野生番茄响应昆虫唾液中FAC诱导物的遗传基础研究

When insects feed on plants, most plants are able to deploy specific defense against insect attack by perceiving some small molecules and proteins, such as the elicitors FAC (fatty acid-amino acid conjugate) in Lepidoptera oral secretions. Although the FAC-induced defense responses have been intensively studied, yet little is known about how plants perceive FAC. Previously we found that the cultivated tomato Solanum lycopersicum does not perceive FAC in insect oral secretions, while the wild tomato S. pennelli is able to perceive FAC specially. One QTL involved in perceiving to FAC was mapped on chromosome 3 using a F2 population derived from the cross between wild tomato and cultivated tomato. Here we name the QTL as RTF (response to FAC). RTF can induce the synthesis of jasmonic acid in plants, and is a dominant gene. In this project, we will plant a large F2 population and select extreme recessive plants with insensitivity to FAC and use the next-generation sequencing technology to assist fine-mapping of RTF. In order to confirm the identity of RTF, wild tomato alleles of the candidate genes will be introduced into cultivated tomato by Agrobacterium-mediated transformation. We will discover its spatial and temporal expression patterns, identify its up-stream and down-stream genes, and unveil the mechanism underlying how this QTL is involved in perceiving herbivore-derived FAC signals using multiple approaches. Our goal is to establish the gene regulatory network in plant perception of herbivore-derived signals, which involves RTF, and to comprehensively understand how plants perceive herbivore-derived signals. Cloning and characterization of RTF will facilitate our understanding of plant-insect interactions.

当昆虫取食植物时，多数植物能够通过识别昆虫唾液中的某些小分子物质或蛋白产生特异的抗虫响应，比如鳞翅目昆虫唾液中的FAC（fatty acid-amino acid conjugate）类诱导物。虽然对FAC诱导的抗虫响应已做了较深入的研究，但是植物如何识别FAC还很不清楚。我们发现栽培番茄不能对昆虫唾液中的FAC成分产生响应，而野生番茄能够特异地响应FAC成分。利用野生番茄/栽培番茄衍生的F2群体，我们在第3染色体定位到一个能响应昆虫唾液诱导物FAC的QTL，我们将它命名为RTF。本申请将在此基础上，利用F2大群体的极端隐性单株，采用图位克隆的方法，同时结合第二代测序技术，将RTF精细定位到30kb。同时利用转基因完成功能互补验证，确证分离该基因。利用分子生物学技术，鉴定上下游调控基因，揭示RTF响应昆虫唾液分泌物FAC的调控机理,将对植物与昆虫之间相互关系的研究具有重要意义。

植物与昆虫共进化过程中，植物针对昆虫取食进化出一套防御机制。植物能够识别昆虫唾液中的某些小分子物质或蛋白产生特异的抗虫响应，比如鳞翅目昆虫唾液中的FAC（fatty acid-amino acid conjugate）类诱导物。我们的研究表明：当昆虫空腔分泌物FAC处理野生番茄（Solanum pennelli）后，其体内的茉莉酸（JA）、茉莉酸异亮氨酸（JA-ILE）以及抗虫次生代谢物蛋白酶抑制剂（PI）的含量显著被诱导；而栽培番茄（Solanum lycopersicum）被FAC处理后，其体内的JA、JA-ILE和PI的含量变化不大，说明野生番茄能够识别FAC，而栽培番茄不能识别FAC。通过昆虫生物测定，生长在野生番茄上的斜纹夜蛾（Spodoptera litura）体重和生长速率都比生长在栽培番茄上的低。本研究采用图为克隆的方法，分离克隆野生番茄识别FAC的基因。首先，利用野生番茄与栽培番茄衍生的192株F2分离群体，结合SSR分子标记技术，我们将目标基因初步定位在第三染色体上，在此基础上我们利用约2100株F2群体，将目标区间精细定位到1.5Mb范围内。利用二代测序技术，结合BSA（Bulked Segregant Analysis）的分析方法，进步精细定位目标基因，最终将目标片段缩小较小的范围，在此区间有87个候选基因。通过转录组分析87个候选基因是否有转录水平的差异，结果表明，有13个基因在野生番茄中能够被FAC诱导表达，其中Sopen03g022530基因编码一个LLR受体蛋白，而且被诱导上调表达约8倍，因此该基因作为候选基因。我们构建了该基因的RNAi载体和正义转化载体，分别转化野生番茄和栽培番茄。

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