热休克蛋白JDP1在油菜自不亲和性的功能分析

Self-incompatibility in the Brassica is controlled by the S-haplotype-specific interaction between pollen ligand (SCR) and its stigmatic receptor (SRK), which triggers a signaling cascade that is proposed to culminate in ARC1-mediated degradation of stigmatic compatibility factors leading to the rejection of self-pollen. A J domain protein 1 (JDP1) was shown to interact with ARC1 and to regulate ARC1 subcellular localization. However, the function of JDP1 in Brassica SI is largely unknown. Here, Brassica napus compatible Westar line and self-incompatible W1 line will be used in this study. We will generate JDP1 RNAi and JDP1, JDP1-Y8F overexpression transgenic lines with stigmatic expression promotor SLR1. Moreover, we also generate JDP1 site-specific genome editing mutant with the CRISPR/Cas9 system. These transgenic lines will be analyzed for flower shape, pollen adhesion, pollen tube growth, pod size and seed set/pod after self and cross pollination.On the other hand, Total JDP1 and phosphorylated JDP1 level against JDP1 Tyr8 will be detected by Western blots. Dynamic localization of JDP1 and ARC1 will be examined by confocal microscope following self-pollination. Phosphorylated modification of JDP1 by SRK and ubiquitinated modification of JDP1 by ARC1 will be tested and analyzed. These results will demonstrate the function of JDP1 in Brassica SI response.

芸薹属植物自交不亲和（SI）是花粉SCR配体与柱头乳突细胞SRK受体特异性结合，通过下游传递因子ARC1介导亲和因子降解，从而导致SI反应。JDP1能够与ARC1相互作用，并且能够影响ARC1的亚细胞定位，但JDP1是否参与SI反应尚不清楚。本项研究以油菜为试验材料，利用柱头特异表达的SLR1启动子获得JDP1和JDP1-Y8F（第8位点的Y突变为F）柱头过量表达植株；通过RNAi技术获得柱头特异抑制JDP1转基因植株；通过CRISPR-Cas9技术获得JDP1特异编辑的基因组突变体植株。检测和分析转基因植株花的形态，授粉后花粉在柱头萌发生长情况；种荚长度及种子数量。另一方面，检测授粉后柱头内总JDP1及第8位点酪氨酸的磷酸化水平变化；观察JDP1与ARC1在柱头内及授粉后的细胞定位变化；分析SRK与JDP1的磷酸化关系，ARC1与JDP1的泛素化关系。全面阐释JDP1在SI的功能。

自交不亲和性（Self-incompatibility, SI）是植物采取的一种促进异交、避免自交的机制，在植物进化过程中起着非常重要的作用。对SI分子机制的研究不仅在植物生殖领域具有重要的科学意义，而且在植物杂交育种等方面具有重要的实际应用价值。本项目通过酵母双杂交分析发现JDP1的X结构域能够与ARC1相互作用，JDP1的J结构域中Tyr8在JDP1-ARC1相互作用中起着调控的作用。通过对SLR1:JDP1-Y8F-RFP的Westar转基因植株进行授粉后花粉/花粉管、种荚大小及种子数量等分析，结果发现过量表达植株具有减少亲和授粉的表型。通过构建pSLR1:JDP1-RNAi干涉表达质粒，获得W1的转基因植株，分析授粉后花粉/花粉管、种荚大小及种子数量等，发现W1 JDP1-RNAi具有打破SI的表型。JDP1在自交授粉10min磷酸化水平特异性的升高，在自交授粉60min恢复到基本水平，这说明JDP1的磷酸化在SI反应中起着重要的作用。SRK1在体外不能够磷酸化JDP1和JDP1-Y8F，表明JDP1的磷酸化水平变化可能不受SRK直接调控，JDP1可能受到其他的激酶调控。在体外的泛素化反应分析中，没有检测到JDP1被ARC1泛素化，说明JDP1并不是ARC1的底物。上述研究结果进一步解析了芸苔属植物SI分子机制，为育种提供了重要的基因和遗传资源。