Clonging And Function Analysis Of SnRK2Family In Potato

The SnRK2(sucrose non-fermenting related protein kinase2) is a gene family coding forSer/Thr protein kinases, and it was believed playing very important roles in linking abioticstresses and metabolic responses of plants. In the present study, we first identified andcharacterized eight StSnRK2gene members from the potato genome, and analysised the genefunction using bioinformation. The subcellular localization was studied by infusion the GFPand StSnRK2. Using real-time PCR to study the StSnRK2gene expression in different potatotissue under abiotic stress. Also, we constructed the characteristics of relatedgene expressiongene expression vecor and the gene function in transgenic tobacco plants.Then the result will proclaim the function, presentation and signal transduction pathway ofStSnRK2in potato. The detail results are as follow.1. A genome-wide search using the cDNA sequences of Arabidopsis, maize and riceSnRK2as queries revealed eight SnRK2homologous sequences in potato genome. Thespecific primers were designed to clone the eight SnRK2homologous cDNA from the potatocultivar Longshu3’. The eight sequences were designated as StSnRK2.1, StSnRK2.2,… toStSnRK2.8and the confirmed full length of cDNA sequences were submitted to NCBI. Thecandidate coding sequences of potato SnRK2were ranged from1008bp to1089bp. Thepredicted protein molecular weight of StSnRK2varied from37.74kDa to41.5kDa. Thepeptide length was distributed from335amino acids to362amino acids. All StSnRK2have aconserved domain of Ser/Thr protein kinase. The peptide lengths of StSnRK2were close tothe SnRK2members which had been reported in Arabidopsisi, rice and maize. An unrootedphylogenetic tree was constructed with the amino acid sequences. The results demonstratedthat SnRK2are highly conserved in plant kingdom. StSnRK2have been related to the othersand could be divided into three distinct groups. The first group included StSnRK2.1,StSnRK2.2, StSnRK2.5, StSnRK2.7and StSnRK2.8. The second group included StSnRK2.4andStSnRK2.6. The third group included StSnRK2.3. All StSnRK2had nine exons exceptStSnRK2.6(seven exons). The cis-element analysis indicated that all members have ABRE,DRE and LTRE, but the number has different.2. Subcellular localization vectors were constructed and the GFP-fusion kinase proteinswere transiently expressed in onion epidermis cells via the Agro-bacterial infiltration. GFP fusion kinase proteins were observed solely to be localized to the plasma membrane andnucleus by a confocal laserscanning microscope.3. The expression was determined at the transcript level by qRT-PCR. The expression ofStSnRK2.1,2.2,2.5and2.6were higher in the root than in the other organs. The expression ofStSnRK2.7was also significantly higher in stem than in the others. The expression ofStSnRK2.3and2.8were higher in the leaf than in the others. The expression of StSnRK2.4was also significantly higher in tuber than in the others.4. The expression of StSnRK2displayed different expression level. After NaCl treat2-4h,the expression of StSnRK2.1、StSnRK2.2、StSnRK2.4and StSnRK2.6have rise, then decline,but the result of48h also high than the CK. The result of48h in StSnRK2.3、StSnRK2.5、StSnRK2.7and StSnRK2.8were lower than the CK. The expression of StSnRK2.1、StSnRK2.2、StSnRK2.3and StSnRK2.4were higher than others after PEG treat48h. The expression ofStSnRK2.5and StSnRK2.6identical to CK, but the StSnRK2.7and StSnRK2.8were lower thanthe CK. Only StSnRK2.3was sensitive to ABA.5. The relative expression levels of8members in Atlantic‘and Longshu No.3‘hadobvious difference after severe water stress. The relative expression of StSnRK2.1、StSnRK2.2and StSnRK2.3were significantly higher than the control, however, obvious difference wasfound in an increase extent of different gene expression. StSnRK2.7had no difference with thecontrol. The expression of StSnRK2.4was9.5fold in Longshu No.3‘, which was themaximum among the8members after water stress. The expression of StSnRK2.1, StSnRK2.2,StSnRK2.3, StSnRK2.4and StSnRK2.6, had a high significantly positive correlation with someof the physiological characteristics. The significantly negative correlation was observedbetween the expression of StSnRK2.5and StSnRK2.8and some of the physiologicalcharacteristics. Expression of StSnRK2.7had no relationship of physiological characteristics.6.Two binary vectors pBI121-35S-StSnRK2s was harboring the35S promoter wereconstructed and introduced into tobacco plants by Agrobacterium tumefaciens-mediatedtransformation. PCR and RT-PCR showed in the transgenic lines. The abiotic related genes(CBL3、NtERD10A、NtERD10B、NtERD10C) were analysis, the transgenic plant ofStSnRK2.7has no different, but the others displayed a very high expression level in transgenicplants. The expressing of StSnRK2.1-StSnRK2.8displayed the different resistance toadversities in tobacco plant. The StSnRK2.1、 StSnRK2.2、StSnRK2.3、 StSnRK2.5andStSnRK2.8displayed actively to water stress, the StSnRK2.4and StSnRK2.6is better than StSnRK2.7, the StSnRK2.7had no different from CK.