Identification Of Trans-Acting Factors For Sm-Nvas Promoter And Functional Study Of Its Homologous Genes In Tobacco

Plant vascular bundle tissue plays a vital role in both nutrition transport and mechanical support.A new gene sm-Nvas related to vascular bundle development has been cloned in our laboratory,and its expression is specifically induced by salicylic acid(SA)and methyl jasmonate(Me JA).A binary expression vector carrying the gene was constructed by using Ca MV35 S as a promoter,and genetic transformation was performed.The transgenic plants showed a series of phenotypic changes,such as dwarfing,vein increasing,stem and vein thickening.The gene was also knocked out by using CRISPR / Cas9 technology.It was found that the number of vascular columns and the density of arrangement had changed in the transgenic plants,which further confirmed that sm-Nvas gene was related to vascular development.In this study,we further studied the gene function of sm-Nvas and its homologous genes.The results are as follows:(1)Screening trans-acting factors in the cDNA library that interacted with the promoter of the sm-Nvas gene using yeast one-hybrid method.A total of 15 candidate c DNA sequences that interact with the promoter sequence were screened out in this experiment.Through bioinformatics analysis,it found that these candidate trans-acting factors are related to disease resistance and damage response,mainly including damage response related factors,MAPK related factors and common transcription factors that interact with promoters to regulate gene expression.It indicated that the sm-Nvas gene may be involved in plant defense responses.(2)Obtaining target protein sm-Nvas by prokaryotic expression.The prokaryotic expression system of sm-Nvas gene was constructed by using p MAL-p5 x vector.The fusion protein with MBP tag was induced and expressed.Then,the target protein sm-Nvas was obtained by cutting with factor Xa and purifying with affinity column chromatography.The purified protein can be used for enzymatic analysis and analysis of sm-Nvas gene at the protein level.(3)Analyzing the expression of sm-Nvas homologous gene induced by exogenous hormones SA,Me JA and injury using real time fluorescence quantitativePCR.After 16 hours of treatment Nicotiana sylvestris and Nicotiana attenuata respectively with SA and MJA,the expression of sm-Nvas homologous genes in vivo changed and reached the peak.The analysis of gene expression showed that homologous genes,similar to sm-Nvas genes,play an important role in plant defense pathway.At the same time,overexpression vectors of homologous genes were constructed and genetic transformation was completed,which provided experimental materials for studying their biological functions in disease resistance.