叶瘿蚊虫害胁迫下荔枝的诱导抗虫性分子机制

Plants can be triggered to produce chemicals to protect themselves from damage after insect herbivory. Using such spontaneous immunity mechanism in plants to control pests is a future ideal strategy. Litchiomyia chinensis is one of the major pests of litchi. Different resistance to this pest among litchi genotypes has been reported, for instance, Litchi chinensis cv. Sanyuehong is strongly resistant to this insect, while Feizixiao and Huaizhi are very susceptive. To elucidate the mechanism of such resistance variation among lithic geneotypes is an interesting topic. We propose a study on the molecular mechanism of induced anti-herbivore defense based on the global gene expression patterns and genome methylations of the three litchi genotypes in response to Litchiomyia chinensis feeding, using cDNA-AFLP and MS-AFLP methods. Polymorphic sites are determined by comparing their differential expression profiles and genome methylation patterns with those induced by herbivory, respectively. Dominant differential fragments are then selected to conduct cloning and sequencing. Functions of these sequences are determined through GeneBank, and insect-induced defense related genes are screened. Induced-defense chemicals and enzyme activity are also determined. The relativity between gene differential expression and DNA differential methylation are also interpreted. Based on all these data sets we can elucidate the molecular mechanism of litchi induced-resistance variation. Linking studies at the level of transcriptomic and heritable phenotypic modification changes with studies on metabolite production and expression of different levels of resistance will provide new insight not only into the mechanisms underlying variation in ecological interactions, but also into the litchi breeding and the pest control.

利用新一代 mRNA和DNA指纹图谱技术（cDNA-AFLP与MS-AFLP），以抗虫性不同的荔枝品种（三月红、妃子笑和淮枝）为材料，大规模比较研究在叶瘿蚊虫害胁迫下的荔枝诱导抗虫性的分子机制。分别在基因表达水平和表观遗传学（DNA甲基化）水平上检测不同抗虫性品种间的遗传变化模式，对相关差异性条带进行筛选、克隆、测序、功能比对，结合化学成分、生理生化数据，揭示差异表达基因与DNA甲基化调控之间的相关性，从基因组水平全面、系统探讨荔枝品种间抗虫性分化的遗传基础和基因表达调控的模式与机制，旨在为抗性品种选育、协调利用作物的自身抗性和诱导抗性进行荔枝虫害的综合防治提供理论基础。

采用生理生化测试及cDNA-AFLP、MS-AFLP和qRT-PCR等分子生物学技术研究了虫害胁迫下的荔枝抗虫性分子机制，旨在为抗性品种选育、协调利用作物的自身抗性和诱导抗性进行荔枝虫害的综合防治提供理论基础。获得了荔枝品种淮枝，妃子笑，三叶红及桂味与虫害诱导机制相关的酶（POD，SOD，PPO，CAT）活性变化数据，在分子水平上得到多个差异基因表达片段（TDF），通过回收克隆测序获得这些片段的序列。对获得的 TDF 序列进行同源性比对分析表明，28个基因分属解毒基因、糖原激酶合成蛋白基因、信号传导蛋白、家族蛋白基因、功能未知基因等。在此基础上设计引物进行qRT-PCR分析荔枝不同品种中与诱导抗性相关的基因表达量的变化，结果表明被害虫取食24h和48h后，不同品种的POD3、萜类吲哚生物碱合成、锌离子结合蛋白、FMD-3色素蛋白、ATP合成基因的表达有一定的诱导上调作用，但48h后POD3、萜类吲哚生物碱、醛脱氢酶表达减弱。值得注意的是，抗性品种三月红的差异基因表达不明显。MS-AFLP分析结果初步表明虫害并没有诱导稳定且特异的植物基因组DNA 胞嘧啶位点的甲基化模式变化，但不同品种间存在表观遗传差异。在此基础上进一步利用cDNA-AFLP技术结合及克隆测序、qRT-PCR研究了外源信号物质茉莉酸甲酯诱导下荔枝桂味的抗性分子机制，对比研究虫害与信号物质茉莉酸甲酯诱导下的荔枝抗性差异表达基因的异同。与虫害胁迫相比，虫害取食和外源茉莉酸甲酯处理荔枝后，荔枝的萜类吲哚生物碱合成及FMD-3色素蛋白基因的表达效果存在差异性，但两者的锌离子结合蛋白、假定蛋白、醛脱氢酶以及POD3基因都有相似的表达效果。除此以外我们还开展了包括荔枝、海南草海桐、互花米草等物种在内的多种热带代表性植物的分子系统学与遗传多样性研究，为这些植物的种质资源的保护和利用提供分子证据。