Cloning And Expression Analysis Of Two WRKY Transcription Factors In Apple (malus Domestica Borkh.)

WRKY transcription factors are one of the largest families of transcriptional regulators in plants which play an important role in plants growth and development, senescence, substance metabolism, resistance and form integral parts of signalling webs that modulate many plant processes. Studies on model organism show that WRKY transcription factors as an important regulator in host-pathogen interaction is an important element in plant defense signaling network which can positively or negatively regulate disease-related gene’s expression through different pathways to make plants have spectrum and durable resistance. In this study, 132 putative WRKY genes in the genomes of apple were identified through bioinformatics analysis and its classification and genetic structure has been detailedly studied. The expression analysis of 42 MdWRKY genes in different tissues were studied and two MdWRKY genes were cloned. Their gene expression analysis in response to abiotic and biotic stress were studied. The main results were shown as follows:1. The apple WRKY proteins were claasified into Group I, Group II and Group III which each contains 24, 79 and 29 members and their zinc-finger motif were CX4CX22-23 HXH, CX4-5CX23 HXH and CX7CX23–24HXC respectively. Group II members could be further divided into five subgroups( II-a, II-b, II-c, II-d and II-e subgroup), which contains 8, 12, 31, 14 and 14 members respectively. The apple WRKY genes were distributed with different densities on 17 chromosomes from 4 to 13. The results of gene structure and conserved motif analysis revealed that most of the WRKY genes contain 2-5 exons and 10 conserved motifs including WRKY domain which were made up of motif 1-6. These results suggested that WRKY domain contain relatively highly conserved motif.2. The RT-PCR results of 42 MdWRKY genes indicated that 12 MdWRKY were expressed in leaves, stems, roots, flowers and fruits at various expression levels. These results suggested that MdWRKY genes may be involved into the regulation of growth and development processes in apple.3. The qRT-PCR results of MdWRKY5 and MdWRKY54 in response to high salinity, simulative drought with PEG, high and low temperature indicated that the two genes were induced by high salinity, high and low temperature obviously. The qRT-PCR results of MdWRKY5 and MdWRKY54 in response to SA, ABA and MeJA indicated that the two genes were induced by plant hormones which MdWRKY5 was induced by the SA obviously and MdWRKY54 was induced by the ABA obviously. These results suggested that these two genes may be involved into the response to abiotic stress.4. The q RT-PCR results of MdWRKY5 and MdWRKY54 in response to Glomerella cingulate indicated that the two genes were induced by the pathogen especially the MdWRKY5 which gene expression growing all the time and reached 14.6 folds of the control at 48 h. These results suggested that these two genes may be involved into the response to biotic stress.