为建立一种新的外源蛋白安全评估方法，构建转外源基因酵母是关键步骤。本研究以来源于转基因作物的抗除草剂合成酶蛋白CP4-EPSPS为研究模板，首先通过基因优化和化学合成获得cp4-epsps基因，优化后基因密码子适用性指数(CAI)为0.85，GC含量为52%。目的基因克隆至毕赤酵母表达载体pPICZb，经鉴定筛选正确的重组载体pPICZb-EPSPS电击转化至毕赤酵母GS115，cp4-epsps基因通过同源重组方式整合至酵母基因组，PCR扩增酵母基因组筛选得到在正确位点发生同源重组的4株阳性菌株。随后，各阳性菌株分别于28 ℃、250~300 r/min培养，0.5%甲醇诱导4 d后，提取各组菌株总蛋白，采用SDS-PAGE和western blotting鉴定CP4-EPSPS表达的正确性。CP4-EPSPS单克隆抗体及载体标签C-MYC单克隆抗体的western blotting结果一致显示cp4-epsps基因在毕赤酵母GS115中成功获得表达，大小约50 ku。本实验成功构建了转cp4-epsps基因的毕赤酵母基因工程菌，为下一步开展转基因作物CP4-EPSPS成份的安全评价奠定基础。

Constructing the genetically modified (GM) Pichia pastoris stains is the key step forestablishing a new method to assess the safety of foreign protein . Based on the research model of the 5-enolpyruvyl shikinmate-3-phosphate synthase derived from Agrobacterium CP4 strain (CP4-EPSPS), the cp4-epsps gene was chemically synthesized after optimizing thecodon usage bias. The codon adaption index (CAI) of optimized sequence was 0.85, and the GC content of optimized sequence was 52%. Cp4-epsps gene was amplified by PCR and cloned to the expression vector pPICZb of Pichia pastoris GS115 through homologous end-joining, and the recombinant vector pPICZb-EPSPS was constructed. The correct recombinant vector was transferred into GS115 strain through electroporation, and four positive strains were screened through the right site via PCR. Then, those positive strains were induced by 0.5% methanol for four days at 28 ℃, 250~300 r/min. Finally, the expression of cp4-epsps in GS115 was identified from CP4-EPSPS monoclonal antibody and C-MYC monoclonal antibody by SDS-PAGE and western blotting. The results showed that the target gene was expressed in GS115 strain and MW of protein was 50 ku, which coincided with the theoretical results. In conclusion, the construction of transgenic Pichia pastoris GS115 strains is beneficial for the safety assessment of transgenic CP4-EPSPS synthase in GM crops.

环保部公益性行业科研专项（201509044）