哈茨木霉几丁质合酶基因ThChsC的克隆及序列分析  

Cloning and Bioinformatics Analysis of Chitin Synthase Gene ThChsC from Trichoderma harzianum

作　　者：冀颖[1] 李梅[1] 田云龙[2] 刘卫德[1] 牛静[1] 蒋细良[1] 

机构地区：[1]中国农业科学院植物保护研究所/农业部作物有害生物综合治理综合性重点实验室,北京100081 [2]中国农业科学院农业环境与可持续发展研究所,北京100081

出　　处：《中国农业科学》2011年第17期3537-3546,共10页Scientia Agricultura Sinica

基　　金：国家自然科学基金项目(30671400;31071728);国家高技术研究发展计划(2011AA10A205);中国农业科学院植物保护研究所基本科研业务费专项(1610142011002);植物病害生物学国家重点实验室开放基金(SKL20100P17;SKL20110P13)

摘　　要：【目的】克隆哈茨木霉几丁质合酶基因全序列,并对序列进行生物信息学分析。【方法】利用多聚酶链式反应(PCR)和染色体步移技术(genome walking)克隆几丁质合酶基因全序列;应用生物信息学软件对获得的基因序列及编码的蛋白序列进行分析;通过同源建模,对蛋白质的三维结构进行预测。【结果】从哈茨木霉(Trichoderma harzianum Th-33)中扩增得到几丁质合酶基因ThChsC(GenBank登录号HQ419000),编码区(CDS)长为2 835 bp,由4个外显子和3个内含子组成,开放阅读框(ORF)长2 688 bp,编码895个氨基酸。序列比对和同源性分析显示,该基因与串珠状赤霉(Gibberella moniliformis,ACY08039.1)和禾生刺盘孢菌(Colletotrichum graminicola,AAL23718.1)几丁质合酶基因相似性最高,分别为80%和81%;相应的氨基酸序列同源性均为80%,系统进化分析表明,ThChsC属于III型几丁质合酶亚家族。几丁质合酶ThChsC含有1个Chitin_synth_1结构域,1个Chitin_synth_1N结构域,1个Chitin_synth_2结构域,3个跨膜结构域,不含信号肽,为膜结合蛋白。对预测的三维结构分析表明,ThChsC含有糖基转移酶的保守DHD结构域。【结论】从哈茨木霉Th-33中克隆得到几丁质合酶基因ThChsC,对该基因的深入研究有助于探索哈茨木霉几丁质合成的机制。[Objective] The objective of this study is to clone and make bioinformatics analysis of the sequence of chitin synthase gene ThChsC from Trichoderma harzianum. [Method] The full length of ThChsC was cloned by polymerase chain reaction （PCR） and genome walking methods, the gene sequence obtained and the putative amino acid sequence were analyzed by bioinformatics software, and the three-dimensional structure of ThChsC was predicted by homology modeling. [Result] ThChsC （GenBank accession number HQ419000） coding sequence spans 2 835 bp and is comprised of 4 extrons inserted by 3 introns. The ThChsC contains an open reading frame （ORF） of 2 688 bp capable of encoding a polypeptide of 895 amino acids and shares 80% and 81% identity to Gibberella moniliformis chsB （ACY08039.1） and Colletotrichum graminicola chs2 （AAL23718.1 ）, respectively. Phylogenetic analysis indicates that ThChsC belongs to the type III chitin synthase subfamily. The conserved Chitin_synth_1, Chitin synth 1N and Chitin_synth 2 domains and three transmembrane domains were found in the predicted ThChsC protein. A conserved DHD domain of glycosyl transferase was found in the three-dimensional structure of ThChsC predicted. [ Conclusion ] The chitin synthase gene ThChsC was cloned for the first time from Trichoderma harzianum Th-33. The further study of ThChsC will have an important significance in exploring the mechanism of chitin synthesis of Trichoderma harzianum.

关 键 词：哈茨木霉 几丁质合酶 基因克隆 序列分析 

分 类 号：Q78[生物学—分子生物学]