Cloning and expression analysis of WRKY family member SmWRKY14 in Salvia miltiorrhiza / 中草药

Objective: To clone a WRKY protein gene SmWRKY14 with full length cDNA from Salvia miltiorrhiza and carry out bioinformatics and expression analysis in different tissues and response to environmental factors and phytohomone. Methods: The PCR was preformed based on the sequence of Unigene (c50007_g1) searched from our transcriptome database, and characteristics of physiochemical properties, conserved domains and structure prediction of the protein were determined using a series of bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed using DNAMAN and MEGA 6.0, respectively. Real-time quantitative PCR was used for gene expression analysis. Results: In this study, the full length cDNA of SmWRKY14 was 1103 bp in size, encoding a 244-aa protein with a molecular weight of 27.6 KDa and an isoelectric point of 8.19. SmWRKY14 was an unstable hydrophilic protein containing characteristic and conserved WRKY domain without signal peptide or transmembrane domain. The main secondary structure of the amino acid sequences was random coil. Moreover, multiple sequence alignments and phylogenetic trees showed that SmWRKY14 protein had high homology with WRKY14 of Diospyros kaki. Quantitative real-time PCR indicated that SmWRKY14 constitutively expressed in the roots, stems, leaves and flowers of S. miltiorrhiza and was strongly induced by methyl jasmonate, abscisic acid, gibberellins, and mechanical wound, which indicated SmWRKY14 could participate in regulation of biosynthesis of tanshinones and defense process. Conclusion: The gene sequences of SmWRKY14 was successfully cloned and the bioinformatics and expression pattern analysis was carried out, which will provide a foundation for further research on the molecular mechanism of regulation of tanshinones synthesis and response to defense process in S. miltiorrhiza.

Cloning of AhWRKY33 from Asarum heterotropoides and its genetic transformation in Arabidopsis / 中国中药杂志

The WRKY family genes, which play an important role in plant morphogenesis and stress response, were selected based on the data of the full-length transcriptome of Asarum heterotropoides. Using AtWRKY33, which regulates the synthesis of the camalexin in the model plant Arabidopsis to compare homologous genes in A. heterotropoides, primers were designed to amplify the open reading frame(ORF) fragment of AhWRKY33 gene by RT-PCR using total RNA of A. heterotropoides leaves as template. Real-time PCR results showed that there was a significant difference between the aerial part and the underground part of A. heterotropoides, the toxic aristolochic acid content is highly expressed in the leaves higher than the root. After verification, the WRKY33 gene of A. heterotropoides is ORF long 1 686 bp, encoding 561 amino acids.AhWRKY33 had two conserved WRKYGQK domains. According to the classical classification, it belongs to group Ⅰ WRKY transcription factor. A. heterotropoides WRKY33 had some homology with amino acids of other species. The study successfully constructed the plant eukaryotic expression vector PHG-AhWRKY33 and transformed Arabidopsis thaliana, the transgenic Arabidopsis was obtained by PCR detection and hygromycin resistant plate screening. It found that the germination of transgenic Arabidopsis seeds was accelerated and the stress resistance was increased. It laid a foundation for further analysis of WRKY transcription factor in the growth and development of A. heterotropoides and the synthesis of secondary metabolites.

GbWRKY1, a novel cotton (Gossypium barbadense) WRKY gene isolated from a bacteriophage full-length cDNA library, is induced by infection with Verticillium dahliae.

WRKY transcription factor proteins play important roles in diverse stress responses. In this study, we first cloned a novel WRKY from our constructed bacteriophage full-length cDNA library for cotton (Gossypium barbadense). The plants were stressed by exposure to a defoliating strain of Verticillium dahliae. The capacity of primary cDNA library was 1.28 × 106 PFU and the titer of the amplified cDNA library was >1010 PFU mL–1. The recombination rate of the library was 94% and average insert size was about 1.1 kb. This novel gene, named GbWRKY1 was 1971 bp long and encodes a protein of 489 amino acids. It contains two characteristic WRKY domains and two zinc finger motifs. The sub-cellular assay indicated that GbWRKY1–GFP fusion protein was localized in the nucleus. Furthermore, Northern blot analysis showed that expression pattern of GbWRKY1 was similar among tissue types (roots, stems and leaves), but differed between pathogen-infiltrated and Czapek medium-infiltrated (untreated control) plants. Quantitative real-time PCR showed that GbWRKY1 could also be induced by salicylic acid (SA), methyl jasmonate (MeJA) and 1-aminocyclopropane-1-carboxylic acid (ACC). These findings clearly suggest that as a pathogen-inducible transcription factor GbWRKY1 plays an important role in plant defense responses.