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[目的]构建甘蓝型油菜叶绿体多顺反子表达载体,以期为油菜叶绿体基因工程研究奠定基础。[方法]根据GenBank中已知的油菜叶绿体DNA序列AF267640和Z50868,设计两对引物,用PCR方法获得了两段甘蓝型油菜叶绿体DNA片段,分别命名为RbcL和ACCD。将这两段甘蓝型油菜叶绿体DNA同源片段克隆到质粒pMD18-T中得到质粒pHBM715,然后再将由壮观霉素抗性基因aadA、甘露聚糖酶基因man和绿色荧光蛋白基因gfp这3个基因串联的表达盒克隆到质粒pHBM715中,从而构建成甘蓝型油菜叶绿体多顺反子表达载体pHBM716,并将该载体在大肠杆菌中进行表达鉴定。[结果]通过平板定性分析对所构建的油菜叶绿体表达载体上的表达盒进行了功能鉴定,表明同一多顺反子的3个基因在大肠杆菌中均得到了表达。[结论]该研究成功构建了甘蓝型油菜叶绿体多顺反子表达载体,为油菜叶绿体基因工程研究奠定了基础。
[Objective] The study aimed to construct a chloroplast polycistronic expression vector in Brassica napus, which laid the foundation for gene engineering of rape chloroplast. [Method] Two pairs of primers were designed according to the known chloroplast DNA sequence AF267640 and Z50868 in GenBank. Two chloroplast DNA fragments of rapeseed (Brassica napus L.) were obtained by PCR and named RbcL and ACCD respectively. The two homologous fragments of chloroplast DNA from Brassica napus were cloned into the plasmid pMD18-T to get the plasmid pHBM715. Then three genes, spectinomycin resistant gene aadA, mannanase gene man and green fluorescent protein gene gfp, The tandem expression cassette was cloned into the plasmid pHBM715 to construct a chloroplast polycistronic expression vector pHBM716 of Brassica napus, and the expression vector was identified in E. coli. [Result] The expression cassette of the constructed expression vector of the chloroplast in rapeseed was identified by the qualitative analysis of the plate, indicating that the three genes of the same polycistronic gene were expressed in E. coli. [Conclusion] The study successfully constructed the chloroplast polycistronic expression vector of Brassica napus, which laid the foundation for the genetic engineering of rape chloroplast.