摘要
[背景]椰心叶甲啮小蜂在防治椰心叶甲危害上取得了重要成果,其耐低温胁迫能力将决定该寄生蜂是否能在低温环境下建立种群并发挥控害效能.筛选椰心叶甲啮小蜂低温胁迫下相关响应基因,可为揭示其耐寒性机理提供重要线索.[方法]采用抑制消减杂交技术,分别以经0℃处理24 h后的试虫与未经低温处理的试虫.DNA互为t.st.r与driver,正反向构建椰心叶甲啮小蜂cDNA消减文库,经蓝、白斑筛选,鉴定阳性差异片段,并分别随机挑选正反向文库224和119个阳性克隆进行序列比对分析.[结果]本试验所构建的cDNA消减文库为椰心叶甲啮小蜂低温胁迫下特异表达的cDNA消减文库;经蓝、白斑筛选,分别得到了40和4个高质量ESTs,经PCR鉴定插入片段长度主要分布于200～700 bp之间;序列分析结果表明,差异表达基因的功能主要涉及新陈代谢、细胞结构、信号传导、氨基酸和蛋白质合成的生理过程,并筛选出5类与啮小蜂耐寒性相关的基因,包括Hsp家族蛋白、海藻糖磷酸合酶、谷氨酰胺合成酶、ATP合成酶β亚基和NADH脱氢酶.[结论与意义]该研究对进一步研究椰心叶甲啮小蜂抗寒性状的表达具有一定的参考价值,为揭示啮小蜂抗低温耐寒机制打下一定的理论基础.
[ Background] Tetrastichus brontispae （Ferriere）, is a parasitoid that progressively improved the prevention of damage caused by the Brontispa longissima （Gestro）. Its cold tolerance plays an important role in its population establishment and control efficiency. Thus, exploring the tolerance genes of this pest induced by cold temperatures could provide important clues for the study of cold tolerance mechanism of this parasitoid. [ Method ] In this study, cDNA libraries was constructed using forward and reverse suppression subtractive hybridization （SSH）. Here, cDNA from cold-acchmated insects were used as testers against the cDNA of none-acclimated insects used as drivers and vice versa, cold-acclimated insects as drivers with none-acclimated insects as testers. I- dentification of positive differential fragments by white-blue plague selection and those confirmed clones were randomly selected for sequencing analysis, of which the results were aligned with GenBank database via BLASTX programme. [ Result ] The specific cDNA subtractive library of T. brontispae under low temperature was successfully constructed. The data showed that 40 and 4 high quality ESTs defined from the forward and reverse suppression subtractive libraries were selected of which the length for the inserted se- quences varied between 200 bp and 700 bp. The 44 high quality ESTs of subject were highly homogenous with that of other known species and the main function of these ESTs involved in metabolism, cell structure, signal transduction, amino and protein synthesis. We speculated that there were at least 5 different candidate genes that might be related to the cold tolerance of T. brontispae, including HSP proteins, trehalose-phosphate synthase, glutamine synthetase, ATP synthase subunit beta, and NADH dehydrogenase （ubiquinone）. [ Conclusion and significance] This study is important for future research on the expression of cold hardness of T. Brontispae, and lays some theoretical basis on the molecular mechanism of cold hardnes
出处
《生物安全学报》
2014年第3期210-216,共7页
Journal of biosafety
基金
国家自然科学基金项目(31101496)
留学人员科技活动择优资助项目(2011年度)
国家科技合作专项(KY201301025)
关键词
椰心叶甲啮小蜂
差异表达基因
抑制消减杂交
Tetrastichus brontispae
differentially expressed gene
suppression subtractive hybridization
