Cloning And Functional Verification Of AmWRKY1-like In Agropyron Mongolicum Keng

Agropyron mongolicum Keng belongs to the cold and dry diploid herbaceous plant of the genus Agropyron.Its drought resistance,salt and alkali resistance genes are excellent gene banks of improved varieties of forage and wheat crops.WRKY transcription factors are a plant-specific transcription factor super family,with a variety of biological functions:They play an important role in plant stress response,and development of metabolic and biosynthetic,but there is little research on WRKY transcription factors involved in regulating A.mongolicum.Based on the RNA-Seq data of A.mongolicum grass under drought stress,this study cloned the AmWRKY1-like gene,and performed preliminary research through sequence analysis,mRNA expression abundance under drought stress,heterologous tobacco subcellular localization,and genetic transformation.The function of Am WRKY1-like gene of A.mongolicum was described.The main results are as follows:1.The type Ⅱd AmWRKY1-like gene was cloned from A.mongolicum RNA-Seq for the first time,with a full-length ORF of 1182bp,encoding 393 amino acids,and a molecular weight of 42318.07Da.2.The qRT-PCR analysis revealed that mRNA expression abundance of AmWRKY1-like gene of A.mongolicum was induced by natural drought and 15%PEG-6000,which were all negatively regulated.3.Using the principle of homologous recombination and seamless cloning technology,the over-expression vector pART-CAM-AmWRKY1-like and transient localization vector pCAM35S-AmWRKY1-like-EGFP of AmWRKY1-like gene were successfully constructed.4.The fluorescent signal GFP detection revealed that AmWRKY1-like protein of A.mongolicum was localized on the nucleus of heterologous tobacco cells.5.Sterile tobacco was transformed by leaf disc method.After screening of Kana+resistant strains and PCR detection,a total of 10 T0 transgenic AmWRKY1-like tobacco lines were obtained.