黄蓝状菌几丁质酶基因tfchi1的克隆、表达及抗菌活性  

Cloning and expression of a chitinase gene tfchi1 from Talaromyces flavus and the antifungal activity of the recombinant enzyme

作　　者：咸洪泉[1,2] 汤伟[2] 张丽青[1] 李安娜[1] 李多川[1] 

机构地区：[1]山东农业大学植物保护学院,泰安271018 [2]青岛农业大学生命科学学院,青岛266109

出　　处：《植物病理学报》2012年第2期146-153,共8页Acta Phytopathologica Sinica

基　　金：国家自然科学基金项目(310717237);转基因生物新品种培育科技重大专项(2008ZX08001-002)

摘　　要：为研究黄蓝状菌(Talaromyces flavus)几丁质酶性质和作用,通过RT-PCR、3'RACE及5'TAIL-PCR技术克隆了一个黄蓝状菌几丁质酶基因tfchi1,该基因全长为2 561 bp,含有6个内含子,包含一个1 194 bp的ORF,编码397个氨基酸,分子量为43.47 kDa,属于糖苷水解酶第18家族。利用基因重组的方法构建酵母分泌型表达载体pPIC9K/tfchi1,并转化毕赤酵母,筛选得到一株高效表达几丁质酶的工程菌GS-TFCHI1-9,甲醇诱导第7 d酶活性最高,达32.29 U.mL-1。SDS-PAGE检测纯化重组蛋白的分子量约44 kDa。重组几丁质酶Tfchi1最适反应温度为35℃,最适反应pH值为5.5,在pH 6～8之间稳定。Zn2+、Cu2+对Tfchi1活性有明显的抑制作用。抑菌活性测定结果显示,Tfchi1可明显抑制敏感植物病原真菌的菌丝生长和分生孢子萌发。该酶在几丁质资源的开发利用和生物防治等领域具有较广的应用前景。In order to study the properties and functions of chitinases from Talaromyces flavus, the full-length DNA of a chitinase gene, tfchil was amplified by RT-PCR, 3＇ RACE and 5＇ TAIL-PCR. It has 2 561 bp in length with six introns, and contains an 1194-bp long open reading frame （ORF） encoding a protein of 397 amino acid residues with the calculated molecular mass of 43.47 kDa. The protein, Tfchil belongs to lycoside hydrolase family No. 18. The plasmid pPIC9K/tfchil was constructed and transformed into Pichia pastoris and a recombinant strain with efficient expression of chitinase, GS-TFCHI1-9 was obtained. Tfchil was secreted successfully with the yield reaching 32.29 U·mL^-1 by the 7-day methanol induction; it had a molecular weight of about 44.0 kDa according to the SDS-PAGE detection, which was close to the predicted molecular mass. The optimal pH and temperature of Tfchil were 5.5 and 35℃ respectively. Tfchil was quite stable at pH ranging from 6 to 8 and its activity was significantly inhibited by Zn^2＋, Cu^2＋. The results of antifungal activity assay showed that Tfchil could inhibit the mycelium growth and spore germination of sensitive plant pathogenic fungi evidently, and the Tfchil produced by T. flavus was considered to have potential application in exploitation of chitin resources and in biological control of plant fungal diseases.

关 键 词：黄蓝状菌 几丁质酶 trchi1 克隆 表达 抑菌活性 

分 类 号：S432.44[农业科学—植物病理学]