Cloning And Expression Analysis Of TaWRKY14 Transcription Factor In Taraxacum Antungense Kitag

The WRKY transcription factor family is almost involved in the process of plant growth and development.In this study,Taraxacum antungense Kitag.was used as experimental material to study the relative expression of key enzymes genes in four phenylpropane metabolic pathways induced by two hormones under two abiotic stresses.A member of the WRKY gene family was cloned and studied.The results were as follows:1.A member of the WRKY gene family was successfully cloned from Taraxacum antungense Kitag.and named Ta WRKY14(Gene login number: MH513939).Ta WRKY14 with a total length of 1056 bp,encoding 351 amino acids.The conserved domain of the amino acid sequence of Ta WRKY14 is the same as that of At WRKY14 protein in Arabidopsis thaliana.Phylogenetic tree results show that the gene obtained by homologous cloning is WRKY IIe subgroup gene and its close relationship is WRKY40 in Salvia miltiorrhiza.2.The tissue specificity of WRKY14 in Taraxacum antungense roots,petioles,leaves and flowers was analyzed by q RT-PCR.The results showed that WRKY14 was most abundant in Taraxacum antungense Kitag.roots,roots > leaves > petioles > flowers.The relative expression of genes in leaves was regarded as 1,the relative expression of genes in roots was 4.8,followed by flowers and petioles,and the expression of WRKY14 in petioles was 0.65.The relative expression of Ta WRKY14 gene in root was 20 times higher than that in flower,4.8 times higher than that in leaf and 7 times higher than that in petiole.After drought stress on Taraxacum antungense Kitag.,the up-regulation trend of Ta WRKY14 in leaves was the most obvious,indicating that Ta WRKY14 was induced by drought stress factors;the expression of Ta WRKY14 increased after treatment with Na Cl aqueous solution of 500 m M;the relative expression of Ta WRKY14 increased after treatment with SA of 100 ?mol/L;and the Ta WRKY14 table in leaves after treatment with Me JA of 100 ?mol/L.There was no significant change in quantity,analysis of the relationship between them and Ta WRKY14.3.The active ingredient(chlorogenic acid and caffeic acid)of Taraxacum antungense Kitag.after stress were determined by high performance liquid chromatography.The results showed that chlorogenic acid and caffeic acid in leaves increased after treated with 100 ?mol/L Me JA,and chlorogenic acid and caffeic acid in leaves also increased after treated with 100 ?mol/L SA.The trend was that caffeic acid decreased first and then increased after treatment with different concentration of salt solution.Quantitative RT-PCR was used to analyze two abiotic stresses.Four key proteases in the phenylpropane metabolism pathway of Taraxacum antungense Kitag.induced by two hormones: the relative expression of C4 H,ANS,HCT and CHI genes were changed.After salt stress,Ta CHI showed a trend of up-regulation and then recovery,which was consistent with the change trend of relative gene expression of Ta WRKY14.After drought stress,Ta C4 H showed a state of up-regulation and then recovery,which was consistent with the change of relative gene expression of Ta WRKY14.4.The yeast one-hybrid vector was constructed by the analysis of promoter elements.The results showed that the promoters of Ta C4 H,Ta CHI could bind in yeast with Ta WRKY14,Ta WRKY14 possible involvement in regulation Ta C4 H and Ta CHI.The prokaryotic expression vector of Ta WRKY14 was constructed.It was found that when OD600=0.6,the induction concentration of IPTG was 0.8 mmol/L,the induction time was 3 h and the temperature was 37 °C,the target protein with molecular weight of 58.5 KDa could be successfully induced,the optimum induction conditions were determined.GFP subcellular localization vectors were constructed,and the results showed that they were located in the nucleus.