cry基因保守序列克隆及其在大肠杆菌Rosetta(DE3)中的表达

Cloning and Expression of Conserved Sequences of cry-type Gene in Rosetta(DE3)

[目的]对cry基因保守序列进行克隆,并使其在大肠杆菌Rosetta(DE3)中进行表达。[方法]根据NCBI数据库信息设计引物序列,采用PCR技术从抗虫棉基因组DNA中扩增出抗虫基因cry的保守序列,构建重组载体并转化到大肠杆菌Rosetta(DE3)菌株中,利用pGEX原核表达系统诱导蛋白表达。同时,分析了不同IPTG浓度及不同诱导时间对蛋白表达量的影响。[结果]扩增出抗虫基因cry的两段保守序列,长度分别为304和853 bp;SDS-PAGE检测结果显示,经IPTG诱导后重组质粒pGEX-4t-1-304和pGEX-4t-1-853成功地表达了大量GST融合蛋白,分子量分别为39和62.4 kDa,与预期结果一致;确定了IPTG最佳诱导浓度为0.15 mmol/L,最佳诱导时间为7h。[结论]为今后检测转Bt基因农作物奠定了基础。

[Objective] The aim was to clone and express the conserved sequence of cry-type gene in Rosetta（DE3）.[Method] Two pairs of special primers were designed according to NCBI database information and the conserved sequences of cry-type gene was amplified by PCR from Bt transgenic cotton.Then recombinant plasmids have been constructed and expressed in Escherichia coli Rosetta（DE3） strain.And the effect of different concentration and inducing time of IPTG on the expression product was analyzed.[Result] Two conserved sequences of cry-type gene were amplified and the length of them was 304 and 853 bp respectively.The result of SDS-PAGE confirmed that the recombinant plasmids pGEX-4t-1-304 and pGEX-4t-1-853 which induced by IPTG could express fusion proteins and the molecular weight was 39 and 62.4 kDa respectively,which was in accordance with predicted results.Then a better protein expression conditions were confirmed that the concentration of IPTG was 0.15 mmol/L and the inducing time was 7 hours.[Conclusion] It prepared the ground for the further detection of Bt transgenic crop.