The Function Of NtWRKY-R1 Response To Damage Signaling Pathway By Topping

WRKY is one of the largest family of transcription factors in plant,which involved in some important physiological process in plants,including the plant development,abiotic and biotic stress and so on.Existing research shows that WRKY transcription factors associated with plant hormones regulation and secondary metabolic pathways.To study the relationship between tobacco topping damage and the nicotine biosynthesis in root,Nt WRKY-R1 was selected from the tobacco SSH-c DNA library before and after tobacco topping,and combining the RNA-sequencing data.The expression of Nt WRKY-R1 was the negative correlation with the nicotine biosynthesis.Our Previous research has shown that Nt WRKY-R1 have the function of the integration of JA and IAA signaling pathway.This study is for further clarify whether the Nt WRKY-R1 could integrate the JA and IAA signal induced by topping damage and target regulation of nicotine biosynthesis.The results are as follows:(1)Analysis of the Nt WRKY-R1 responses to topping damage,JA,IAA signal pathway and tissue-specific express.Detecting expression level of Nt WRKY-R1,PMT,IAA13 and PDF1.2 by qPCR were detected for the four treatments,including daub NAA at topping stem;spray Me JA on upper leaves of no topping and before and after topping,the results showed that: after topping,the expression level of PMT,IAA13 and PDF1.2 were significantly increased,while Nt WRKY-R1 decreased significantly;Both in NAA and Me JA two treatments.topping decreased the expression of Nt WRKY-R1 have been alleviated,but the sensitivity of Nt WRKY-R1 to NAA was greater than that of Me JA,the expression of PMT has no obviously difference between the two treatments and no-topping treatment.Results indicate that JA-and IAA signaling pathways were trigged by topping and mediated the regulation of nicotine biosynthesis.Tissue specific expression analysis showed that Nt WRKY-R1 expression was higher in roots.(2)Clone the Nt WRKY-R1 transcription factors from tobacco root.Using electronic cloning combined with RT-PCR,obtained the full length of Nt WRKY-R1 transcription factor gene from tobacco root.Bioinformatics analysis showed that the gene has 915 bp ORF sequence,153 bp 5'-terminal and 311 bp 3'-terminal UTR.There is a highly conserved WRKYGQK motif,suggesting that the gene belongs to the WRKY gene family.Phylogenetic tree analysis revealed that it belongs to the class WRKY IIe subfamily.(3)Analysis of Nt WRKY-R1 activation structure domain.Using yeast two-hybrid system shows that Nt WRKY-R1 has obvious self-activation function.Partial deletion analysis shows that the 80 amino acid residues in N-ternimal and the amino acid residues at C-ternimal of the protein were activated.(4)Analysis of Nt WRKY-R1 interacting proteins.Study on the interaction protein of Nt WRKY-R1 by yeast two-hybrid system,total of 15 positive clones were obtained.The results of sequencing were carried out on the NCBI by comparing the sequences of nucleic acids and proteins,and the related information of the selected genes was obtained.Shows that 40 percent of interacting proteins are actin-depolymerizing factor/actin-binding protein(ADF).ADF is considered as primary regulatory factor for the reorganization of the actin cytoskeleton,which is involved in various cellular and developmental activities,as well as in response to biotic and abiotic stresses.