黄瓜miRNAs在根结线虫寄生与巨型细胞发育中的功能解析

miRNAs play an important role and have been shown to be involved in the regulation of plant- nematode interactions, yet the underlying role of cucumber miRNAs in defense responses to root-knot nematodes remains unclear. The present study first intends to screen and identify miRNAs and target genes related to the resistance to root-knot nematodes through high-throughput sequencing and bioinformatics analysis, and the wild cucumber with high resistance to the pest will be selected as plant material to generate miRNAs and degradation libraries derived from this incompatible interactions. This will be followed by the generation of an integrated network regulated by miRNAs to target genes, through analysis of miRNAs and target genes expression and its regulatory mode. Afterwards, the mode of target genes regulated by miRNAs will be detected through tobacco co-transformation of these two types of genes, and the location of target genes in cells and its transactivation function will be documented using the onion epidermal cell transformation and yeast transactivation respectively, on the basis of the cloning for miRNAs and target genes candidates. Ultimately, miRNAs overexpression and target genes silencing will be conducted to investigate the roles of cucumber miRNAs involved in root-knot nematodes parasitism and its giant cells development. The expected results obtained from the study will provide the foundation to improve molecular mechanisms in an incompatible interaction between cucumber and root-knot nematodes, and the related outcomes obtained here will provide a technical support for cucumber nematode resistance improvement through genetic manipulation of miRNAs, and this should have potential application in controlling nematodes in the future.

miRNAs在植物-线虫互作中具有重要的调控作用，而有关黄瓜miRNAs在根结线虫防卫反应中的作用目前尚不清楚。本项目拟以高抗根结线虫的野生黄瓜为试材，构建黄瓜与根结线虫非亲和互作的miRNAs和降解组文库，通过高通量测序与生物信息学分析，筛选鉴定与根结线虫抗性密切相关的miRNAs及其靶基因；通过miRNAs与靶基因表达和调控模式分析，创建黄瓜miRNAs与靶基因的调控网络；克隆候选miRNAs及靶基因，利用烟草共转化检测miRNAs与靶基因的调控模式，采用洋葱表皮细胞转化与酵母转录激活确定靶基因在细胞中的定位与转录活性；通过miRNAs过表达与靶基因沉默分析，解析黄瓜miRNAs及靶基因在根结线虫寄生与巨型细胞发育中的作用。该项研究将为完善黄瓜与根结线虫非亲和互作的分子机理奠定基础，相关成果可为通过miRNAs的遗传操作来改良黄瓜的根结线虫抗性提供技术支撑，具有潜在的应用前景。

miRNAs在植物-线虫互作中发挥调控作用，有关黄瓜miRNAs在根结线虫防卫反应中的作用未见报道。本项目通过角瓜与南方根结线虫非亲和互作的sRNA测序，确定了miRNAs占sRNA的0.3～0.5%，包含565个已知miRNAs，归属75个miRNAs家族。筛选获得10条显著差异表达的角瓜miRNAs，这些miRNAs与角瓜-南方根结线虫非亲和互作密切相关。构建了miRNAs与靶基因的调控网络，包含10条miRNAs与61个靶基因，一条miRNA可调控多个靶基因，同一miRNA的靶基因同源性较高，miRNAs靶基因的功能涉及转录激活、参与植物生长发育和调控以及编码信号传导和细胞代谢相关蛋白等。从角瓜中克隆获得5条完整的miR396前体序列，5条前体序列剪切产生3种不同的成熟序列。克隆获得3条角瓜GRF基因的完整ORF序列，GRF在进化上较为保守，经RLM-5´RACE验证miR396对3条GRF的mRNA均进行单一剪切，GRF为角瓜miR396的靶基因。通过亚细胞定位研究，将miR396a靶标GRF11、GRF42、GRF92定位于细胞核中。构建了单基因（GRF92）与双基因（miR396a/GRF92）过表达载体，通过农杆菌侵染花序法进行了拟南芥转化，经Kan抗性筛选获得了转基因拟南芥植株。采用qPCR对转基因植株中miR396及其靶标GRF92进行表达分析，miR396在转基因植株中表达量显著高于野生型，而转基因植株中GRF92呈下调表达，证实了miR396靶向调控GRF92的表达。过表达miR396抑制了根系中GRF92表达，根结线虫取食与发育可能受到抑制，根结线虫数目将会减少。这些结果为完善黄瓜与根结线虫非亲和互作的分子机理奠定了基础，有关miRNAs的遗传操作为改良黄瓜的根结线虫抗性提供了技术支撑。