The Whole Genomic Identification,Expression Analysis Of SNRK2Gene Family In Grapevine And Function Verification Of VVSNRK2.2Gene

Grape (Vitis vinifera L.), is deciduous vine in the vitaceaefamily, and is one of the most economically important fruit trees worldwide. With the development of economy and explision of population, which leads to environmental deterioration, drought, salinity, high and low temperature and oxidative stresses which influence plant growth and development seriously, and then influence the productivity and quality of crops.Therefore, improving the drought tolerance has becoming one of the main objectives of the agriculture and horticulture. SnRK2(Sucrose non-fermenting1-related protein kinases2), is a class of Ser/Thr proteins that is specific to plant. Studies had shown that SnRK2s play an important role in plant growth and development, including cell division, plant metabolism, intracellular signal transduction and reponses to drought, extreme temperature and salinity. This study mainly includes3aspects:1.Using the method of bioinformatics to identify SnRK2s in grapevine at genomic level, and predict, analyze the evolutionary relationship, physical and chemical characteristics, secondary structures, gene structure, motif, C-terminus sequences and EST;2. Employing gene microarray and RT-PCR to analyze the expression patterns SnRK2s in tissues/organs development and different biotic/abiotic stress treatments;3. Clone VvSnRK2.2gene in5BB and construct the vector sucsessfully, then transform the Arabidopsis to understand the functions of VvSnRK2.2. The main results are as follow:1. We identified6SnRK2members in grape genome (VvSnRK2.1-VvSnRK2.6) employing the HMM search and phylogenetic tree. We then predicted and analyzed physical and chemical characteristics, secondary structures, gene structure, motif, C-terminus sequences and EST. The results showed that the genes identified in grapevine are hydrophilic protein, secondary structure are mainly a-helix and irregular curl. The VvSnRK2genes are classified into three subfamilies. The gene structure analysis showed that the exons and introns are conserved, all the genes have9exons, and almost all the exons are0phase expect VvSnRK2.3which is1phase. The motif and C-terminus sequence analyses showed that grape SnRK2protein kinases are highly conserved. The EST analysis showed that6SnRK2s are unevenly expressed in10tissues, maily expressed in fruit, cell suspension, leaf and flower.2. With analysis of mocroarray data, we determined the expression patterns of6VvSnRK2s in tissues/organs development and biotic/abiotic stress treatments. The tissue development microarray data showed that VvSnRK2s are involved in seed development, fruit development and senescence, and they are also induced by biotic and abiotic stresses. In seed development, VvSnRK2.1,VvSnRK2.2and VvSnRK2.3are mainly up-regulated, VvSnRK2.5are down-regulated, in fruit development and senescence, most VvSnRK2s are down-regulated, especially in VvSnRK2.5; In biotic/abiotic stress microarray analysis, VvSnRK2.6are induced by powdery mildew and heat stress, but VvSnRK2.1are induced by water stress and salt; the group I are not induced by ABA, the group II are weakly induced by ABA, and the groupIII are induced by ABA intensely.3. In order to analyze the function of6VvSnRK2s (VvSnRK2.1-VvSnRK2.6), we analyzed the expression patterns of6VvSnRK2genes under ABA, drought, NaCl, heat stress and cold with RT-PCR. The results suggest that VvSnRK2are widely induced by drought, ABA, NaCl and extreme temperatures, among them, VvSnRK2.1are up-regulated significantly by drought and NaCl, consistant to the microarray data; VvSnRK2.2and VvSnRK2.4are induced by heat stress; VvSnRK2.3is induced by cold; VvSnRK2.1and VvSnRK2.5are induced by ABA.4. To further verify the function of grape VvSnRK2s, we cloned VvSnRK2.2from5BB, a highly stress-tolerant rootstock clone, constructed plant binary expression vetor (35S:VvSnRK2.2), then transformed into Agrobacterium tumefaciens strain EHA105, then used for Arabidopsis transformation. We obtained6lines by GUS staining assay, and detected L34and L42by RT-PCR. Taking the seeds and seedlings of T3lines as material, we conducted the analysis of abiotic resistance assay. The result suggested that on ABA treatment medium, L34and L42transgenic lines are more sensitive than WT seedlings, and the leaves of transgenic lines grow normally, but the leaves of WT are upturned at6days after treatment. These results may suggest this gene’s association with stomatal movement or leaf tissue structure. Drought tolerance test confirmed that VvSnRK2.2can improve drought tolerance; but under NaCl treatment, transgeninc lines are more sensitive than WT. This extopic expression suggests that the grapevine SnRK2.2plays a significant role in drought response and regulation.