圆锥节丛孢菌AC-XJ株几丁质酶AC-chi6195基因的克隆表达及生物学活性研究  

Cloning,expression and bioactivity analysis of chitinase AC-chi6195 gene from Arthrobotrys conoides AC-XJ strain

作　　者：王水怡 刘丹丹 杜少磊 刘雨桐 姜冰冰 朱慧茹 巴音查汗[1,2] 张杨 李永畅[1,2] 张伟 WANG Shuiyi;LIU Dandan;DU Shaolei;LIU Yutong;JIANG Bingbing;ZHU Huiru;Bayinchahan;ZHANG Yang;LI Yongchang;ZHANG Wei(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China;Xinjiang Key Laboratory for Research and Development of New Herbivore Drugs,Urumqi,830052,China)

机构地区：[1]新疆农业大学动物医学学院,新疆乌鲁木齐830052 [2]新疆草食动物新药研究与创制重点实验室,新疆乌鲁木齐830052

出　　处：《畜牧与兽医》2025年第5期64-74,共11页Animal Husbandry & Veterinary Medicine

基　　金：中央引导地方科技发展专项基金项目(ZYYD2023C03-2);新疆维吾尔自治区重大科技专项项目子课题(2022A02013-2-6)。

摘　　要：旨在探究圆锥节丛孢菌几丁质酶的生物学功能。在圆锥节丛孢菌新疆株AC-XJ全基因组测序基础上,选取几丁质酶基因AC-chi6195为研究对象,对其进行克隆与分子特征分析并原核表达获得重组蛋白,采用3,5-二硝基水杨酸(DNS)比色法测定该重组蛋白的酶活性,分析该重组蛋白在不同pH值、温度、底物、金属离子及化学试剂条件下的酶学特性,将其作用于秀丽隐杆线虫及马圆形线虫三期幼虫并分析其生物学功能。结果:AC-chi6195基因全长1453 bp,有2个内含子序列,共编码398个氨基酸,AC-chi6195属于糖基水解酶18家族且三级结构为典型的几丁质酶三磷酸异构酶桶形结构;遗传进化分析显示其与少孢节丛孢菌XJ-A1株几丁质酶AO-379亲缘关系相对最近;SDS-PAGE分析显示,重组蛋白AC-chi6195分子质量约为42 kDa;采用Ni柱层析法结合Western blot检测显示,获得了纯化的目的蛋白;DNS还原糖法检测显示,该重组几丁质酶活性为102 U/mg;酶学特性分析显示,该酶的最适反应条件为pH=7,40℃,1,4-二硫代苏糖醇(DTT)、SDS、Cu^(2+)对其有抑制作用,该酶可降解胶体几丁质和少量壳聚糖;用重组几丁质酶AC-chi6195分别处理秀丽隐杆线虫和马圆形线虫三期幼虫12、24、36 h后,秀丽隐杆线虫死亡率分别为65%、93%、100%,马圆形线虫三期幼虫死亡率分别为75%、97%、100%。本研究结果为捕食性真菌高效杀线虫酶制剂的研发提供了依据。To explore the biological functions of chitinases from Arthrobotrys conoides,this study was focused on the cloning and molecular characterization of chitinase gene AC-chi6195 from the Xinjiang strain(AC-XJ)of Arthrobotrys conoides,which was previously sequenced by our research group.The gene was cloned and expressed in prokaryotes to obtain recombinant proteins.The enzymatic activity of the recombinant chitinase was determined using the DNS method;and its enzymatic properties under various conditions such as pH,temperature,substrates,metals,and chemical reagents were analyzed.Furthermore,the biological function of the recombinant chitinase was assessed by treating the nematodes Caenorhabditis elegans and Strongylus equinus third-stage larvae.The results showed that the AC-chi6195 gene was 1453 bp in length,and that it contains two intron sequences and encodes 398 amino acids.AC-chi6195 belonged to the glycosyl hydrolase 18 family and had a typical chitinase three-dimensional structure in the form of a barrel.Phylogenetic analysis revealed that it was relatively closely related to the chitinase AO-379 from the fungus theArthrobotrys oligosporaXJ-A1 strain.The SDS-PAGE results indicated that therecombinant chitinase AC-chi6195 had a molecular weight of approximately 42 kDa.The purified target protein was verified through purifica-tion by Nickel column chromatography and identification by Western blot.The DNS assay demonstrated that the recombinant chitinase had anactivity of 102 U/mg.The enzymatic characterization analysis showed that the optimal reaction conditions for this enzyme were pH=7 and40℃,and DTT,SDS;and Cu^(2+)exhibited inhibitory effects.The enzyme degraded colloidal chitin and a small amount of chitosan.Aftertreating the third-stage larvae ofC.elegansandS.equinuswith the recombinant chitinase AC-chi6195 for 12,24,and 36 hours,the mortal-ity rates were 65%,93%,and 100%,respectively,forC.elegans;and 75%,97%,and 100%,respectively,forS.equinusthird-stagelarvae.These findings contributed to the developmen

关 键 词：圆锥节丛孢菌 几丁质酶 酶学特性 生物学活性 

分 类 号：S852[农业科学—基础兽医学]