舞毒蛾Ldcht10基因序列的克隆及其功能分析
Cloning and functional analysis of the Ldcht10 gene sequence in Lymantria dispar larvae
刘建红** 张 常 赵秋勇 范晓军***
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DOI：10.7679/j.issn.2095-1353.2015.143
作者单位：太原理工大学化学化工学院生物与制药工程系，太原 030024
中文关键词：舞毒蛾，几丁质酶，实时荧光定量PCR，RNA干扰
英文关键词：Lymantria dispar, chitinases, qRT-PCR, RNAi
中文摘要：【目的】 研究舞毒蛾Lymantria dispar几丁质酶基因的时空表达特性及其在蜕皮发育过程中的生物学功能，筛选在舞毒蛾发育过程中致死性的几丁质酶基因，为实现基于RNAi的舞毒蛾有效控制提供重要的基础数据。【方法】 设计简并引物克隆舞毒蛾几丁质酶基因Ldcht10关键序列，通过使用实时荧光定量PCR方法检测该基因在舞毒蛾不同龄期与组织中的相对表达量，选取部分序列的双链RNA（dsRNA）研究该基因功能。【结果】 本研究克隆了长度为2 057 bp的舞毒蛾几丁质酶基因Ldcht10序列；各组织与不同龄期RT-PCR结果表明Ldcht10的时空表达特性存在明显差异，Ldcht10在各个龄期均表达活跃且在前肠与后肠中的表达水平最高；Ldcht10的RNA干扰试验表明：注射Ldcht10 dsRNA后Ldcht10的表达受到极大抑制，该基因被沉默后24.3%的舞毒蛾幼虫因无法完成蜕皮或化蛹而死亡。【结论】 舞毒蛾中几丁质酶基因Ldcht10在各个龄期与组织中的表达存在差异，且在舞毒蛾蜕皮过程中具有十分重要的生物学功能，该基因被沉默后部分舞毒蛾因无法完成蜕皮而导致死亡。
英文摘要： [Objectives] In order to provide a basis for effective pest control based on RNAi, the temporal and spatial expression, and the biological functions of the chitinase family of genes in Lymantria dispar were investigated and chitinase genes causing death during development were screened. [Methods] Degenerate primers were designed to clone the critical sequence of LdCht10 and the real-time quantitative PCR method was used to detect the relative expression level of LdCht10 in different instars and tissues of Lymantria dispar larvae. A fragment of the cloned sequence was chosen to study gene function using the RNA interference method. [Results] A critical sequence of LdCht10 of 2 057 bp in length was successfully cloned. The RT-PCR results show that the temporal and spatial expression patterns of LdCht10 were significantly different, and that the LdCht10 gene was expressed in all instars and tissues, with the highest expression occurring in the foregut and hindgut. RNA interference showed that 24.3% of test larvae died as a result of not completing molting and pupation after the gene was silenced. [Conclusion] The temporal and spatial expression profiles of LdCht10 were distinct in different instars and tissues. Based on the RNAi results, LdCht10 could play an important role in the molting process of L. dispar, and gene silencing can block ecdysis causing death in some larvae.