莲草直胸跳甲寄主专一性的分子机制及相关基因筛选

Alligatorweed, Alternanthera philoxeroides (Mart.) Griseb (Amaranthaceae) is an amphibious perennial plant. It has been recognized as a prominently problematic weed all over the world and listed as one of 9 seriously invasive species in China. Biological control is a crucial tool in the battle against biological invasions. Agasicles hygrophila (Coleoptera: Chrysomelidae) was introduced into China as a biological control agent of invasive weed A. philoxeroides. It has been proven to be efficient in the control of this aquatic invader in the southern parts of China and all over the world. This project will study the effects of different test plant on enzyme activities and expression responses of single gene and illustrate the mechanism of host specificity in Agasicles hygrophila. First, various digestive and metabolic enzyme activities will be tested in feeding different host plants. Second, we will obtain the transcriptome information of Agasicles hygrophila by RNA-seq and compare gene expression difference between feeding target and non-target plant of Agasicles hygrophila by digital gene expression (DGE) analysis. Then, we will choose ten differentially expressed unigenes to detect their expression between feeding target and non-target plant of Agasicles hygrophila using quantitative RT-PCR. Our research will focus the roles of differentially expressed unigenes in host specificity by using RNA interference (RNAi). By these means, we will illustrate the mechanism of host specificity in Agasicles hygrophila in transcript level and biochemical level. The foundation of transcriptome database of Agasicles hygrophila will provide important information platform for gene expression and functional genomics research and provide a lot of information reference for follow-up study.

喜旱莲子草是中国首批9种重要生物入侵植物之一，生物防治是控制入侵生物的一种重要手段。莲草直胸跳甲被公认为是生物防治喜旱莲子草最有效的专食性天敌。本项目以莲草直胸跳甲为研究对象，研究取食不同寄主植物对其体内酶活性及基因表达的影响，进而阐明莲草直胸跳甲寄主专一性的分子机制。主要研究内容如下：1）研究其在取食不同寄主植物后体内各种消化酶和代谢酶的酶活性变化；2）通过转录组测序技术获得莲草直胸跳甲的转录组信息，并通过数字基因表达谱的测序分析取食不同寄主植物后的基因表达差异。3）选择10个差异表达的unigenes，用real-time PCR验证在取食不同寄主植物后莲草直胸跳甲中的表达，用RNA干扰技术对差异表达基因的功能进行检测，明确差异表达基因在莲草直胸跳甲寄主专一性中的功能和作用机制。同时转录组数据库的建立还可为该物种基因表达、功能基因组研究提供重要的信息公共平台，为后续研究提供信息参考。

莲草直胸跳甲Agasicles hygrophila Selman & Vogts，是恶性杂草喜旱莲子草Alternanthera philoxeroides (Mart.) Griseb的专食性天敌，是目前防治喜旱莲子草最有效的生物防治措施，目前已在美国、澳大利亚、中国等国家引进并推广。莲草直胸跳甲的寄主专一性分子机理是怎样的，相关的研究报道很少。为了阐明莲草直胸跳甲寄主专一性的分子机制，本研究针对代谢酶和消化酶开展了一系列研究：（1）利用生化分析检测了四种植物营养物质、次生物质及含水量的差异，并获得莲草直胸跳甲取食不同植物及饥饿后的酶活性变化数据。（2）利用第二代高通量测序技术对莲草直胸跳甲的转录组进行测序，测序质量较高，组装得到46,151 条unigene，并对搜索获得的代谢酶、消化酶和气味结合蛋白基因进行聚类分析，为研究莲草直胸跳甲寄主专一性的分子机制提供了序列信息。(3)利用RNA-Seq技术对取食喜旱莲子草、莙荙及饥饿的莲草直胸跳甲进行数字基因表达谱分析，获得差异表达基因，可能这些基因在莲草直胸跳甲选择寄主取食过程中承担重要的任务。(4)对转录组数据库进行搜索，获得内参基因并利用geNorm和NormFinder软件综合分析了10个候选内参基因在莲草直胸跳甲取食不同植物及饥饿条件下mRNA的表达稳定性，筛选出了适用于不同条件下莲草直胸跳甲基因表达分析的内参基因。（5）利用荧光定量PCR方法检测差异基因表达，并对部分基因进行了全长克隆，从分子水平论述了代谢酶和消化酶的表达变化与取食不同植物之间的关系，为阐明莲草直胸跳甲取食专一性提供理论依据。