中国农业科学 ›› 2013, Vol. 46 ›› Issue (7): 1359-1369.doi: 10.3864/j.issn.0578-1752.2013.07.006

• 植物保护 • 上一篇    下一篇

斜纹夜蛾SpLPDI的克隆、表达及其 对莱氏野村菌的免疫应答分析

 李燕, 王中康, 陈环, 冯二艳, 殷幼平   

  1. 重庆大学生命科学学院/重庆市杀虫真菌生物农药工程技术中心,重庆 400030
  • 收稿日期:2012-09-28 出版日期:2013-04-01 发布日期:2012-10-23
  • 通讯作者: 通信作者殷幼平,Tel/Fax:023-65120489;E-mail:ypy128@vip.sina.com
  • 作者简介:李燕,Tel/Fax:023-65120489;E-mail:sungq520@yeah.net
  • 基金资助:

    国家公益性行业(农业)科研专项(201103002)、国家“863”计划(2011AA10A201)、重庆大学研究生创新基金(CDJXS112300)

Molecular Cloning and Expression Pattern Analysis of a Protein Disul?de Isomerases (SpLPDI) in Spodoptera litura

 LI  Yan, WANG  Zhong-Kang, CHEN  Huan, FENG  二Yan, YIN  You-Ping   

  1. School of Life Sciences, Chongqing University/Chongqing Engineering Research Center for Fungal Insecticides, Chongqing 400030
  • Received:2012-09-28 Online:2013-04-01 Published:2012-10-23

摘要: 【目的】研究斜纹夜蛾(Spodoptera litura)蛋白质二硫键异构酶基因(SpLPDI)的表达特性以及在莱氏野村菌入侵过程中的可能作用,为深入探讨斜纹夜蛾应答莱氏野村菌侵染的免疫分子机制奠定基础。【方法】根据斜纹夜蛾脂肪体抑制差减杂交文库(SSH)中筛选的PDI 的EST序列设计特异引物,结合SMART RACE技术,克隆SpLPDI的cDNA全长,并进行生物信息学分析;运用半定量RT-PCR和定量qPCR进行该基因的组织分布与差异分析;用qPCR研究SpLPDI被莱氏野村侵染诱导后的表达特性;用原核表达来研究其蛋白特征。【结果】斜纹夜蛾SpLPDI的cDNA全长为2 367 bp,包含1个1 485 bp的ORF,编码494个氨基酸。生物信息学分析表明,SpLPDI含有1个17个氨基酸残基的信号肽,2个硫氧还蛋白的活性域和1个RDEL内质网定位信号肽,且具有典型的PDI蛋白家族a-b-b′-a′的结构特点;同源序列对比分析发现SpLPDI蛋白在a和a′结构的活性域和内质网定位信号肽均极为保守;进化分析表明,斜纹夜蛾SpLPDI与家蚕的亲缘最近,与隐孢子虫和黑曲霉的亲缘较远。表达谱分析表明,SpLPDI在所选组织中均有表达,在血液中表达量最高,脂肪体次之,头部最低。经莱氏野村菌诱导后,SpLPDI在脂肪体和中肠中表达均有显著上调表达,但是在脂肪体的上调幅度比中肠的大。克隆的SpLPDI在大肠杆菌中成功表达出大量可溶蛋白。【结论】从斜纹夜蛾中克隆出蛋白质二硫键异构酶基因SpLPDI,在莱氏野村菌的诱导下有显著上调表达,可能与斜纹夜蛾相关的免疫应答有关。

关键词: 斜纹夜蛾 , 蛋白二硫异构酶基因 , 莱氏野村菌 , SpLPDI

Abstract: 【Objective】The objective of this study is to analyze the mRNA expression characteristics and immune related function of protein disulfide isomerase gene (SpLPDI) from Spodoptera litura, and to provide a foundation for further study of S. litura immune molecular mechanism in response to Nomuraea rileyi infection.【Method】The full-length cDNA of SpLPDI was amplified by specific primers based on the EST from the fatbody suppression subtractive hybridization (SSH) library and SMART RACE cloning technology and further analysed by bioinformatics software. The expression patterns of SpLPDI in different tissues were analyzed by semi-quantitative RT-PCR and quantitative PCR. The mRNA expression of SpLPDI upon N. rileyi challenge was studied by qPCR and the protein characteristics by prokaryotic expression was analyzed.【Result】The full length cDNA of SpLPDI was cloned which was 2 367 bp, including 1 485 bp ORF encoding 494 amino acid proteins. The SpLPDI protein had two thioredoxin domains and an ER retention signal (KDEL) with a signal peptide of 17 amino acid residues containing an a-b-b′-a′ domain organization. The result of multiple alignment indicated that a, a′ domains and ER retention signal were highly conserved. Phylogeny analysis showed that SpLPDI had a closest genetic relationship with Bombyx mori while a distant genetic relationship with Cryptosporidium parvum and Aspergillus niger. Expression profile analysis showed that SpLPDI was expressed in all detected tissues, but highest expressed in hemocytes, followed in fatbodies, and lowest in heads. Induction expression analysis showed that mRNA levels of SpLPDI were significantly increased in fat bodies and midguts upon N. rileyi challenge but the rate of increase in fat bodies higher than that in midgut. SpLPDI was successfully expressed in E. coli, producing a soluble fusion protein. 【Conclusion】The full length cDNA of SpLPDI was cloned from S. litura and changes of PDI mRNA expression were observed in different tissues of S. litura after N. rileyi injection. It suggested that SpLPDI might be related to immune response of S. litura to N. rileyi.

Key words: Spodoptera litura , protein disulfide isomerase , Nomuraea rileyi , SpLPDI