昆虫抗真菌肽基因的分泌型表达及活性鉴定

 【目的】黑腹果蝇(Drosophila melanogaster)抗真菌肽Drosomycin (Drs)及其同系物Drosomycin-like C (Drs-lC)和斑腹刺螠蝽(Podisus maculiverntris)抗真菌肽Thanatin对丝状真菌具有广谱高效的抑杀作用。实现抗真菌肽基因在酵母中的高效分泌型表达，对探讨利用转基因酵母的发酵液直接进行果蔬、食品和农产品防腐保鲜的生物技术研发有重要意义。【方法】将Drosomycin基因（Drs）及其同系物基因Drs-lC和Thanatin基因分别与酵母分泌型表达载体pPICZαA重组，构建成重组表达质粒pPICZαA-Drs、pPICZαA-Drs-lC和pPICZαA–Thanatin。利用电转化将重组质粒转化毕赤酵母GS115，经表型筛选和PCR鉴定获得的重组毕赤酵母转化子，在甲醇诱导下进行抗真菌肽的分泌表达。【结果】3种抗真菌肽基因的酵母表达产物对6个供试真菌中的5个有明显的抑制作用，Thanatin同时对供试细菌有抗菌活性。【结论】Drs、Drs-lC和Thanatin抗真菌肽基因成功地转化毕赤酵母，并实现其分泌型表达。

Secretive Expression of Insect Antifungal Peptide Genes in Pichia pastoris and Antifungal Activity Assay for the Expressed Products

The antifungal peptides drosomycin (Drs) and its isoform drosomycin-like C (Drs-lC) from Drosophila melanogaster and thanatin from Podisus maculiventris have broad and potent activity to filamentous fungi. The secretive expression of these antifungal peptide genes in yeast will provide the possibility to use their expressive products as protective regents on fruit, vegetable, food and agricultural products directly. The three genes of drs, Drs-lC and thanatin were cloned into pPICZα, a secretive vector of yeast. The recombinant vectors which were named pPICZαA-drs、pPICZαA-Drs-lC and pPICZα-Thanatin were integrated into the genome of Pichia pastoris by electric transformation method. The recombinant Pichia pastoris was induced to express antifungal peptide by methanol. The expressive products of the three recombinant showed antifungal activity to 5 of 6 tested fungi species and the expressive product of thanatin also had strong activity to the tested bacteria.