蛇白蔹白藜芦醇合酶基因CNRS2的克隆与原核表达

Cloning and its expression in prokaryote of RS gene CNRS2 Ampelopsis Japonica (Thunb.) Makino

白藜芦醇合酶(resveratrol synthetic enzyme,RS)是植物白藜芦醇合成途径中的关键酶,利用已知的葡萄RS基因(AF274281)序列设计并合成了一对引物,以蛇白蔹基因组DNA为模板,PCR扩增得到包含RS完整基因在内的一段序列,测序与序列分析表明:该克隆片段全长1 536bp,其中包含一个内含子及两个外显子。采用悬挂延伸PCR法克隆了目的基因,命名为CNRS2。序列分析表明该基因的开放读码框1 170 bp,编码389个氨基酸残基。同源性比较发现,CNRS2与已知葡萄RS基因序列的同源性达93%～98%。CNRS2与pET-30a(+)构建原核表达载体,经IPTG诱导后可表达获得相对分子量约为46 kD的外源融合蛋白。以上结果证实CNRS2属葡萄RS基因家族成员,为今后进一步对该基因的研究利用打下基础。

Resveratrol synthetic enzyme（RS） is a key enzyme in resveratrol synthesis pathway of plant.To uncover and characterize the function of the RS in plant developmental process,we isolated a RS gene by PCR using total DNA of Ampelopsis Japonica（Thunb.）Makino.The sequence analysis results indicated that the length of the determined nucleotide sequence was 1 536 bp,comprise intron and two exon.The cDNA of RS gene CNRS2 was cloned by overhan-gextension PCR,The results showed that the CNRS2 had a 1 170 bp open reading frame encoding 389 amino acid polypeptide,and exhibiting high similarities with other members of RS genes family.The recombinant CNRS2 protein product with the molecular weight about 46 kD could also be detected in E.coli protein expression system.These results indicated that the CNRS2 was a member of RS family.