栝楼性别分化中功能microRNAs的鉴定及其作用机制的初步研究

Trichosanthes (Trichosanthes kirilowii Maxim.) is an important type of Chinese medicine crops, different sex plants have different application and economic value. The preliminary study found that the key sex differentiation candidate genes were regulated by microRNAs. In order to further explore the mechanism of sex differentiation and reveal the mechanism of microRNAs in sex differentiation of trichosanthes, this study will construct sRNA libraries of male and female flowers and leaves based on the previous analysis of mRNA, identify key microRNAs interrelated to sex differentiation and to verifiy its target gene function. In the experiment, the flowers and leaves of male and female plants will be taken as samples and the samples will be collected at 3 periods: before, during and after flower bud differentiation, respectively. The main details are as follows: At first, total RNA of flowers and leaves will be extracted and then the sRNA transcription libraries of flowers and the leaves from different stage were established by Illumina sequencing. we will search for microRNAs associated with sex differentiation. Secondly, combined with the results of mRNA analysis, the RLM-RACE technology will be used to verify the key microRNAs target genes. Thirdly, the over expression vector and RNAi vector of the target genes will be constructed, then transformed to Arabidopsis and Trichosanthes callus, so as to verify the function of the target genes. The study will unravel the underling molecular mechanism of sex determination of Trichosanthes and also provide a theoretical basis for gender regulation and seeds seedling of Trichosanthes kirilowii Maxim.

栝楼是我国重要的中药类经济作物，不同性别植株具有不同的应用价值。申请者前期研究发现栝楼性别分化关键候选基因受microRNAs调控。为进一步揭示microRNA在栝楼性别分化中的作用机制，本项目拟在前期mRNA研究的基础上，构建栝楼雌雄株花及叶的sRNA文库，结合转录组鉴定植物性别分化关键microRNAs并对其靶基因功能进行验证。试验以雌雄株花和叶为材料，分别于花芽分化前、中、后3个时期取样。研究：①利用Illumina-seq测序，分别构建栝楼雌雄株花和叶的microRNA文库，寻找关联性别分化的microRNAs；②结合生物信息学及转录组数据库（前期已构建），筛选microRNAs可能的靶基因并通过RLM-RACE验证靶基因；③构建靶基因过表达载体和RNAi载体，转拟南芥和栝楼愈伤组织，初步验证其功能。本项目为揭示栝楼性别分化机制，从而进行栝楼定性育苗及植物性别调提供理论依据。

栝楼是我国重要的中药类经济作物，不同性别植株具有不同的应用价值。前期研究发现栝楼性别分化关键候选基因受microRNA调控，为进一步探究栝楼性别分化机制，揭示microRNA在栝楼性别分化中的作用机制，开展了如下研究：.1.再次对栝楼雌雄花芽进行转录组测序，测序数据量10G/样。最终筛选到11个性别差异候选基因：MYB80，MYB108，CER1，CBL9，ABCB19，SERK1，HSP81-3，ACS9，SEP3，AUX1和YUC6。本次测序筛选到了前期未筛到的候选基因，如ACS9，ACS2，这些候选基因是葫芦科植物性别分化关键决定基因。这为后期确定其分化机制提供了新思路和方向。.2.利用Illumina-seq测序，构建栝楼雌雄株花和叶的microRNA文库，寻找关联性别分化的microRNAs；通过mRNA与sRNA测序联合分析，发现22个性别分化关键miRNA负调控75个靶基因；32个性别分化关键miRNA正调控88个靶基因；KEGG分析也证实这些关键miRNA极有可能参与了性别分化过程。.3.结合生物信息学及转录组数据库，筛选microRNAs可能的靶基因并通过RLM-RACE验证靶基因：构建了miRNA和靶基因的调控网络，对关键miRNA和靶基因进行了分析，重点解析了miR166、miR172、miR396、miR169、miR157、miR156之间复杂的调控关系。特别关注miR156和miR396，采用RLM-RACE 证实了miR156对靶基因tkSPL13B和tkSPL18的直接调控作用；miR396对靶基因tkU2AF65B的直接调控作用。.4.构建过表达载体，转拟南芥和栝楼愈伤组织，初步验证功能。分别构建miR156、miR396、靶基因tkSPL13B、tkSPL18、tkU2AF65B过表达载体；拟南芥表型观察发现过表达miR396的2株转基因拟南芥与野生型相比，叶长、叶宽减小，开花时间延迟；过表达miR156的3株转基因拟南芥与野生型相比植株矮小，且雌蕊缩短，这证实miR156确实参与了性别分化过程；靶基因功能验证还没有得到最后的栝楼表型植株。.本项目从microRNA角度揭示栝楼性别分化机制，并对关键microRNA及靶基因进行功能验证，证实miR156、miR396确实参与了植物性别调控，但其调控机理还未清晰，还需进一步研究