油菜cDNA表达文库构建及核盘菌致病因子互作蛋白的筛选和鉴定

核盘菌 Sclerotinia sclerotiorum (Lib.) de Bary] 是引起油菜菌核病的病原真菌。核盘菌侵染植物细胞早期,可分泌一些细胞壁降解酶, 如不同类型的多聚半乳糖醛酸酶(polygalacturonase, PG)。这些酶对病菌在植物组织上的定植起关键作用, 同时酶活性的高低也决定病原菌的毒性或致病性。PG可以激活细胞的防卫反应而与其酶活性无关。为了解PG的信号转导途径, 利用RT-PCR方法克隆了核盘菌的PG基因, 将PG的编码区与酵母GAL4的DNA结合功能区融合, 构建到酵母诱饵蛋白表达载体PGBKT7中; 然后在酵母中构建了油菜cDNA表达文库。通过酵母双杂交方法在油菜cDNA表达文库中对与PG互作的蛋白进行了筛选, 分离到一个与PG互作的蛋白, 测序及BLAST分析表明该蛋白含有一个与钙离子结合的结构域, 称为C2结构域(C2-domain), 推测该蛋白是一种钙依赖的膜结合蛋白。该蛋白与拟南芥中一个含C2结构域功能未知蛋白的氨基酸同源性高达80.24%。利用半定量PCR分析了核盘菌诱导后该基因在油菜花、茎、叶中的表达, 结果表明该基因在叶片中表达量最高。克隆到的C2蛋白与其他物种中的C2蛋白比较发现, 与钙离子结合的天冬氨酸残基很保守。

Construction of cDNA Expression Library of Oilseed Rape and Screening and Identification of Interaction Partner of PG, A Virulence Factor from Sclerotinia sclerotiorum

Sclerotinia sclerotiorum (Lib.) de Bary is the fungus that causes stem rot disease in oilseed rape. To overcome plant cell wall, during early pathogenesis, the fungus secretes several types of cell wall-degrading enzymes such as polygalacturonases (PGs). The activity of these enzymes is crucial for the colonization of plant tissues, and these enzymes can be determinants of pathogenicity or virulence. PG can activate defense reactions in plants, having no relation to the enzyme activity. In order to understand PG signaling pathway, PG gene was cloned from Sclerotinia sclerotiorum by RT-PCR, the encoding region of PG was fused to the yeast GAL4 DNA-binding domain in yeast expression vector PGBKT7 and an oilseed rape cDNA expression library was constructed in yeast. PG-interacting proteins were screened in the library with PG as bait by yeast two-hybrid assay. An interacting protein was isolated, sequencing and BLAST analysis revealed that the protein contains a C2-domain and was predicted to be a Ca²⁺ binding domain. The protein shares 80.24% identity in amino acids with a C2-domain protein in Arabidopsis with unknown function. The expression levels of the C2-domain protein in floral, leaf and stem organs of oilseed rape were investigated by semi-quantitative RT-PCR, the results showed that the C2-domain protein was highly expressed in leaves after induced by Sclerotinia sclerotiorum. Comparison in C2-domain proteins between oilseed rape and other species suggested that aspartatic acid residues involving in Ca²⁺ binding were conserved.