菌寄生真菌黄蓝状菌(Talaromyces flavus)几丁质酶基因的克隆与生物信息学分析  

Cloning and Bioinformatics Analysis of Chitinase Gene from Mycoparasitic Talaromyces flavus

作　　者：咸洪泉[1,2] 汤伟[2] 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：《农业生物技术学报》2011年第6期1089-1098,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.310717237);国家海洋可再生能源专项(No.SDME2011SW01);转基因生物新品种培育科技重大专项(No.2008ZX08001-002)共同资助

摘　　要：菌寄生真菌的几丁质酶有很强的降解几丁质能力,在控制植物病害方面起着重要的作用。为克隆和研究菌寄生真菌黄蓝状菌(Talaromyces flavus)几丁质酶基因(tfchi1),本研究根据真菌几丁质酶基因的保守序列设计扩增基因中间片段的简并引物,采用RT-PCR、3'-RACE及5'-TAIL的方法获得了该基因的DNA和mRNA序列(GenBank:GU361769,GU361770)。tfchi1长为2 561 bp,具有6个内含子,长度分别为129、78、68、65、53和59 bp,包含1 194 bp的ORF,编码一个由397个氨基酸组成的蛋白。推导的tfchi1氨基酸序列以及蛋白质结构生物信息学分析表明,该蛋白具有典型的几丁质酶催化区保守序列SXGGW和DGXDXDWE,属于糖苷水解酶18家族几丁质酶,与柄篮状菌(Talaromyces stipitatus ATCC 10500)几丁质酶(XP_002480365)氨基酸序列同源性为96%,分子量为43.47 kD,等电点为4.97。该蛋白无信号肽序列,有15个潜在的N-糖基化位点,Tfchi1的二级结构以无规卷曲和α-螺旋为蛋白的主要结构元件,三级结构中有(α/β)8的圆桶形结构。tfchi1转化毕赤酵母(Pichia pastoris)GS115,酵母工程菌可分泌具几丁质酶活性的表达产物,重组蛋白的分子量与理论值相符。结果说明,本研究已从T.flavus中正确克隆了1个糖苷水解酶18家族几丁质酶基因。Mycoparasitic chitinases play an important role in resisting plant diseases for their strong capacity of degrading chitin.In order to clone and study chitinase gene（tfchi1） fromTalaromyces flavus,a pair of degenerate primers were designed according to the conserved sequences of chitinase genes in other fungi.The full-length DNA and cDNA of tfchi1 were cloned by using RT-PCR,3＇-RACE and 5＇-TAIL（GenBank： GU361769,GU361770）.The full-length DNA of tfchi1 was 2 561 bp,containing six introns with the length of 129,78,68,65,53 and 59 bp,respectively,ORF was 1 194 bp and it encoded 397 amino acids.Based on the predicted amino acid sequence of tfchi1 and the bioinformatic analysis of protein structure,two prevalent conserved catalytic domains with the sequences of SXGGW and DGXDXDWE were identified,Tfchi1 belonged to family 18glycoside hydrolase,and shared 96% homology with the sequence of chitinase（XP-002480365） from Talaromyces stipitatus ATCC 10550.Its predicted molecular weight and isoelectric point were 43.47 kD and 4.97,respectively.Tfchi1 had no signal peptide sequence but 15 potential N-glycosylation sites.Random coils and α-helices were the major structural elements in secondary structure of Tfchi1,while（α/β） 8 rounded buckets were evident in tertiary structure.A recombinant protein with chitinase activity was obtained after tfchi1 was transformed into Pichia pastoris GS115 and its molecular weight was also consistent to the theoretical value.The results show that a chitinase gene,belonging to family 18 glycoside hydrolase,is correctly cloned from T.flavus in this study.

关 键 词：黄蓝状菌 几丁质酶 基因克隆 序列分析 

分 类 号：S572[农业科学—烟草工业]