蝉拟青霉类枯草杆菌蛋白酶基因的克隆及其序列和蛋白质分析

doi:10.3969/j.issn.1004-1524.2019.10.12

浙江农业学报 ›› 2019, Vol. 31 ›› Issue (10): 1663-1670.DOI: 10.3969/j.issn.1004-1524.2019.10.12

蝉拟青霉类枯草杆菌蛋白酶基因的克隆及其序列和蛋白质分析

陈官菊, 柴一秋^*, 厉晓腊, 方鸣, 刘又高, 金轶伟

浙江省亚热带作物研究所,浙江温州 325005

收稿日期:2019-02-22 出版日期:2019-10-25 发布日期:2019-10-30
通讯作者: *,柴一秋,E-mail: chai-yiqiu@yeah.net
作者简介:陈官菊(1979—),女,湖北天门人,硕士,助理研究员,从事虫生真菌及植物保护方面的研究工作。E-mail: guanju_chen14@163.com
基金资助:
浙江省自然科学基金(LY12C14004)

Cloning of sublitisin-like protease gene cDNA from Isaria cicadae and characterization of its sequence and deduced protein

CHEN Guanju, CHAI Yiqiu^*, LI Xiaola, FANG Ming, LIU Yougao, JIN Yiwei

Zhejiang Institute of Subtropical Crops, Wenzhou 325005, China

Received:2019-02-22 Online:2019-10-25 Published:2019-10-30

摘要/Abstract

摘要： 蝉拟青霉是一种常见的昆虫病原真菌,被广泛应用于生物防治。本研究根据GenBank中已登录的淡紫拟青霉、粉拟青霉、球孢白僵菌和金龟子绿僵菌的类枯草杆菌蛋白酶基因序列的同源性比较,以它们高度保守的核苷酸序列设计一对引物,采用RT-PCR和3'/5'-RACE相结合的方法,从蝉拟青霉中克隆出完整的类枯草杆菌蛋白酶(subtilisin-like protease)cDNA基因序列。cDNA全序列为2 031 bp,该序列5'-端和3'-端的非编码序列长度分别为170 bp和262 bp,开放阅读框为1 599 bp,编码532个氨基酸,信号肽长度为18个氨基酸,前肽长度为133个氨基酸。该基因编码的蛋白序列与虫草棒束孢(Isaria farinosa)、球孢白僵菌(Beauveria bassiana)、蛹虫草(Cordyceps militaris)和哈茨木霉(Trichoderma harzianum)类枯草杆菌蛋白酶有较高的同源性,同源性分别为88%、88%、89%和71%。成熟蛋白具有典型的丝氨酸蛋白酶活性位点,同时有5个半胱氨酸,4个潜在的N-糖基化位点,位于细胞的液泡中与分泌途径相关,二级结构中以α-螺旋和无规则卷曲为主。

关键词: 虫生真菌, 蝉拟青霉, 类枯草杆菌蛋白酶基因, 克隆

Abstract: Isaria cicadae is one of the most common species of entomopathogenic fungi, and has been used as biocontrol agents against pests. In order to obtain a complete cDNA of sublitisin-like protease from I. cicadae, a pair of primers was designed using MegAign of DNAStar analysis software according to the high conserved nucleotide sequences in GenBank, and RT-PCR, 3'/5'-RACE PCR were used. The whole sequence of cloned cDNA was 2 031 bp, and the results showed that it had 170 bp and 262 bp on the 5'-terminal and 3'-terminal respectively, encompassed an open reading frame (ORF) with 1 599 bp encoding 532 amino acid (aa) residues, including 18-aa signal peptide, 133-aa propeptide. Alignments with the deduced amino acid of proteins in the species of Isaria farinose, Beauveria bassiana, Cordyceps militaris and Trichoderma harzianum showed a homology of 88%, 88%, 89% and 71%, respectively. The sublitisin-like protease of I. cicadae was located in the vacuoles of cells associated with the secretory pathway, with 5 cycteines and 4 putative N-glycosylation sites. The secondary structure of the protein was mainly the α-helix and randon coil.

Key words: Entomogenous fungi, Isaria cicadae, sublitisin-like protease gene, cloning

中图分类号:

S476.12

陈官菊, 柴一秋, 厉晓腊, 方鸣, 刘又高, 金轶伟. 蝉拟青霉类枯草杆菌蛋白酶基因的克隆及其序列和蛋白质分析[J]. 浙江农业学报, 2019, 31(10): 1663-1670.

CHEN Guanju, CHAI Yiqiu, LI Xiaola, FANG Ming, LIU Yougao, JIN Yiwei. Cloning of sublitisin-like protease gene cDNA from Isaria cicadae and characterization of its sequence and deduced protein[J]. , 2019, 31(10): 1663-1670.

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蝉拟青霉类枯草杆菌蛋白酶基因的克隆及其序列和蛋白质分析

Cloning of sublitisin-like protease gene cDNA from Isaria cicadae and characterization of its sequence and deduced protein

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