渐狭蜡蚧菌的鉴定及其几丁质酶基因 LACHI1的克隆  

Identification of Fungus Lecanicillium attenuatum and Cloning of Its Chitinase Gene LACHI1

作　　者：赵洋[1] 陈德鑫[2] 王凤龙[2] 黄化刚 武侠[1] 

机构地区：[1]青岛农业大学农学与植物保护学院、山东省植物病虫害综合防控重点实验室,山东青岛266109 [2]中国农业科学院烟草研究所,山东青岛266101 [3]贵州省烟草公司毕节市公司,贵州毕节551700

出　　处：《华北农学报》2014年第4期63-70,共8页Acta Agriculturae Boreali-Sinica

基　　金：中国烟草总公司资助项目(110200902065-4);山东烟草专卖局资助项目(201001);农业部科研项目(2130108);山东省“泰山学者”建设工程专项

摘　　要：克隆渐狭蜡蚧菌几丁质酶基因,为进一步明确该菌产生的几丁质酶对定居性根结类和孢囊类线虫卵孵化抑制作用提供理论依据。采用分离自黑龙江大豆孢囊线虫孢囊寄生真菌CGMCC5328,通过形态学特征及ITS序列比较分析,鉴定该菌株为渐狭蜡蚧菌。通过简并引物设计和RACE技术,克隆渐狭蜡蚧菌中几丁质酶基因,利用生物学软件分析该基因序列及其编码的氨基酸序列。首次从渐狭蜡蚧菌中克隆得到一个几丁质酶基因LACHI1,该基因DNA序列长1 743 bp,含3个内含子,包含1 272 bp开放阅读框,编码423个氨基酸,理论分子量45.9 kDa,等电点5.90。LACHI1基因编码的几丁质酶,属于糖基水解酶18家族几丁质酶。同源性比对表明该菌产生的几丁质酶与昆虫寄生真菌和食线虫真菌产生的几丁质酶有很高的同源性。To clone chitinolytic genes from Lecanicillium attenuatum and provide theoretical basis for the inhibi-tion of the chitinases on egg-hatching of root knot and cyst nematode .In this study ,an nematode egg-parasitic fungus CGMCC5328 was isolated from Heterodera glycines infecting soybean in Heilongjiang Province .Based on morphological characters and molecular analysis of ITS-rDNA,the strain was identified as L.attenuatum.The chitinase gene LACHI1 from L.attenuatum was cloned using the degenerate PCR primers and RACE techniques .The analysis of the gene LA-CHI1 and its amino acid sequence was by biology software .We have cloned a chitinase gene LACHI1 from L.attenua-tum for the first time.The gene is 1 743 bp in length and contains three putative introns .The ORF of LACHI1 is 1 272 bp in size with encoding protein of 423 aa,molecular mass of 45.9 kDa and pI of 5.90.The chitinase deduced by LA-CHI1 belongs to glycosyl hydrolase family 18 chitinase .Comparison of the chitinase amino acid sequence with other chitinases from entomopathogenic fungi and nematophagous fungi revealed that the enzymes were highly similar .

关 键 词：渐狭蜡蚧菌 几丁质酶基因 克隆 系统发育分析 

分 类 号：Q78[生物学—分子生物学]