酵母双杂交系统筛选番茄SlMPK1互作蛋白

Screening of tomato SlMPK1 interacting proteins by yeast two-hybrid system

促分裂源活化蛋白激酶(MAPK)在植物生长发育及响应环境胁迫过程中发挥着重要作用。采用酵母双杂交技术,用番茄SlMPK1作为诱饵,从番茄cDNA文库中筛选能与SlMPK1相互作用的蛋白。结果表明:SlMPK1全长序列成功构建至pGBKT7载体,获得诱饵质粒pGBKT7-K1,其对酵母细胞无毒性,且20 mmol·L^-13-AT能有效抑制其自激活活性。将pGBKT7-K1与含pGADT7-cDNA文库的质粒共转入Y2H Gold酵母中,对阳性克隆进行鉴定,共筛选到155种候选蛋白可能与SlMPK1相互作用,其中SlSPRH1和SlMKK4是已知能与SlMPK1相互作用的蛋白。分析显示,这些蛋白主要参与蛋白折叠、胁迫响应等生物学过程。

The mitogen-activated protein kinase(MAPK)plays an important role in plant growth and development and in environmental stress response.Previous studies in our laboratory have found that tomato SlMPK1 is involved in stress response.In order to further explore the mechanisms of SlMPK1 function,yeast two-hybrid(Y2 H)technology was used to screen the proteins interacting with SlMPK1 from tomato cDNA library using SlMPK1 as a bait.The results showed that the full-length sequence of SlMPK1 was successfully constructed into pGBKT7 vector to obtain bait plasmid pGBKT7-K1,which was not toxic to yeast cells.Besides,20 mmol·L^-13-AT could effectively inhibit its auto-activation activity.The pGBKT7-K1 and the plasmid containing the pGADT7-cDNA library were co-transfected into Y2 H Gold yeast,and the positive clones were identified.A total of 155 candidate proteins had been screened to interact with SlMPK1,and SlSPRH1 and SlMKK4 were known to interact with SlMPK1.Biological process(BP)analysis showed that these proteins were mainly involved in biological processes such as protein folding and stress response,providing a theoretical basis for further study of the function of SlMPK1.