分别为2400、610、440和110bp的含有不同调控元件的HMW谷蛋白基因启动子,与质粒pBI101.1的报告基因GUS重组,构建了四个嵌合质粒pWG2400、pWG600、pWG440和pWG100.经基因枪、微束激光和PEG等方法将pWG2400转化玉米胚乳悬浮细胞,GUS基因获得表达.这为进一步研究HMW谷蛋白基因的调控机理奠定了基础.
By site-directed mutagenesis, three bases were changed between the TATAbox upstream of the 5 ’upstream regulatory sequence of wheat high molecular weight (HMW) glutenin gene and the gene translation initiation codon ATG to create a new BamHI endonuclease site. In the regulatory sequence, four suitable endonucleases were selected and the upstream sequence was deleted. Four HMW glutenin gene promoters with different regulatory elements of 2400, 610, 440 and 110 bp respectively were isolated and compared with the plasmid pBI101 .1 reporter gene GUS recombination to construct four chimeric plasmids pWG2400, pWG600, pWG440 and pWG100. PWG2400 was transformed into suspension cells of maize endosperm by gene gun, microbeam laser and PEG. The GUS gene was expressed. This laid the foundation for further study on the regulation mechanism of HMW glutenin gene.