miR1030/160调控丛枝菌根诱导番茄抗根结线虫的分子机理研究

Arbuscular mycorrhizal fungi (AMF) can induce systemic resistance in plants against root-knot nematodes (RKN). However, the mechanisms of such systemic signaling and its regulation are largely unknown. Plant miRNAs function as systemic signals in response to biotic stress. Our previous studies have revealed that AMF-induced resistance against RKN is closely related to the expression of miRNA. 92 known miRNAs and 63 new miRNAs were screened based on Solexa sequencing. AMF affected the expression of miR1030/160 in tomato infected with RKN. Moreover, overexpression of miR1030/160 suppressed and enhanced tomato tolerance to RKN, respectively. Based on the previous study, the project will identify the target mRNAs of miRNAs on a whole genome scale by using high throughput degradome sequencing approach, and then further validation by RLM 5’-race. The involvement of potential signal pathways in the regulation of miR1030/160-targeted genes will be analyzed. Then the changes in miR1030/160, their target mRNAs and RKN resistance response to altered signals will be determined using chemical methods to Change the level of important signal molecules. Finally, the changes in downstream signaling cascades and key gene expressions as influenced by miR1030/160 overexpression and target gene silencing in plants will be analyzed to verify the roles of miRNA involved in feedback communication of AMF against RKN infection. Thus, the project aims to elucidating the role of miR1030/160 signal in AMF-induced resistance to RKN in tomato. Eventually, this study will provide a new useful tool for the regulation of RKN resistance in vegetable production.

丛枝菌根(AMF)可有效提高植物对根结线虫的抗性，但调控机制尚不清楚。miRNA在植物逆境胁迫应答中起重要的调节作用。前期研究发现，AMF诱导的番茄根结线虫抗性与miRNA差异表达密切相关。接种线虫后AMF可调控miR1030/160及其预测靶基因的表达，过表达miR1030和miR160分别降低和提高了番茄对线虫抗性。在此基础上，首先利用降解组测序获得靶基因，并利用5’-RACE 对所鉴定的靶基因验证。分析miR1030/160及其靶基因可能参与的信号途径，通过化学手段改变激素等重要信号分子水平，分析线虫抗性进而明确其上游信号分子。然后利用转基因和VIGS技术，分析过表达miR1030/160和沉默相应靶基因对关键信号分子及基因表达的影响，揭示其下游信号传导途径。研究可望阐明miR1030/160在AMF诱导的线虫抗性中的调控机制，为根结线虫的抗性调控提供理论与实践基础。

丛枝菌根可有效提高植物对根结线虫的抗性。项目研究了miR1030/160在AMF诱导的线虫抗性中的调控作用。接种AMF后，番茄根系的根结数较对照显著降低，番茄根系的根结数最少，根结指数显著降低。AMF处理对二龄幼虫有较好的控制效果，防治效果达74.63%，显著高于对照。构建番茄小RNA文库，利用Solexa测序技术对小RNA文库进行测序分析。共筛选出92个已知miRNA，预测到63个新miRNA。与对照相比，枯萎病侵染处理的番茄根系中有62个miRNA表达发生显著变化。通过靶基因功能对参与线虫胁迫响应的保守miRNA家族的miRNA功能进行了初步分析。利用qRT-PCR对筛选出的9个差异表达已知miRNA的表达结果进行测序结果验证。转基因结果表明，过表达miR1030和miR160分别降低和提高了番茄的线虫抗性。构建了抑制miR160a-5p1功能的植物转化载体。通过农杆菌介导法遗传转化获得转基因植株。在过量表达miR160a-5p1的转基因番茄植株中，靶基因ARF16表达量显著下降。接种根结线虫后，过表达miR160a-5p1的降低了AMF诱导的番茄线虫抗性，根结数、H2O2、O2-、NO、硫醇RSH和MDA增加，JA合成和PAL、PPO等次生代谢相关基因表达量下调。而过表达靶基因ARF16则显著提高了AMF诱导的番茄植株的抗性，酚类、类黄酮、木质素、纤维素等次生代谢物的积累明显增加。过表达靶基因ARF16植株的防御相关酶活性和相关基因（PAL、C4H、4CL、CAD、LPO、CCOMT、Tpx1和G6PDH）的表达显著高于未转化RKN处理。与接种RKN的未转化番茄根系相比，过表达靶基因ARF16植株接种RKN可显著提高AMF诱导的致病相关蛋白β-1, 3-葡聚糖酶、几丁质酶、蛋白酶和淀粉酶的活性。这些结果表明，miR160a-5p1及其靶基因ARF16通过调控番茄根系次生代谢和防御相关酶的活性和转录，在诱导番茄抗线虫病中发挥了积极作用。项目执行期间，发表论文10篇，获河南省科技进步奖二等奖1项，授权发明专利2项。