| CDPKs (Calcium-dependent protein kinase) have only been identified in plants and some protozoans. CDPKs are a unique class of Serine/Threonine (Ser/Thr) protein kinases. CDPKs play an essential role in plant Ca2+-mediated signal transduction, growth and development, biotic or abiotic stress responses. But the underlying mechanism remains largely unknown. Seed deterioration was often happen during seed growth, development and storage because it’s high protein content (about40%) and high oil content (about20%), In the previous study, we have identified resistance varieties of soybean seed, we have analysed seed deterioration between resistance and sensitive soybean cultivars (cv.) with proteomics, CDPK-SK5protein (NCBI GenBank:LOC547885) showed up-regulated expression patterns in response to HTH stress (high temperature and humidity stress) in sensitive soybean cultivars (cv.) NingzhenNo.1. herein, CDPK-SK5gene and promoter sequence was isolatied, bioinformatic, expression pattern analyses and subcellular localization were conducted. The main results are as follows:1. We have isolated the gene and promoter sequence of CDPK protein gene CDPK-SK5. The full ORF sequence of CDPK-SK5was1527and it encoded a57.11KD protein with508amino acids. The analysis of bioinformatic indicated that the hydrophilicity of CDPK-SK5protein had no transmembrane sites, contained multiple potential Ser, Thr, Tyr phosphorylation site, CDPK-SK5protein might have localized in the chloroplast. We identified the cis-elements in responsive to stress in promoter region of CDPK-SK5genes with prediction software contained TATA box, W box, CAAT box, GTAC, ABRE-like, MYC, MYB, ERE; Seed-specif ic promoter cis-elements of soybean contained RY repeat, SEF1motif, SEF4motif, E box; Light regulation promoter cis-elements contained GT-1sites, I box and GATA-box.2. Real-time quantitative PCR analysis showed that CDPK-SK5expression level in HTH, low tempature and organ specificity. Seed deterioration resistance of different varieties soybean seeds between Ningzhen No.1and xiangdou No.3CDPK-SK5gene expression was differences after HTH, both in0and168h did not show significant difference, both24and96h show significant difference. All for the performance of the seed deterioration resistance varieties Xiangdou No.3expression level was significantly higher than sentive varieties Ningzhen No.1, at24and96h difference reached a significant level. CDPK-SK5gene after low temperature stress, with the extension of time, Seed deterioration resistance of different varieties soybean seeds between Ningzhen No.1and Xiangdou No.3CDPK-SK5gene expression was differences. Both in7and24h expression level was no significant difference; At0h and19h, Ningzhen No.1expression level was significant higher than Xiangdou No.3; At14h, Xiangdou No.3expression level was significant higher than Ningzhen No.1. And CDPK-SK5gene expression at different growth stages also have significant differences. CDPK-SK5gene in vegetative growth period and reproductive growth period were emerged the first down-regulated after up-regulated trend; in vegetative growth period, the expression of S period expression level was significantly lower than R and L period, in Reproductive growth period, MS period expression level was significant higher than F to GS period, but F to GS period expression difference was not significant.3. Transient expression of the CDPK-SK5protein in onion epidermal cells showed that CDPK-SK5was localized in cell membrane.In this study, we isolatied CDPK-SK5cDNA, DNA and promoter sequences, bioinformatics characteristic analysis, transient expression analysis for further investigation on the gene function research and regulation mechanism research laid the molecular basis. At the same time, to further illustrate CDPK-SK5gene in resistance to seed deterioration, signal transduction and seed chemical transformation of the accumulation provide theoretical foundation. |
PDF Full Text Request