响应胶孢炭疽菌侵染的茶树microRNA及其靶基因的网络调控机制

Anthracnose disease is one of the most severe diseases caused by Colletotrichum gloeosporioides in tea plant. Understanding the molecular response mechanism is the basic premise to deal with anthracnose resistance of tea plant. However, the functional regulation of microRNAs in tea plant during C. gloeosporioids infection remains unclear. In this study, we have sequenced microRNAs and mRNA transcripts using high-throughput approach. Degradome analysis has been also conducted to identify target genes and site of microRNAs in response to infection of C. gloeosporioides. Besides, metabolites in response to C. gloeosporioides will be identified by using GC-MS and LC-MS method. We intend to conjointly analyze the differential metabolites and transcripts to identify metabolites related to microRNA target genes. Antimicrobial activity of the metabolites identified from tea leaves will be detected by using bio-autographic method. Artificial microRNAs will be acquired via overlapping PCR technology. In order to verify the splicing effect of pre-microRNAs and mature microRNAs on target genes, microRNAs, artificial microRNAs and target genes will be co-transformed or transient-expressed in tobacco leaves. This study will considerably broaden the investigation of the tea genome encoded miRNA function during stress responses and might be applied as a new RNA interference based crop improvement strategy in tea. Our results will aid to overcome the yield and tea quality problems associated with this important economic crop.

胶孢炭疽菌是引起茶树炭疽病的主要病原菌，了解茶树炭疽病防御机制是应对茶树炭疽病的基础，而茶树miRNAs及其靶基因响应胶孢炭疽菌侵染的机制仍不清楚。申请人利用高通量测序获得响应茶树炭疽病菌侵染的基因和miRNAs，结合降解组测序确定miRNAs的靶基因，分析miRNAs的靶向作用位点。在此基础上，利用GC-MS和LC-MS方法测定响应茶树炭疽病菌侵染的代谢物，并与转录组联合分析，确定与靶基因相关的茶树抗炭疽病代谢物，用生物自显影法检测其抗菌活性。用overlapping PCR技术获得amiRNAs序列，分别将候选miRNAs、amiRNAs及靶基因共转化烟草，以验证miRNAs前体和成熟体对靶基因的剪切作用。这将在分子水平上了解响应胶孢炭疽菌侵染的茶树miRNAs及其靶基因的网络调控机制，发展新的基于RNA操作的降低杀菌剂使用、提高产量和品质的茶叶品种改良策略和技术提供新的科学依据。

炭疽菌（Colletotrichum spp.）是一类常见的植物病原性真菌，对全球农业生产造成严重影响。茶树炭疽病（Anthracnose）是我国茶园中受炭疽菌侵染引起的一种主要病害，全国约有30~60%的茶园受到炭疽病的影响，茶叶生产受损严重。本项目以龙井43和中茶108为研究材料，通过施加外源咖啡因以及壳聚糖，探索茶树中C. gloeosporioides响应性miRNAs和防御响应性miRNAs的作用机制。研究发现外源咖啡因诱导的miRNA介导的靶基因积极参与JA介导的通路调控，改变了LJ43对抗性环境的性质，也增强了ZC108对C. gloeosporioides的抗性性质。采用UPLC–ESI-MS/MS技术探究在胶孢炭疽菌接种(CgI)的条件下，感病品种(LJ43)和抗病品种(ZC108)中外源壳聚糖对防御相关的次级代谢产物积累的影响。结果发现在LJ43中施用壳聚糖，通过降低活性氧的积累，显著诱导了抵抗胶孢炭疽菌侵染的水平，提高了活性氧清除酶（POD和CAT）的活性，并且增加了防御相关基因（PR1和PPO）的表达。外源壳聚糖在ZC108炭疽菌接种叶上可以提高与茶叶品质相关代谢产物（儿茶素和咖啡碱）的含量。CgI+CHT处理还显著诱导了与木质素、咖啡碱、儿茶素和花青素生物合成有关的关键基因和次级代谢产物的增加。这些结果为茶树抗C. gloeosporioides提供了一个理论基础。