摘要
为研究粉红粘帚菌(Clonostachys rosea)生防机制并获得生物防治相关基因,文章利用RT-PCR方法克隆粉红粘帚菌1个几丁质酶基因T-ech42,对该基因进行生物信息学分析,并在大肠杆菌原核表达系统中进行外源表达。该基因长度为1 263 bp,编码420个氨基酸。推导T-ech42氨基酸序列以及蛋白质结构的生物信息学分析表明,该蛋白等电点为5.85,理论分子质量为45.13 ku,N端含信号肽,长度为20个氨基酸;T-ech42属于糖基水解酶18家族内切几丁质酶,包含SIGGW底物结合位点和FDGIDXDWE活性位点。将该基因构建到原核表达载体p ET-22b(+)上,转化至大肠杆菌BL21(DE3)菌株中,经终浓度1 mmol·L-1的IPTG诱导4 h后,可见酶蛋白活性表达。
In order to study the biocontrol mechanisms of Clonostachys rosea and get genes associated with biological control, the chitinase cDNA gene of T-ech42 from Clonostachys rosea was cloned by using RT-PCR, which was performed bioinformatics analysis and exogenous expression in prokaryotic expression system. The length of the gene was 1 263 bp and it encoded 420 amino acids. Based on the predicted amino acid sequence of T-ech42 and the bioinformatic analysis of protein structure, Its isoelectric point and predicted molecular weight were 5.85 and 45.13 ku, respectively and it had 20 N-terminal signal peptide. T-ech42 belonged to family 18 glycoside hydrolase, which contained substrate binding site SiGGW and active site FDGIDXDWE. There was some expression of the activity of enzyme protein after the gene constructing into the prokaryotic expression vector of pET-22b（＋）, transforming into E. coli BL21 （DE3） strain and was induced for 4 h by IPTG（1 mmol.L-1）.
出处
《东北农业大学学报》
CAS
CSCD
北大核心
2015年第8期9-14,共6页
Journal of Northeast Agricultural University
基金
教育部高校博士点专项科研基金(20092325110001)
教育部教学科学技术研究重点项目(211043)
黑龙江省教育厅新世纪优秀人才项目(1251-NCET-004)
关键词
粉红粘帚菌
几丁质酶
基因克隆
表达
Clonostachys rosea
chitinase
gene cloning
expression
