透明颤菌血红蛋白基因(vgb)与monellin基因在毕赤酵母中的联合表达  

Co-expression of Vitreoscilla hemoglobin gene and monellin gene in Pichia pastoris

作　　者：王清路[1] 张玉军[1] 窦烨[1] 李俏俏[1] 周彩霞[1] 

机构地区：[1]山东万杰医学院生物化学教研室,山东淄博255213

出　　处：《中国酿造》2008年第8期37-40,共4页China Brewing

基　　金：山东省教育厅科技计划(J06L77)

摘　　要：根据酵母偏爱密码子合成了monellin甜蛋白基因和vgb基因,然后构建了2种不同方式表达的载体,胞内表达的pPIC3.5KV和分泌表达的pPIC9KM。电击转化得到重组子GS115/pPIC9KM和GS115/pPIC9KM/pPIC3.5KV,通过PCR、SDS-PAGE及western blotting检测发现monellin甜蛋白基因和vgb基因成功整合进毕赤酵母基因组并成功表达,通过品尝和CO-差示光谱法证实其表达产物具有活性,用southern blotting对6株高产菌株进行拷贝数分析并未发现拷贝数与分泌蛋白产量之间存在明显的正相关。对比发现,含vgb基因菌株比未含菌株高甜度monellin的产量最高提高了20%,菌体密度平均提高了9%。According to Pichia pastoris preference codon, the monellin gene and Vitreoscilla hemoglobin gene （vgb） were synthesized. Two expression vectors, pPIC3.SKV for intracellular expression and pPIC9KM for extracellular expression were constructed. And the recombinant Pichia pastoris stains GS 115/pPIC9KM and GS 115/pPIC9KM/pPIC3.SKV were constructed via electroporation. The results of PCR, SDS-PAGE and westem-blotring indicated that the vgb gene and monellin gene had integrated into the genome of Pichia pastoris GS 115 and expressed in efficient level. The activities of vgb gene and monellin gene products were verified respectively by taste and CO-difference spectrum analysis. The positive correlations of copy number and monellin or vgb gene production were not found by southem-blotting analysis. With comparason, the yield of high sweet monellin invgb-containing strain raised by 20% that of the origin strain and the average cell density raised 9%.

关 键 词：透明颤菌血红蛋白基因 甜蛋白基因 毕赤酵母 拷贝数 外源基因 

分 类 号：Q78[生物学—分子生物学] TQ92[轻工技术与工程—发酵工程]