水稻多聚半乳糖醛酸酶抑制蛋白OsPGIP2显著提高甘蓝型油菜抗菌核病的分子机理研究

Sclerotinia sclerotiorum is a necrotrophic and non-host specific fungal pathogen that infects important oil crops such as oilseed rape. Stem rot in oilseed rape (Brassica napus L.) caused by S.sclerotiorum is one of the most devastating diseases for yield and quality. The relationship between Major QTLs for Scleratinia stem rot and the genes responding to pathogen inoculation are largely unknown. Breeding of Sclerotinia-resistant varieties is confronted with two major difficulties at present. First, no immune or highly resistant germplasm in B.napus and its close relatives has been reported so far. Second, the molecular mechanism of the interaction of the interaction of pathogenic infection and resistance reaction in host plants is pooly understood. Polygalacturonase-inhibiting proteins are ubiquitous plant cell proteins that are directed against fungal polygalacturonase (PGs), which are important pathogenicity factors...With the long-term goal to develop an effective strategy for genetic important of the resistance to S.sclerotiorum, our study has transformed rice OsPGIP2 to Brassica napus susceptible material P61-5 and parital resistant material T45 and 7-5. The inhibiting activity of OsPGIP2 directly reduces the aggressive potential of S.sclerotiorum in detached leaf inoculation and increased resistance to soft rot in field stem inoculation with mycelia agar plugs in consecutive growing seasons from 2011 to 2013. Our study aim to investigate whether OsPGIP2 constitutively expression leads to a series of the changes of systemic defense pathways such as oxidative burst, SA-mediated and JA/ET-mediated pathways, defense enzymes peroxidase and phenylalnine ammonialyase activity detection, WRKY transcription factor mediated pathway. Sclerotinia sclerotiorum releases a battery of polygalacturonases (PGs) during infection. To study the interaction between S.sclerotiorum PGs and OsPGIP2, 7 S.sclerotium PGs (SSPG1-7) and OsPGIP2 were expressed in Pichia pastoris system. In addition, SSPG1-7 expression in uninfected, margin and necrotic zones from the leaf were detected in order to discover the concerted expression patterns of SSPGs during infection initiation and lesion expansion. Finally, evaluation of yield and quality from the improved transgenic lines will give us the best candidate for S.sclerotium-resistance varieties...Through this project, we obtain the improved resistance/tolerance germplasm of Sclerotinia stem rot in B.napus. Furthermore, the molecular mechanism of the interaction of pathogenic infection factors SSPGs and defense reactions leaded by OsPGIP2 expression in vivo will provide valuable insights for PGIP-PG differentiation between monocot and dicot species.

菌核病是影响油菜产量和品质的重要病害之一。现今未知的抗菌核病遗传规律限制了油菜抗性资源的发掘。在水稻等模式植物中，多聚半乳糖醛酸酶抑制蛋白（PGIP）能够阻止病原菌对植物细胞壁的降解，增强植物的抗性。然而油菜内源PGIP超表达植株抗菌核病并不显著。本课题组前期工作中发现，水稻OsPGIP2基因在甘蓝型油菜中过表达使植株苗期和成株期都能增加对菌核病的抗病性。在此基础上，本项目将以OsPGIP2转基因油菜为材料，研究核盘菌侵染后激活的自身防卫途径，对病原物的识别、信号传递、防御相关基因的表达调控；分析OsPGIP2与核盘菌侵染基因SSPG1-7在体内和体外的互作；此外，以受体材料为对照，分析转基因家系的产量与品质，从中筛选出具有育种价值的新种质。通过本项目的实施，既获得抗菌核病的油菜新种质，又揭示外源基因OsPGIP2在油菜体内激活的防卫途径以及OsPGIP2与核盘菌PG类蛋白的互作规律。

菌核病是由核盘菌引起严重威胁油菜产量和品质的真菌性病害。由于甘蓝型油菜中缺乏菌核病抗源，难以通过常规杂交育种来培育抗菌核病的品种。本研究将水稻多聚半乳醛糖酸酶基因OsPGIP2转入3种不同遗传背景的甘蓝型油菜，对苗期和成株期的T2至T4代转基因家系进行抗性鉴定，发现转基因家系菌斑长度显著小于非转基因家系，自然条件下的发病率显著降低，证明转化OsPGIP2基因能够改良油菜恢复系7-5、T45以及P61-5的抗病性，考察成株期接菌后的转基因家系的千粒重和品质性状，发现转基因家系千粒重和含油量都显著高于非转基因家系，受到核盘菌影响程度较小。为深入挖掘OsPGIP2基因介导的油菜抗性增强的分子机理，利用RNA-seq技术聚类分析了7-5D和T45B两个不同背景下的转基因家系相比非转基因家系差异表达基因，发现OsPGIP2介导的抗病反应主要依赖于调节与RLKs（Receptor-like kinases）、Ca2+离子信号、氧化还原反应、JA/ET及生长素、病原菌相关的防卫反应、细胞壁结构、CYP/GST和次生代谢等相关植物天然免疫反应路径。活性氧积累和Evans blue检测实验显示转基因家系H2O2的积累显著性高于非转基因家系，坏死细胞数量显著低于非转基因家系，证明转基因家系通过延缓细胞死亡来提高抗性。为深入研究转基因家系与核盘菌互作机理，定量PCR分析茎杆接菌和叶片接菌时核盘菌致病基因Oxalogenesis、SsPG1、SsPG3、SsPG7、SsCutA、SsHADV-1、endo-β-1,4-D-glucanases和exo-β-1,3-glucanases的表达，结果显示转基因材料中核盘菌的致病基因在接菌的组织中都表现出延迟表达或低表达。在毕赤酵母系统表达的核盘菌致病性蛋白SsPG6对转基因家系的组织侵染程度下降，核盘菌在加入OsPGIP2转基因植株叶片提取物的培养基上生长显著延缓。以上实验证明转基因家系能够通过延迟或者降低核盘菌致病相关基因的表达来削弱病原菌的致病能力。此外对转基因家系茎秆木质素和糖含量进行测定显示木质素含量在二者间无显著差异；阿拉伯糖/木糖（Ara/Xyl）比值随花期的发展降低，在转基因家系中，在盛花期和终花期Ara/Xyl的比值显著性高于非转基因家系。以上结果说明转基因家系中OsPGIP2可能介导改善糖类组成相关途径来提高菌核病抗病性。