苏云金芽胞杆菌N-酰基高丝氨酸内酯酶基因(aiiA)在毕赤酵母中的优化表达  

Optimized Expression of N-acylhomoserine Lactonase Gene(aiiA) from Bacillus thuringiensis in Pichia pastoris

作　　者：简思美 何晶晶[1] 毛惠民[1] 杨梅[1] 

机构地区：[1]福建师范大学生命科学学院,福州350108

出　　处：《农业生物技术学报》2014年第11期1347-1356,共10页Journal of Agricultural Biotechnology

基　　金：福建省自然科学基金(No.2012J01123);福建省教育厅项目(No.JK2012009)

摘　　要：苏云金芽胞杆菌(Bacillus thuringiensis)N-酰基高丝氨酸内酯酶基因(auto inducer inactivation A,aiiA)编码的N-酰基高丝氨酸内酯酶(N-acylhomoserine lactonase,AiiA)能够水解植物病原菌群体效应的信号分子N-酰基高丝氨酸内酯(N-acylhomoserine lactone,AHL),进而使革兰氏阴性细菌群体感应受到抑制,减弱病原菌的致病性。本研究将aiiA基因连接至分泌型穿梭表达载体pPICZαB,获得重组表达质粒p PICZαB-aiiA,线性化后电击转化毕赤酵母(Pichia pastoris)GS115,获得重组工程菌GS115-p PICZαBaiiA。利用定点突变技术,对aiiA基因进行密码子优化,获得重组工程菌GS115-p PICZαB-MaiiA。以终浓度为1%的甲醇、28℃条件下诱导表达,重组工程菌GS115-p PICZαB-aiiA和GS115-p PICZαB-MaiiA均成功表达并分泌出AiiA蛋白。抗病性分析表明,分泌表达的AiiA蛋白能有效抑制胡萝卜软腐欧文氏杆菌(Erwinia carotovora)的致病性。AiiA蛋白在毕赤酵母中的分泌表达,拓宽了AiiA蛋白的获取途径,为AiiA蛋白的产业化提供理论依据。密码子优化为今后改造AiiA蛋白,提高AiiA蛋白的表达效率提供了新的思路。Many phytopathogens use N-acylhomoserine lactone （AHL） as quorum sensing （QS） signals, inducing the expression of virulence factors to destroy plant. N-acylhomoserine lactonase （AiiA） can degrade AHL, resulting in the disruption of AHL-mediated QS system in gram-negative bacteria. In this study, auto inducer inactivation A（aiiA） gene was firstly inserted into the shuttle expression vector of pPICZαB and introduced to GS115 strain of Pichia pastoris, resulting in a recombinant yeast strain GS115-pPICZαB-aiiA. On the other hand, codon optimized aiiA gene, named as MaiiA, was carried out by using site-directed mutagenesis to adapt to P. pastoris expression system betterly, and was also inserted into the expression vector and introduced into yeast, and constructed GS115-pPICZαB-MaiiA. Both GS115-pPICZαB-aiiA and GS115-pPICZαB-MaiiA were successfully induced to express secreted AiiA proteins by the addition of 1%methanol at 28 ℃. Antimicrobial activity analysis showed that the AiiA protein inhibited the virulence of Erwinia carotovora. The expression of secreted AiiA protein in P. pastoris, broadening the acquisition route of Aii protein, provides a theoretical basis and new idea for AiiA industrialization. Codon optimization provides a new way of thinking for the future reconstruction and the efficient expression of AiiA protein.

关 键 词：N-酰基高丝氨酸内酯酶蛋白(AiiA) 分泌表达 密码子优化 

分 类 号：S432.4[农业科学—植物病理学]