Induced Expression Analysis Of Soybean GmWRKY Gene And Functional Study Of GmWRKY148

WRKY transcription factors play a crucial role in plants stress responses.We used AtWRKY44 gene as the probe to screen the homolog in soybean.GmWRKY148(gene no.is Glyma.14G199800)was cloned and analyzed by bioinformatics methods.The transcript expression of GmWRKY148 mRNA in various tissues and postinoculation by P.sojae is examined.The CDS sequence of GmWRKY148 was cloned,ligated into the vector pBINGFP2(OE-GmWRKY148).OE-GmWRKY148 and empty vector are transformed into soybean hairy roots by high-efficiency Agrobacterium rhizogenes-mediated transformation,respectively.The positive hairy roots are inoculated by P.sojae.Then the lesion length and biomass of P.sojae and germination of oospores are analyzed.The research contents and results were listed as follows:1.The induction and expression analysis of AtWRKY44 homologous genes of arabidopsis thaliana in soybeanUsing AtWRKY44(AT2G37260.1)as a probe,BLAST in the online website Phytozome(http://www.Phytozome.net/),search with the homologous genes in soybean.The results showed that the homologous gene is too much,choose GmWRKYgenes which score more than or equal to 100 for next research.Using P6497 infected soybean cultivars Williams(sensitive)and Williams82(resistance),analysis expression to these GmWRKY genes.GmWRKY148 gene with significant difference in expression before and after inoculation was selected for cloning and functional verification.2.Cloning of GmWRKY148The results showed that there were too many homologous genes of AtWRKY44(AT2G37260.1).After the induction of P6497,the gene with large variation of expression was selected,namely GmWRKY148.The coding sequence of GmWRKY148 was isolated from the roots of Williams 82.Its cDNA is 1328bp,including the CDS sequence of 999 bp,5' non-coding region is 249 bp and 3' non-coding region is 80bp,GmWRKY148 encodes 332 amino acids,and the isoelectric point is 7.61.3.Protein structure,promoter and phylogenetic analysis of GmWRKY148GmWRKY148 contained seven alpha helix and four beta folds,which was highly conserved.Some cis-acting elements involved in defense and stress were predicted in GmWRKY148 promoter regions.Phylogenetic analysis indicated that the GmWRKY148 from Glycine max and other Legumes plants were highly similar.4.Tissue specific expression and infection induced by P.Sojae of GmWRKY148GmWRKY148 was constitutively expressed in the roots,stems,leaves,and cotyledons.It was significantly induced by P.sojae.After the infection of P.Sojae,In the susceptible variety(Williams)and resistant variety(Williams 82),the expression of GmWRKY148 presented the trend of overall upward expression,but increased higher in Williams.5.Subcellular localization of GmWRKY148GmWRKY148 is likely to be located in the nucleus and thus regulate the expression of the corresponding gene,according to the online prediction website.The CDS sequence of GmWRKY148 was combined with GFP protein to construct pBIN-GmWRKY148-GFP vector.The results show that GmWRKY148 is a nuclear localization protein.6.GmWRKY148 enhanced the resistance to P.sojaeThe positive hairy roots of OE-GmWRKY148 post inoculation in Williams showed the shorter lesion length and lower P.sojae biomass accumulation.The mycelia and oospore formation were significantly decreased in OE-GmWRKY148 transgenic hairy roots than that in EV.The results indicate that GmWRKY148 is a positive regulator in soybean P.sojae interaction.