农业生物技术学报
農業生物技術學報
농업생물기술학보
JOURNAL OF AGRICULTURAL BIOTECHNOLOGY
2014年
11期
1347-1356
,共10页
简思美%何晶晶%毛惠民%杨梅
簡思美%何晶晶%毛惠民%楊梅
간사미%하정정%모혜민%양매
N-酰基高丝氨酸内酯酶蛋白(AiiA)%分泌表达%密码子优化
N-酰基高絲氨痠內酯酶蛋白(AiiA)%分泌錶達%密碼子優化
N-선기고사안산내지매단백(AiiA)%분비표체%밀마자우화
N-acylhomoserine lactonase (AiiA)%Secretory expression%Codon optimization
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pPICZαB-aiiA，线性化后电击转化毕赤酵母(Pichia pastoris)GS115，获得重组工程菌GS115-pPICZαB-aiiA。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-pPICZαB-MaiiA。以终浓度为1%的甲醇、28℃条件下诱导表达，重组工程菌GS115-pPICZαB-aiiA和GS115-pPICZαB-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。
囌雲金芽胞桿菌(Bacillus thuringiensis)N-酰基高絲氨痠內酯酶基因(auto inducer inactivation A, aiiA)編碼的N-酰基高絲氨痠內酯酶(N-acylhomoserine lactonase, AiiA)能夠水解植物病原菌群體效應的信號分子N-酰基高絲氨痠內酯(N-acylhomoserine lactone, AHL)，進而使革蘭氏陰性細菌群體感應受到抑製，減弱病原菌的緻病性。本研究將aiiA基因連接至分泌型穿梭錶達載體pPICZαB，穫得重組錶達質粒pPICZαB-aiiA，線性化後電擊轉化畢赤酵母(Pichia pastoris)GS115，穫得重組工程菌GS115-pPICZαB-aiiA。利用定點突變技術，對aiiA基因進行密碼子優化，穫得重組工程菌GS115-pPICZαB-MaiiA。以終濃度為1%的甲醇、28℃條件下誘導錶達，重組工程菌GS115-pPICZαB-aiiA和GS115-pPICZαB-MaiiA均成功錶達併分泌齣AiiA蛋白。抗病性分析錶明，分泌錶達的AiiA蛋白能有效抑製鬍蘿蔔軟腐歐文氏桿菌(Erwinia carotovora)的緻病性。AiiA蛋白在畢赤酵母中的分泌錶達，拓寬瞭AiiA蛋白的穫取途徑，為AiiA蛋白的產業化提供理論依據。密碼子優化為今後改造AiiA蛋白，提高AiiA蛋白的錶達效率提供瞭新的思路。
소운금아포간균(Bacillus thuringiensis)N-선기고사안산내지매기인(auto inducer inactivation A, aiiA)편마적N-선기고사안산내지매(N-acylhomoserine lactonase, AiiA)능구수해식물병원균군체효응적신호분자N-선기고사안산내지(N-acylhomoserine lactone, AHL)，진이사혁란씨음성세균군체감응수도억제，감약병원균적치병성。본연구장aiiA기인련접지분비형천사표체재체pPICZαB，획득중조표체질립pPICZαB-aiiA，선성화후전격전화필적효모(Pichia pastoris)GS115，획득중조공정균GS115-pPICZαB-aiiA。이용정점돌변기술，대aiiA기인진행밀마자우화，획득중조공정균GS115-pPICZαB-MaiiA。이종농도위1%적갑순、28℃조건하유도표체，중조공정균GS115-pPICZαB-aiiA화GS115-pPICZαB-MaiiA균성공표체병분비출AiiA단백。항병성분석표명，분비표체적AiiA단백능유효억제호라복연부구문씨간균(Erwinia carotovora)적치병성。AiiA단백재필적효모중적분비표체，탁관료AiiA단백적획취도경，위AiiA단백적산업화제공이론의거。밀마자우화위금후개조AiiA단백，제고AiiA단백적표체효솔제공료신적사로。
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 AiiA 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.
