畜牧与兽医2025，Vol.57Issue(5)：64-74,11.
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
摘要
Abstract
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 recom-binant chitinase was determined using the DNS method;and its enzymatic properties under various conditions such as pH,temperature,sub-strates,metals,and chemical reagents were analyzed.Furthermore,the biological function of the recombinant chitinase was assessed by trea-ting the nematodes Caenorhabditis elegans and Strongylus equinus third-stage larvae.The results showed that the AC-chi6195 gene was 1 453 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 the Arthrobotrys oligospora XJ-A1 strain.The SDS-PAGE results indicated that the recombinant 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 an activity of 102 U/mg.The enzymatic characterization analysis showed that the optimal reaction conditions for this enzyme were pH=7 and 40 ℃,and DTT,SDS;and Cu2+exhibited inhibitory effects.The enzyme degraded colloidal chitin and a small amount of chitosan.After treating the third-stage larvae of C.elegans and S.equinus with the recombinant chitinase AC-chi6195 for 12,24,and 36 hours,the mortal-ity rates were 65％,93％,and 100％,respectively,for C.elegans;and 75％,97％,and 100％,respectively,for S.equinus third-stage larvae.These findings contributed to the development of efficient chitinase biopesticides from predatory fungi.关键词
圆锥节丛孢菌/几丁质酶/酶学特性/生物学活性Key words
Arthrobotrys conoides/chitinase/enzymatic properties/biological activity分类
农业科技引用本文复制引用
王水怡,张伟,刘丹丹,杜少磊,刘雨桐,姜冰冰,朱慧茹,巴音查汗,张杨,李永畅..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[J].畜牧与兽医,2025,57(5):64-74,11.基金项目
中央引导地方科技发展专项基金项目(ZYYD2023C03-2) （ZYYD2023C03-2）
新疆维吾尔自治区重大科技专项项目子课题(2022A02013-2-6) （2022A02013-2-6）
