少孢节丛孢菌XJ-A1几丁质酶AO-483基因的克隆及生物学活性  

Gene cloning and bioactivity analysis of chitinase gene AO-483 from Arthrobotrys oligospora XJ-A1

作　　者：贡莎莎 孟庆玲[1] 乔军[1] 钟文强 黄运福 张国武 陈英 才学鹏[2] GONG Shasha;MENG Qingling;QIAO Jun;ZHONG Wenqiang;HUANG Yunfu;ZHANG Guowu;CHEN Ying;CAI Xuepeng(Department of Animal Science & Technology,Shihezi University,Shihezi 832003,China;Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832003 [2]中国农业科学院兰州兽医研究所,甘肃兰州730046

出　　处：《浙江农业学报》2019年第2期222-228,共7页Acta Agriculturae Zhejiangensis

基　　金：国家自然科学基金(31460654;31260601);新疆自治区研究生科研创新项目(XJGRI2015038)

摘　　要：为弄清少孢节丛孢菌几丁质酶的生物学功能,对少孢节丛孢菌XJ-A1几丁质酶AO-483基因进行克隆及分子特征分析,并在大肠埃希菌中进行诱导表达;采用DNS还原糖法检测经镍柱纯化的重组几丁质酶活性,将其作用于秀丽隐杆线虫幼虫,分析其生物活性。结果显示,AO-483基因编码309个氨基酸,与少孢节丛孢菌标准株(ATCC 24927)的几丁质酶AO-483基因序列的同源性为96. 88%,氨基酸序列同源性为97. 73%;AO-483含有1个Glyco-hydro-18结构域,位于20～297位氨基酸,属于糖苷水解酶18家族,还含有特征序列GMLGG和LDGLDLDVE;三级结构为典型的三磷酸异构酶桶形结构。SDS-PAGE分析结果表明,重组蛋白AO-483分子质量约为52 ku,与预测大小一致; Western blot分析证实,该重组蛋白能与小鼠抗少孢节丛孢菌多克隆血清抗体发生特异性反应。经DNS还原糖法检测,该重组几丁质酶活性为223. 31 U·m L^(-1);重组几丁质酶对秀丽隐杆线虫Ⅰ期和Ⅳ期幼虫有较强的降解活性。In order to study the functions of chitinase,chitinase gene AO- 483 of nematode-trapping fungi Arthrobotrys oligospora XJ-A1 was cloned,analyzed and expressed in prokaryotic expression system Escherichia coli.The pure prokaryotic expression recombinant fusion protein was purified by Ni column and the chitinase activity was determined by DNS method,and the biological activity of recombinant protein AO-483 was verified by dealing with larvae of Caenorhabditis elegans.The results showed that chitinase AO- 483 gene encoded 309 amino acids,the homology of its gene sequence was 96.88% and the amino acid sequence was 97.73% respectively with Arthrobotrys oligospora standard strain (ATCC 24927).The chitinase AO-483 belonged to the family 18 glycoside hydrolase with a Glyco-hydro-18 structure domained at amino acid sequence of 20~297 and had two prevalent conserved catalytic domained with the sequence of GMLGG and LDGLDLDVE.The tertiary structure was a typical triosephosphate isomerase barrel structure.The protein identified by SDS-PAGE showed that the recombinant protein had a molecular weight of about 52 ku which was consistent with prediction.Western blot revealed that recombinant protein can specifically react with anti- Arthrobotrys oligospora polyclonal antibody.The activity of recombinant chitinase AO-483 was determined by the DNS method,with a value of 223.31 U·mL^-1.The recombinant AO-483 owned stronger biological activity that can degrade Caenorhabditis elegans larvae stage 1and stage 4.

关 键 词：少孢节丛孢菌 几丁质酶 克隆 生物学活性 

分 类 号：Q78[生物学—分子生物学]