茶树被茶尺蠖取食诱导的根部防御反应分子机制及其信号传递特征研究

Foliar herbivory of tea plants by Ectropis oblique is one of the important problems of affecting the quality and safety of production in tea industry, hence, many attentions have always been focused on the induced defence mechanisms activated in response to herbivory on tea plants. In spite of the shoot, the root is also the important plant organ involved in the systemically induced defence to herbivory. However, the molecular mechanism of systemic defence in roots and characteristics of signal transmission from shoot to root induced by pest feeding on shoots has not been studied yet. In this project, the RNA sequencing technology (RNA-Seq), microarray and quantitative real-time PCR or Northern hybridization techniques, combined with bioinformatics will be utilized to screen differentially expressed genes in roots responsive to pest feeding on shoots as well as study their dynamic differential expression profilings in the root of tea plant and the correlations of differential expression profilings among different tea varieties attacked by Ectropis oblique. Furthermore, based on above-mentioned genes, their correlations of differential expression profilings in roots will also be analyzed after JA, SA, ET, auxin treatments and pest feeding on the leaves, respectively. These researches will be helpful for elucidating that the molecular mechanism of systemic defence in roots and characteristics of signal transmission from shoot to root induced by pest feeding on shoots, which could reveal the biological roles of roots with the indction of systemic defence responsive to herbivory-stress on the shoots at the transcriptional level. It will provide and perfect the theory of induced defence mechanisms activated in response to herbivory on tea plants, as well as have an important significance for future molecular breeding of insect-resistant tea varieties, screening the insect-resistant genes and integrated pest control.

茶树叶部虫害是困扰我国茶产业优质、安全生产的重要问题之一。茶树被害虫取食诱导的防御机制研究一直备受关注，而根部为系统防御中的重要组成部分，叶部被害虫取食诱导根部产生的分子防御机制及其涉及的系统信号传递特征尚无相关研究。本项目应用转录组深度测序（RNA-Seq）、基因芯片和qRT-PCR等技术，结合生物信息学手段，获得茶树被茶尺蠖取食诱导的根部差异表达基因；研究差异基因在被取食茶树根中的时空表达模式，及在不同抗性品种间的差别；比较分析JA、SA、ET、生长素和茶尺蠖取食处理茶树叶片后根部差异基因时空表达模式间的相互关系。通过这些研究，将明确茶树被茶尺蠖取食诱导的根部防御反应分子机制和从叶到根的信号传递特征，在转录水平上揭示出茶树根部响应叶部虫害胁迫产生系统诱导防御的生物学意义，从理论上丰富和完善了茶树抗虫的诱导防御机制，同时也为茶树抗虫品种选育、抗性基因发掘和害虫综合防治等方面提供理论依据。

茶树叶部虫害是困扰我国茶产业优质、安全生产的重要问题之一。茶树被害虫取食诱导的防御机制研究一直备受关注，而根部为系统防御中的重要组成部分，叶部被害虫取食诱导根部产生的分子防御机制及其涉及的系统信号传递特征尚无相关研究。本项目应用转录组深度测序（RNA-Seq）技术对被茶尺蠖取食前后的茶树叶和根部进行深度测序，共获得52.7Gb数据，基于生物信息学分析手段，组装获得102,845条unigene，其中根部产生特异的差异表达基因（9,568个DEG）明显多于叶部（5,084个DEG）。分析发现叶和根中受诱导的应答、信号传导网络及启动防御功能相关DEG的数量和类型均有差异，并用qRT-PCR技术验证了信号传导和抗性相关DEG在叶和根中的差异表达模式，从而在转录水平上证实了茶尺蠖取食茶树叶部不仅诱导叶部产生局部防御反应，还诱导根部产生大规模的系统防御反应，但两者防御反应的产生机制和发挥作用的防御性物质有差异。基于LC-MS技术分析叶部和根部激素JA、SA、ABA受诱导后的含量变化，并结合qRT-PCR分析激素信号相关基因的时空表达模式，发现JA和MeSA作为重要的激素信号参与了茶树根部的系统性防御。进一步采用HPLC分析其在山茶科的茶树和油茶中的抗虫性次生代谢物类黄酮和咖啡碱的含量差异，发现茶树叶部儿茶素类（类黄酮）和咖啡碱含量比油茶高1.5~173倍。比较分析茶树和油茶叶部转录组数据，并结合qRT-PCR技术鉴定了DFR、LAR和ANR基因是决定非酯型儿茶素合成的关键基因，而TCS基因是决定咖啡碱合成的最关键基因。此外，比较分析发现茶树中儿茶素、咖啡碱的含量显著高于供试的山茶属其他4个野生近缘种，分析其RAD-Seq数据获得15,444个SNP，结合转录组数据鉴定24 个SNP位点与类黄酮合成相关基因相关，一个SNP位点与抗性基因 LRR receptor-like kinase 2相关。这些研究结果明确了茶树被茶尺蠖取食诱导的叶-根系统防御反应的分子机制差异和系统信号分子的传递特征，进一步从理论上丰富和完善茶树抗虫的防御机制，同时也为茶树抗虫品种选育、抗性基因发掘和害虫综合防治等方面提供理论支持。