极细链格孢激发子PeaT1受体及抗逆关键基因的研究与鉴定

Protein elicitor can induce an immune response in plant, but the inducing mechanism is not comprehensive accurate understanding. The first step of plant immune defense reaction was the recognition between plant cell receptors and elicitor,this is also the key point to understand the induction mechanism. Protein elicitor PeaT1 was isolated from Alternaria tenuissima in our lab, it can induce the defense reaction in rice, improve its ability to resist against adverse reaction. Our preliminary work show that we had screened protein OsMIOX which can interact with elicitor from rice. The OsMIOX can interact with the PeaT1 by yeast two-hybrid system. In this project, pull down, bimolecular fluorescence complementation (BiFC)were applied to test the interaction with PeaT1 in vitro and in vivo; subsequently, Got the rice mutant of the candidate receptor Applied by the technology of CRISPR-CAS9,then, analyzed the change of defense reaction,the difference express of relative genes, combined with the over-expresson to identify the relationship of receptor and key genes for disease-resistant. expounded the function of key genes in the progress of plant defense reaction.This research will provide theoretic foundation to understand the molecular mechanism of PeaT1 induced defense responses in plants.

激发子能诱导植物免疫反应，但对其诱导机制尚缺乏全面精确的认识。植物细胞受体对激发子识别是植物免疫防御反应的第一步，鉴定激发子的识别受体是明确其诱导机制的关键所在。本项目申请团队从极细链格孢菌中筛选获得蛋白激发子PeaT1，能诱导水稻免疫抗性，提高抗逆能力。并从水稻中筛选到PeaT1的互作蛋白肌醇加氧酶，克隆肌醇加氧酶（OsMIOX）基因并通过酵母双杂交验证能与PeaT1互作。本项目在此基础上，利用体内与体外互作验证方法结合验证PeaT1与候选受体肌醇加氧酶的互作；利用CRISPR-CAS9基因组定点编辑技术，获得候选受体的水稻突变体植株，通过候选受体基因定点剪切和修复等技术分析相关基因的表达差异与抗性的变化，结合基因过表达技术验证激发子的受体与抗逆关键基因的关系，阐明抗病关键基因在抗逆过程中的机制，为揭示链格孢激发子蛋白诱导免疫抗性与抗逆机理研究提供依据。

诱导抗性是植物生长过程中抵御各种胁迫的重要手段。蛋白激发子具有诱导植物产生抗逆性的特点，在生物防控中具有重要的应用前景。本课题组前期从极细链格孢(Alternaria tenuissima)中分离得到一个蛋白激发子PeaT1，并获得了异源表达PeaT1水稻纯合株系，发现转PeaT1基因的水稻抗旱能力明显提高，然而其作用机制尚不清楚。在本基金项目的资助下，首先分析了抗旱相关物质包括脱落酸、叶绿素和脯氨酸含量发现在抗旱过程中这些物质的含量显著提高，丙二醛含量显著下降。qRT-PCR检测证明，抗旱相关基因OsCPK9，OsSKIPa，OsNCED2，OsNAC9，OsTPKb，和OsEREBP1在转PeaT1水稻中的表达量显著高于野生型。利用Label free技术在野生型和转基因水稻中共鉴定出蛋白4493个，达到显著差异水平蛋白57个，其中上调32个，下调25个。GO分析表明差异蛋白主要参与新陈代谢、胁迫反应、蛋白代谢、细胞活动及生物进程等过程，其中参与胁迫反应的蛋白SKIP/SNW变化量最大，且在转基因中表达量较高。通过酵母双杂筛选水稻叶片cDNA文库，初步获得9个候选互作蛋白，进一步验证发现肌醇加氧酶(OsMIOX) 与PeaT1在酵母中特异性结合，并利用双分子荧光互补 (BIFC) 和Pull-down技术对互作真实性进行验证。OsMIOX在转PeaT1水稻抗旱过程中表达量显著上调；原位杂交显示OsMIOX主要在叶肉细胞和花药中表达。OsMIOX-GFP与内质网Marker蛋白共定位，表明OsMIOX蛋白在内质网上发挥作用。构建OsMIOX过表达载体和敲除载体，获得过表达阳性水稻株系10株，基因敲除水稻株系6株。对过表达OsMIOX水稻进行抗旱胁迫，发现有743个显著差异基因，其中上调基因534个，下调基因209个。GO与KEGG分析结果表明差异基因主要富集植物激素脱落酸，茉莉酸和糖代谢途径。综上所述，激发子PeaT1通过与OsMIOX互作并影响逆境胁迫相关基因表达来提高水稻的抗旱性。此研究不仅为激发子转基因抗性机制研究提供了一定的理论参考，也对水稻抗旱分子辅助育种的遗传改良具有重要的意义。