The WRKY Family Genes And Its Response To Cold Stress In Grape

How to improve the cold resistance of grapevine is an urgent and arduous problem for grape industry in China. At present, the response mechanism to cold stress in grape is still in the initial stage. The expression profile analysis by our research group showed that the expression of several WKRYs in grape increased under low temperature treatment, suggesting that the WKRY genes may be involved in the response to cold in grape. WRKY transcription factors are one of the largest families of transcriptional regulators that modulate many processes in plants. Members of WRKY family were found to play significant roles in biotic and abiotic stresses. The research on this gene family was conducted not only in model plants, but also in grape since its productivity is largely limited by diseases and abnormal environmental conditions.6WRKYs have identified in grapes, and the functional research focused on biological stress, a little on the response to cold stress.Here, a complete WRKY family was identified based on12×genome sequences from Vitis vinifera cv’Pinot Noir’PN40024. Transcription levels of VvWRKYs were detected in different tissues. A comparative analysis of expression of WRKYs under cold treatment in V. vinifera cv’Muscat Hamburg’and V. amurensis was finished. Two genes induced by cold were overexpressed in Arabidopsis to research their functions. The main results are as follows:1、According to the full-length amino acids sequences of all WRKY proteins in Arabidopsis thaliana, a total of59VvWRKYs were identified with a BLASTp searching. Chromosomal location analysis indicated that VvWRKYs are unevenly distributed in18of the19chromosomes. Phylogenotic analysis allowed categorizing59VvWRKYs into four large groups according to the conserved amino acid sequences of WRKY domain and zinc-finger structure.2、Transcription levels of VvWRKYs were detected in seven tissues including young leaf, mature leaf, tendril, stem apex, young fruit, ripened fruit and root of’Muscat Hamburg’. The majority of VvWRKYs were expressed in more than one tissue. Publicly available microarray data analysis suggested that a subset of VvWRKYs were activated in respond to diverse abiotic and biotic stresses. These results imply that WRKY genes may play fundamental roles during life cycle and in all kinds of stress response process in grape. 3、Quantitative real-time PCR (qRT-PCR) results show that the expression levels of36VvWRKY genes changed over2fold after cold induction in’Muscat Hamburg’, A comparative analysis of microarray data in public database, transriptome data and qRT-PCR results allowed to identifying15Vv WRKYs that show identical expression patterns at least in two methods. And6of them can also be induced by exogenous abscisic acid (ABA), which may indicate its responses to cold signal by ABA-dependent signal transduction pathway. A total of33WRKYs were in accord with gene expression pattern after cold treatment in ’Muscat Hamburg’ and V. amurensis, and the expression levels of42VaWRKY genes changed over2fold after cold induction in V. amurensis.4、We chosed WRKY28and WRKY43for the following cold-related functional analysis in transgenic Arabidopsis thaliana. Transgenic Arabidopsis over-exp ressing WRKY28and WRKY43had higher chilling tolerance and survival ab ility, suggesting WRKY28and WRKY43may participate in cold signal trans duction pathways in grape.In summary, we identified the whole WRKY gene family in grape, compared expression patterns of WRKYs under cold treatment in two kinds of grape material, and the primary function research of2WRKY genesin this dissertation. Through comprehensive analysis of different experimental data, we obtained15WRKY genes related to cold stress (including two WRKY gene used for function research), which laid the foundations for the further study on the function analysis involved in the low temperature-related signal pathways in grape. A comparative analysis of expression of WRKYs under cold treatment in’Muscat Hamburg’and V. amurensis provided clues for the research on WRKYs for high cold resistance in V.amurensis.