昆虫抗真菌肽基因的分泌型表达及活性鉴定  

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

作　　者：桑延霞[1,2] 邓小娟[1] 杨婉莹[1] 刘文权[1] 王文献[3] 黄亚东 曹阳[1,2] 

机构地区：[1]华南农业大学动物科学学院蚕丝科学系 [2]西南大学农业部蚕桑学重点实验室重庆400716 [3]华南农业大学资源环境学院昆虫学系 [4]曁南大学医药生物技术研究开发中心

出　　处：《中国农业科学》2007年第4期842-849,共8页Scientia Agricultura Sinica

基　　金：国家重点基础研究发展计划(973计划)项目(2005CB121000);广东省科学技术计划重点项目(2003C104042);广东省自然科学基金资助项目(032256;04020553)

摘　　要：【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。[ Objective ] The antifungal peptides of Drosomycin （Drs） family from Drosophila melanogaster and Thanatin from Podisus maculiventris have broad and potent activity against filamentous fungi. The secretive expression products of Drs, Drs-lC and Thanatin in yeast can be applied as the antifungal reagent for food freshening, such as the freshening of fruit, vegetable and other agricultural products. [Method] Three genes, Drs, Drs-lC and thanatin, were cloned into pPICZα, a secretive vector of yeast. The recombinant vectors, pPICZαA-Drs, pPICZαA-Drs-IC and pPICZα-Thanatin, were integrated into the genome of Pichiapastoris via electric transformation method. The recombinant Pichia pastoris, which was selected by phenotype and identified by PCR amplification, was induced by methanol for the expression of the target antifungal peptide genes. [Result]The expressed products of the three genes showed antifungal activity against five of six tested fungi strains. Moreover, the expressed product of thanatin showed strong activity against not only fungi but also tested bacteria. [Conclusion] Three antifungal peptide genes, Drs, Drs-lC and Thanatin, were transformed into Pichia pastoris successfully. The expression products were secreted into the culture medium of yeast and showed antifungal activity against tested fungi.

关 键 词：昆虫 抗真菌肽 毕赤酵母 分泌型表达 抗真菌活性鉴定 

分 类 号：Q963[生物学—昆虫学]