小麦高分子量麦谷蛋白5亚基基因的克隆及其表达载体的构建

摘要

Using the PCR amplification technology, a full-length nucleotide sequence (2 750 bp) of wheat high molecular weight glutenin subunit 5 (or Dx5) gene(Glu-1D-1d) and its promoter sequence (630 bp), were cloned from "Cheyenne" wheat. By the selection and reparation of corresponding sites of restriction enzymes, a chimaeric gene, comprising the promoter (Psub5), the structure gene(Glu-1D-1d) and the nopaline synthase (Nos) gene terminator, i.e. pCAMBIA1301-PHGBy9-sub5-nos, was successfully constructed and verified after five transitional plasmids being produced.%小麦麦谷蛋白5亚基直接影响面包的烘烤品质,但我国大部分小麦品种的蛋白中缺少这种亚基.通过引物设计、PCR扩增,从Cheyenne中得到了小麦麦谷蛋白5亚基结构基因(sub5)的全长核苷酸序列(2 750 bp)和小麦麦谷蛋白5亚基基因启动子(Psub5)的核酸序列(630 bp).测序结果表明:得到了小麦籽粒中特异表达启动子--Psub5和用于改良小麦品质的结构基因--sub5.通过选择和改变相应的酶切位点,在构建6个中间载体的基础上,最后得到了含有目的基因的表达载体pCAMBIA1301- Psub5-sub5-nos.酶切电泳及PCR鉴定表明:已成功地合成了sub5的表达载体.有希望通过基因工程的方法将该表达载体用于小麦的品质改良.