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莱氏野村菌Cq菌株几丁质酶基因的克隆与表达分析
引用本文:王金芳,刘光英,翟逸,万永继.莱氏野村菌Cq菌株几丁质酶基因的克隆与表达分析[J].微生物学报,2010,50(4):493-499.
作者姓名:王金芳  刘光英  翟逸  万永继
作者单位:西南大学生物技术学院,重庆,400716
基金项目:西南大学博士基金(100082);国家现代农业产业技术体系建设专项资金
摘    要:【目的】为揭示昆虫病原真菌分泌的几丁质酶对宿主感染致病时的作用,对莱氏野村菌Cq菌株几丁质酶基因进行了克隆与表达,并检测了表达产物的活性。【方法】采用CTAB法提取菌体DNA,设计特异性引物,多次PCR扩增克隆莱氏野村菌Cq菌株几丁质酶基因全序列,并克隆基因的ORF片段chit1,与载体pPIC9K相连接,构建表达载体pPIC9K-Chit1,转入毕赤酵母感受态细胞中,然后通过1.5mg/L浓度的G418筛选及PCR验证,将阳性转化子进行诱导培养,对发酵液分别进行酶活性测定试验、几丁质酶透明圈验证试验和SDS-PAGE电泳检测。【结果】莱氏野村菌Cq菌株几丁质酶基因全长序列为2756bp(NCBI登录号:EU795711),PCR扩增得到开放阅读框ORF片段chit1为1827bp,其中包含3个内含子,5′端非编码区长76bp,3′端非编码区240bp,编码424个氨基酸的几丁质酶前体,理论信号肽剪切位点在Gly(20)与Leu(21)之间;毕赤酵母重组细胞发酵液中几丁质酶活性随着发酵时间的延长而增加,72h达到最大值482.5U/100μL,透明圈活性验证试验显示,在含1%的几丁质平板上可出现明显的透明圈,表达产物SDS-PAGE电泳检测其分子量为41.0kDa。【结论】本研究克隆到莱氏野村菌Cq菌株几丁质酶基因,其ORF成功重组到毕赤酵母中并表达出有活性的几丁质酶。基因表达产物的利用对进一步研究病原真菌染病昆虫的机制等具有重要意义。

关 键 词:关键词:莱氏野村菌  几丁质酶  克隆  表达  产物活性
收稿时间:2009/9/22 0:00:00
修稿时间:2010/1/23 0:00:00

Cloning and expression analysis of the chitinase gene Chit1 from entomopathogenic fungus Nomuraea rileyi CQ strain
Jinfang Wang,Guangying Liu,Yi Zhai and Yongji Wan.Cloning and expression analysis of the chitinase gene Chit1 from entomopathogenic fungus Nomuraea rileyi CQ strain[J].Acta Microbiologica Sinica,2010,50(4):493-499.
Authors:Jinfang Wang  Guangying Liu  Yi Zhai and Yongji Wan
Institution:College of Biotechnology, Southwest University, Chongqing, BeiBei 400716, China;College of Biotechnology, Southwest University, Chongqing, BeiBei 400716, China;College of Biotechnology, Southwest University, Chongqing, BeiBei 400716, China;College of Biotechnology, Southwest University, Chongqing, BeiBei 400716, China
Abstract:ObjectiveIn order to understand the role of the entomopathogenic fungal chitinase in invasion and pathogenic process,we cloned and expressed a chitinase gene from Nomuraea rileyi CQ strain,and detected chitinase activity in recombinant Pichia pastori fermentation supernatant.MethodsSpecific primers were used to amplify the complete sequence of chitinase gene and open reading frame(ORF) from genomic DNA of N.rileyi Cq Strain extracted by CTAB.The fragment of ORF was linked with vector plasmid pPIC9K to const...
Keywords:Keyword: Nomuraea rileyi  chitinase  clone  expression  product activity
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