小麦蓝矮植原体RNA沉默抑制子互作蛋白的筛选与分析

Screening and analysis of interaction proteins of RNA silencing suppressor in WBD phytoplasmid

【目的】揭示小麦蓝矮植原体(WBD phytoplasma)的致病机理及其与寄主的互作关系,为植原体病害防治提供理论依据。【方法】以SWP16为诱饵,将SWP16构建到诱饵载体pBT3-STE和pBT3-SUC上,在小麦酵母双杂交膜系统cDNA文库中筛选与SWP16互作的蛋白,并确定最佳的筛库条件。通过α-半乳糖苷酶活性检测试验对筛选到的互作蛋白进行二次验证,并运用Uniprot对其进行Gene Ontology(GO)注释分析。【结果】以pBT3STE-SWP16作为筛库的诱饵蛋白,确定20 mmol/L 3-AT为筛选文库的最佳浓度,从小麦cDNA文库中筛选到20种互作蛋白,包括Rieske蛋白、细胞色素b5、CASP蛋白等。经过复筛和α-半乳糖苷酶活性检测试验进一步验证了互作关系,其中14种互作蛋白参与运输、pH调节、光合作用、内质网应激反应、蛋白质泛素化等生理过程;18种互作蛋白存在于线粒体、内质网、细胞膜、细胞质、叶绿体和细胞壁等6种细胞组分中;20种互作蛋白的分子功能主要包括几丁质酶活性、反转运蛋白活性、脱水酶活性、转移酶活性和转录因子活性等。【结论】筛选获得20种与小麦蓝矮植原体效应蛋白SWP16互作的蛋白,有助于推动小麦蓝矮植原体与寄主互作关系的研究。

【Objective】To reveal the pathogenic mechanism of wheat blue dwarf phytoplasma(WBD phytoplasma)and its interaction with its host,so as to provide a theoretical basis for the control of phytoplasma diseases.【Method】SWP16 was constructed into two bait vectors(pBT3-STE and pBT3-SUC)for wheat cDNA library screening.The optimal screening conditions were determined.The screened interaction proteins were confirmed byα-galactosidase activity and the Gene Ontology(GO)analysis was performed by Uniprot.【Result】Using pBT3STE-SWP16 as the bait protein,20 mmol/L 3-AT was determined as the optimal concentration for screening the library.A total of 20 interacting proteins including Rieske protein and cytochrome b5 and CASP protein were obtained from the wheat cDNA library.The interaction was further verified by re-screening andα-galactosidase activity assay.Among them,14 interacting proteins are involved in physiological processes such as transport,pH regulation,photosynthesis,endoplasmic reticulum stress response and protein ubiquitination.18 interacting proteins were found in 6 cell components such as mitochondria,endoplasmic reticulum,membrane,cytoplasm,chloroplast and cell wall.The molecular functions of 20 interacting proteins mainly include chitinase activity,antiporter activity,dehydratase activity,transferase activity and transcription factor activity.【Conclusion】A total of 20 proteins interacting with wheat blue dwarf phytoplasma effector protein SWP16 were obtained through yeast two-hybrid experiment.It is helpful to promote the study on the interaction between wheat blue dwarf phytophyte and its host.