miR482在番茄响应生物胁迫中的作用及机制研究

Tomato is often threatened by pathogen during its growth and development stage, causing great damage to its production and quality. miR482 is one kind of newly discovered miRNAs, whose major targets are NBS-LRRs containing the largest number of resistant genes in plants. By using bioinformatic prediction, small RNA library construction and molecular biology experiment, we detected that miR482 had a huge change of expression quantity in tomato and P.infestans interaction. Therefore, based on the analysis of promoters and target genes as well as the characteristics of all miR482 family members, we are intended to identify miR482*s and their major target genes in tomato. Meanwhile, via qRT-PCR, Northern blot, etc., we will also analyze the expression characteristics of some related genes under stress of bacteria, fungus and virus. Simultaneously, over-expression and RNAi vectors will be constructed followed by transformed into tomato. By means of resistance，physiological and biochemical characteristics and gene expression profile analysis to transformants, we will define the function of these genes in order to uncover the regulator network relationships among miR482 related gene groups under biotic stress. The project will lay foundations for the improvement of tomato varieties and the efficient usage of genetic resources, as well as provide new thought and theory basis for comprehensive and systematic researches of miRNAs.

番茄在生长发育期内经常受到病原微生物的危害，对产量和品质影响甚大。miR482是近年来发现的miRNA，其主要靶标NBS-LRR是植物体内数量最多的一类抗病基因。我们通过生物信息学预测、小RNA建库以及分子生物学实验，检测到番茄受病菌侵染后miR482的表达量变化剧烈。因而，分析了番茄miR482的启动子、靶基因以及家族各成员的特征特性，在此基础上，本项目拟进一步鉴定番茄miR482*s及其主要靶基因；通过qRT-PCR、Northern blot等技术研究生物胁迫下miR482关联主要基因的表达特性；构建这些基因的超表达载体和RNA干扰载体；转化番茄；借助对转化株进行的抗性、生理生化特性以及表达谱的分析，确定各基因的功能；揭示番茄在生物胁迫下体内miR482相关基因群体间的网络调控关系。为番茄基因资源的有效利用和品种改良奠定基础；也为全面系统研究miRNA提供新的思路和理论依据。

番茄在其生育期内不可避免地会受到多种病原物的侵袭，影响其产量和品质。了解和掌握番茄抗病的分子机制，有助于培育优良品种、治理病害、增产增收等。近年来，miRNA强大的调控作用倍受关注，我们的研究也发现miR482通过靶向植物体内数量最多的一类抗病基因NBS-LRR调控着番茄的抗性。.过表达miR482a、miR482b、miR482c、miR482e和miR482e*的5种番茄，离体叶片喷施病原菌后，死细胞数和病斑直径均显著高于野生型；整株喷施病原菌后，病斑面积、病情指数以及病原孢子数也都显著高于野生型，植株的抗病性降低。利用STTM技术和人工反义技术分别沉默miR482a和miR482b的番茄，喷施病原菌后，病斑面积、病情指数以及病原孢子数都显著低于野生型，MDA显著高于野生型，植株的抗病性增强；利用CRISPR/Cas9技术同时敲除miR482b和miR482c的番茄，病情指数及叶片的死细胞数都显著低于对照，活性氧清除能力显著提高，植株的抗病性增强。.利用生物信息学预测、qRT-PCR、降解组测序及5’RACE等技术手段，鉴定了miR482s和miR482*s的靶基因共23个。过表达miR482b的靶基因Solyc02g036270.2（一种NBS-LRR基因）的番茄，显著提高了病程相关蛋白PR1、PR2和PR5的表达水平；整株喷施病原菌后，叶片的死细胞数、病原孢子数和病斑面积都显著减少，病情指数显著降低，MDA和相对电导率显著升高；抗病性得到增强。利用VIGS沉默Solyc02g036270.2的效果正好相反，植株的抗病性减弱。.对过表达miR482b的材料进行转录组分析后发现，miR482b能影响746个基因的表达，这些基因主要包括在半胱氨酸和蛋氨酸代谢、α-亚麻酸代谢、植物与病原物互作和MAPK相关的信号通路中。此外，研究还发现miR482a不仅在番茄抵御病菌侵染过程中发挥着重要的作用，而且它还与非生物胁迫有关，沉默miR482a后，番茄的抗旱、耐盐能力显著提高。.本项目较好地阐明了miR482在番茄响应生物胁迫中的作用；既有利于揭示番茄抗病的分子机制；又可为番茄基因资源的有效利用和品种改良提供必要的信息；同时，还可为全面系统地研究miRNA提供新的思路。