陆地棉抗黄萎病基因GhENODL6互作蛋白筛选

Screening for the Interacting Proteins with GhENODL6 in Gossypium hirsutum by Yeast Two-Hybrid System

研究发现陆地棉ENODL6基因具有抗黄萎病功能。为进一步揭示其在棉花抗黄萎病过程中发挥的作用,通过Nimble Cloning技术将GhENODL6插入酵母表达载体pNC-GBKT7,构建了重组质粒pNC-GBKT7-ENODL6。重组质粒转入酵母菌Y2HGold后可在DDO培养基上正常生长,但不能生长于QDO/X/A培养基,表明GhENODL6蛋白对酵母宿主无毒害作用,没有自激活活性。将携带pNC-GBKT7-ENODL6诱饵载体的Y2HGold酵母菌与cDNA文库进行杂交筛选,结果获得一个能够显示蓝色的菌落。通过PCR扩增,在蓝色酵母菌中获得一段526 bp的非载体插入片段,与陆地棉基因组内基因WRKY47序列高度一致。通过同源扩增,从陆地棉中克隆到WRKY47开放读码框,全长1 587 bp,编码528个氨基酸残基。再次利用酵母双杂交技术确认了WRKY47与GhENODL6之间存在互作关系。综上,构建了重组载体pNC-GBKT7-ENODL6,并鉴定到与GhENODL6互作的蛋白WRKY47。

GhENODL6 has been discovered to be involved in the resistance of Gossypium hirsutum to Verticillium dahliae.To further reveal its role in cotton resistance against Verticillium wilt,GhENODL6 open reading frame was inserted into the yeast expression vector pNC-GBKT7 using Nimble Cloning technology to construct the recombinant plasmid pNC-GBKT7-ENODL6.The resulting plasmid was successfully transferred into yeast Y2HGold and exhibited normal growth on DDO medium,while failing to grow on QDO/X/A medium,indicating that the GhENODL6 protein did not exhibit toxicity towards the yeast host and lacked autoactivating activity.Subsequently,the Y2HGold yeast carrying the pNC-GBKT7-ENODL6 decoy vector was subjected to hybridization with a cDNA library and screened for colonies displaying blue coloration.Through PCR amplification,a 526 bp non-vector insert was obtained in these blue yeasts,which showed high sequence consistency with the WRKY47 gene within G.hirsutum genome.By employing homologous amplification techniques,we successfully cloned the WRKY47 open reading frame from upland cotton,which spanned 1587 bp in length and encoded 528 amino acid residues.Furthermore,a yeast two-hybrid assay confirmed an interaction between WRKY47 and GhENODL6.To summarize our findings,the study constructed the recombinant vector pNC-GBKT7-ENODL6 and identified WRKY47 as an interacting protein of GhENODL6.