荔枝miRNA的鉴定及其在饥饿胁迫诱导落果过程中的作用机制

The relationship between microRNA and organ shedding has not so far been documented both in model plants and cultivated crops. The organ shedding is associated with plant abiotic stress and senescence that is regarded as being involved in miRNA-mediated regulation, and carbohydrates and endogenous hormones are the two key factors controlling fruit abscission. So we speculate that miRNA might be as key regulators in the process of organ abscission by affecting IAA, ethylene metabolism and signal transduction. In this project, the litchi which is originated and largest cultivated in south China is selected and the experiment of fruit abscission induced by carbohydrate starvation-stressed through defoliation plus girdling treatment will be conducted as entry point. Twenty-one small RNA libraries and one degradome libraries will be constructed，and high-throughput sequencing and degradome analysis are employed to identify known and novel miRNAs and their targets in the litchi; different expressed miRNA profiling among samples (0hr,6hr,12hr,1d and 2d) with and without defoliation plus girdling treatment will be analyzed by informatics, miRNAs and their targets response to starvation will be screened and miRNA related to fruitlet abscission will also be discovered , which will be validated by qRT-PCR and analyzed in relation to the dynamic changes of fruit drop and endogenous hormones (IAA and ethylene in fruit, ACC in abscission zone, IAA in proximal and distal abscission zones). Finally, the selected key miRNA and its targets related to fruit abscission will be tested using another experimental system like spraying IAA and ethylene. These results will provide valuable information to understand the mechanism of miRNA-mediated signaling involved in fruit abscission induced by starvation stress in litchi. Therefore, this proposal aims not only at fruit abscission which is a key scientific issue affecting yield in fruit production, but also explores in the relationship between miRNA and organ shedding which is the forefront of academic disciplines.

在模式植物和栽培作物上均未见miRNA与器官脱落关系的文献报导。器官脱落是一个与胁迫和衰老相关的生物学现象，基于miRNA参与植物胁迫和衰老过程调控以及碳素营养和内源激素是控制果实脱落的两大关键原因的认识，推测miRNA可能通过调控IAA、乙烯代谢和信号转导参与果实的脱落。本项目以荔枝为试材，通过碳水化合物饥饿胁迫诱导果实脱落为切入点，采用小RNA和降解组测序技术和生物信息分析等方法，构建荔枝物种和响应饥饿胁迫诱导的 miRNA及其靶基因两个数据库；筛选参与调控IAA、乙烯代谢和信号转导相关联的 miRNA及靶基因，并分析它们在落果过程中动态表达变化及其与落果率、果实中 IAA 和乙烯、近轴端和远轴端中离区 IAA和ACC含量等指标关系，进而确定与落果相关的候选miRNA及靶基因；设计外喷IAA和乙烯利的试验对候选miRNA进行验证，最终找出与果实脱落相关的miRNA并揭示其作用机制。

本研究通过小RNA测序，降解组测序和转录组测序，结合生物信息学分析以及分子生物学实验，对荔枝miRNA和phasiRNA的调控通路进行了全基因组鉴定。此外，设计 “环剥+去叶” 处理营造碳水化合物胁迫诱导荔枝幼果脱落实验，获得了显著性生物和生理效果，筛选得到与落果过程显著相关的 miRNA及其靶基因。本研究结果为荔枝物种的小 RNA 分子机制研究，特别是荔枝幼果脱落的转录后水平分子调控机制研究提供重要参考价值。主要研究结果如下：.1．分别获得已知和新miRNA 家族42个和40个，分别包含239个和51个成员。其中32个已知miRNA家族（214个成员）和13个新miRNA家族（20个成员）获得395个靶基因验证。.2．获得515个PHAS位点，具有编码能力有420 个，非编码位点有95个。在进行phasiRNA的介导子miRNA鉴定发现6条植物典型miRNA-phasiRNA途径和1条荔枝特异miRNA-phasiRNA途径（miRN1-NB-LRR）。.3. 发现荔枝中miR482/2118家族功能呈现多样化，可以耦合可变剪切/选择性多聚腺苷化产生不同的phasiRNA，进而扩大了下游phasiRNA所调控靶基因的范围。据此，提出了miRNA耦合可变剪切/选择性多聚腺苷化产生不同phasiRNA的调控机理模型。.4．碳水化合物胁迫显著促进了花后 20d 荔枝的幼果脱落，其生理原因是通过改变幼果中内源 IAA、ABA 和乙烯含量及其之间的平衡来实现的。.5. 分别筛选到碳水化合物胁迫诱导荔枝落果过程表达差异显著miRNA 45个，其中与饥饿胁迫诱导荔枝落果相关的差异miRNA 22个。通过对这些miRNA 及靶基因表达模式进行了相关性分析发现，呈 “Mix”（混合相关）的 miRNA/靶基因对最多，单独符合 “FN”（整个过程负相关）和 “FP”（整个过程正相关）的 miRNA/靶基因对只占一小部分。说明碳水化合物胁迫诱导荔枝落果过程中 miRNA 对靶基因的调控是十分复杂的。.7. 筛选和鉴定出10条可能参与了碳水化合物胁迫诱导荔枝落果的 miRNA 调控通路，其中miR160-ARF10/16/17是饥饿胁迫诱导荔枝落果过程中 miRNA参与的重要代谢途径及调控网络，并据此提出了碳水化合物胁迫诱导荔枝落果过程中可能的miRNA的作用机制。