植物防御素PD5基因在毕赤酵母中的分泌表达

Secreted Expression Plant Defensin PD5 Gene in Pichia pastoris

将植物防御素PD5基因克隆到载体pPIC9的XholⅠ和EcoRⅠ位点之间,得到重组载体pPIC9/PD5。pPIC9/PD5经BglⅡ线性化,电转化法转入PichiapastorisGS115,MD和MM平板筛选表型为His+MutS的转化子,PCR鉴定阳性转化子。转化子D24用BMGY培养至OD600为6·0时,经BMM诱导,上清液用Tricien-SDS-PAGE可检测到目标条带。同时经平板抑菌试验显示,D24的诱导发酵上清液可很好的抑制香蕉枯萎菌(Fusariumoxysporumf·sp·Cubense)及甘蓝黑腐菌Xanthomonacampestris(Smith)Dovosen的生长。植物防御素PD5基因在毕赤酵母中得到表达。

The plant defensin PD5 gene was cloned into the restriction site between XholⅠ and EcoRⅠ of plasmid pPIC9 successfully. The recombinant vector pPIC9/PD5 was linearized with enzyme Bgl Ⅱ and was transformated into Pichia pastoris GS115 by electransformation. The transformants were cuhurated with MD and MM medium plates, and His^＋ Mut^S transformants were isolated. Using PCR method identifed true transformants. Transformant D24 was cultured with BMGY medium and induced with BMM medium until the cell density reached an OD600 = 6. 0. The recombinant protein could be detected in the cultured supernatant by Tricien-SDS-PAGE. Plate test indicated that the supematant could inhihite the growth of Fusarium oxysporum f. sp. Cubense and Xanthomona campestris （Smith） Dovosen evidently. Secreted expression plant defensin PD5 gene in Pichia pastoris was successful.