Cloning And Functional Analysis Of The StWRKY-1 Gene From Solanum Torvum

Verticillium Wilt,caused by the vascular fungus Verticillium dahliae Kleb,is one of the most serious diseases during eggplant growth,which caused the production of eggplant to reduce greatly.At present,it is lack of high resistance germplasm in common cultivated varieties of Solanum.However,its near wild species,Solanum torvum has high resistance to Verticillium Wilt.So Solannum torvum becomes an important experimental material for the study of the molecular mechanism of Verticillium Wilt of eggplant.In our study,Up-regulated expression WRKY transcription factor was selected from transcriptome sequencing data ofSolanum torvum infected by Verticillium Wilt and entitled as StWRKY-1,Quantitative Real time qRT-PCR was used to analyze its tissue and induction expression pattern.At the same time,transient infection on tobacco was used to analyse subcellular localization and identificate disease resistance.In addition,the function of StWRKY-1 was verified by virus induced gene silencing(VIGS)and transgenic analysis to study the role of StWRKY-1 gene in the resistance of Verticillium Wilt.The main results were as follows:In this study,StWRKY-1 gene,which was induced by Verticillium Wilt,was cloned from Solanum torvum.And the length of the open reading frame was 1650 bp,encoding a protein of 550 amino acids.Comparison of StWRKY-1 with peptide sequences of WRKY transcription factors protein family and phylogenetic analysis revealed highest homology of StWRKY-1 with CaWRKY6 and CaWRKYl in Capsicum annuum.It revealed by qRT-PCR that the expression of StWRKY-Iwas high in roots but very low expression in leaf.In addition,StWRKY-1 was induced by Verticillium dahliae inoculation,the expression was reached highest level at 24 h.We synthesized a sub-cellular localization ’vector to get the transient expression in Nicotiana benthamiana.By confocal laser scanning microscopy it was observed that the fusion protein was localized in nucleus.The VIGS was conducted in the Solanum torvum and the expression level of StWRKY-1 was decreased.The silenced plants showed more sensitive to V.dahlia.The expression level of V.dahlia in the silenced plants was about 3 fold compared with control at 20 d after inoculation.At the same time,the leaves appeared some abnormal phenotype,such as warping and shrinking.Furthermore,we infected transgenic Nicotiana benthamiana with Phytophthora parasitica,the lesion area of transformed plant was 1.13 cm2 while that of the Control plant was 3.14 cm2 after Phytophthora parasitica infection.And it was obvious that StWRKY-1 enhanced the disease resistance in plant by triggering the expression of pathways of defense response plant hormone genes.The gene was transformed into wild-type Arabidopsis,After screening,Homozygous T3 seeds of StWRKY-1-transgenic Arabidopsis and wild-type lines were selected for futher analysis.The growth of transgenic plants is normal.At the same time,wild-type lines grow slowly to the contrary.All the results showed that StWRKY-1 confered the resistant function to Verticillium dahliae and Phytophthora parasitica,,which can be applied to new germplasm creation for disease resistance.