Genome-Wide Identification, Expression Analysis Of Bzip Transcription Factor Family And Functional Characterization Of VVBZIP45/VVBZIP08 (AREB/ABFS) Gene In Grapevine (Vitis Vinifera Spp.)

Transcriptional regulation is an important mechanism of gene expressional regulation in eukaryota and regulate numerous biological processes. Transcription factors (TFs) play important roles in transcriptional regulation, especially playing critical roles in plant responsing to environmental stress. Therefore, the research of plant TF is help to get insight into the biological processes and mechanisms of plant in response to environmental stress.Basic leucine zipper (bZIPs) transcription factor family is one of the largest and most diverse families, which widely involved in numerous biological processes, such as seed maturation, flower development, seed maturation, vascular morphogenesis and development stress signalling and pathogen defence and so on, particularly playing an important roles in response to biotic and abiotic stress. Presently, most well characterized bZIP transcription factors were mainly from the model plant Arabidopsis, and the following were rice and maize, but limited research in wood plant, and their the genome-wide identification and analysis of bZIP gene family is not report yetGrapevine (Vitis spp.) is a popularly cultivated fruit crop throughout the world, people’s favoritor and the grapevine is famous over the world. On the evolution, grapevine is the earliest diverging lineage of rosids, the genome informations suggested that it has not undergone recent genome duplication, which was named as "palaeo-hexaploid" plant, and which is important for plant evolution and the research of gene functional characterization. In our present study, we identified and analyzed the grapevine bZEP gene family at whole genome level using bioinformatics method, and then performed a deeply research and analysis in the evolution and the expression in response to abiotic stress of this gene family, and transformed VvAREBl gene and the promoters of the paralogous gene pairs (pVvbZIP45(TvAREB1)/VvbZIP08:GUS) into Arabidopsis for functional characterization and analysis of AREB/ABFs. The main results were listed as follows:1. Identified 57 bZIP genes from grapevine (Vitis vinifera) genome, and divided them into 10 groups according to the topological structure and the phylogenetic relationship with those in Arabidopsis. The chromosome distribution and the collinearity analyses suggest that expansion of the grapevine bZIP family is greatly contributed by the segment/chromosomal duplications, which may be associated with the grapevine genome fusion events. Nine kinds of intron patterns within the bZIP domain and the additional conserved motifs are identified among all VvbZIP proteins, named as a to i, and showed a high group specificity. The predicted specificities on DNA-binding sites indicated that some highly conserved amino acid residues exist across each major group in the tree of land plant life. The expression patterns of VvbZIP genes across the 54 tissues/organs/developmental stages based on microarray (with some validation by qualitative real time (qRT-PCR) suggest that VvbZIP genes are widely involved in grapevine organ development, especially seed development. Expression analysis based on qRT-PCR indicated that VvbZIP genes are extensively involved in drought-and heat-responses, with possibly two different mechanisms.2. Genome-wide identification of the bZIP gene family was carry out across plant kingdom using bioinformatics method based on the numberous information of plant genome sequencing and then the evolution and expression profiles of the intronless bZIP genes were analyzed, the results shown that the intronless bZIP genes mainly distributed in group C and configurated a intronless subfamily, which seems to have a special course of evolution. The distribution of the member of this intronless group showed that this group was derived from the early stage of the land plant and the feature of the intronless was derived from the stage of vascular plant. Furthermore, this subfamily has got a rapid expansion after their appearance, and the chromosome distribution of this subfamily in grapevine indicated that this intronless subfamily was very conserved and rarely suffered the purifying selection and loss. The expression of this subfamily suggested that the intronless subfamily has strong tissue specificity and widely induced by various abiotic stresses and may play important roles in plant withstand abiotic stress.3. Cloned the grapevine VvAREBl gene, and which was constructed into a plant over-expression vector successfully, and it was transformed into Arabidopsis using the floral dip method. Afterwards, we identified the the phenotypes of the transgenic Arabidopsis in response to various abiotic stress and analyzing the expression of the downstream gene of AREB/ABFs type, the result showed that the Arabidopsis transformed with VvAREBl gene was sensitive to exogenous ABA, indicating that this gene is invoved in leaf morphogenesis and the stomatal movement. The experiment of NaCl treatment suggested that the transgenic lines showed a significantly resistance to salt stress. However, it not showed a significantly resistance to drought stress. In addition, we determined the molecular level changes of the Arabidopsis after transformed the grapevine VvAREBl gene, the result shown that no significant change was observed in the downstream genes expression of the AREB/ABF type under non-treatment, but which were significantly up-regulated after treated with exogenous ABA, dehydration and salt stress comparing with the wide type Arabidopsis, suggesting that ABA and the stress signals is necessary for VvAREB1 gene in regulating various abiotic stress.4. We cloned the promoter of the paralogous gene pair VvbZIP45/VvbZIP08 and constructed the GUS expression vectors, and transformed them into Arabidopsis, then identified the expression profiles of VvbZIP45/VvbZIP08 promoters using tissue stain of the GUS gene, the results shown that a significant difference of the tissue expression between this gene pair was observed in the transgenic Arabidopsis, indicating that this paralogous gene pairs have been diverged in their evolution history, and suggested that the fate of the duplicated gene pair maybe blong to RD or RN type. Further, the GUS stain of the Arabidopsis treated with exogenous ABA and various abiotic stress shown that the promoter of VvbZIP45/VvbZIP08 were induced by ABA and various abiotic stress. While the GUS stain of the Arabidopsis treated with SA> JA and ETH shown that the expression of the VvbZIP45/VvbZIP08 gene promoter was strongly inhibited by plant defence signals, supposing that this inhibition was contributed by the antagonism between plant defence signals and ABA, Furthermore, this result indicated the importance of ABA signaling to VvbZIP45/VvbZIP08 expression.