Cloning And Functional Analysis Of Puccinellia Tenuifolra SnRK2 Gene (PutSnRK2)

SnRK2 (Sucrose non-fermenting 1-related protein kinases 2) is a kind of protein kinase which exists only in plant. As a member of family of SnRK family (SnRK1, SnRK2, SnRK3), SnRK2s had been cloned in many plants.The members of SnRK family, encoding Ser/Thr like protein kinase,can response to different stress signals in plant, which play an improtant role in plant stress resistance physiological.In this study, we cloned a SnRK2 gene from Puccinellia tenuifolra cDNA library dealted with NaHCO3. The full-length of PutSnRK2 gene was 1726bp, with an open reading frame (ORF) of 1077bp. This gene encoded a protein of 358 amino acids, with a predicted molecular of 41.1 KDa, and an isoelectric point of 5.63. BLAST at NCBI, we found PutSnRK2 has high homology to SnRK2 genes in Arabidopsis, rice and maize. The protein sequence of PutSnRK2 shared 91% identities with rice SAPK7 and maize ZmSnRK2.7. The acticity domain of SnRK2 members were relatively conservative, but their C-terminal showed high diversity.Semi-quantitative RT-PCR was conducted to analysis of the expression of PutSnRK2 gene at mRNA level. The results showed that PutSnRK2 gene expressed in each tissue. The transcriptional levels of the PutSnRK2 genes were effected by salt (NaCl, NaHCO3, Na2CO3) and drought (ABA, PEG6000), and changed with the time course, which suggested that there existed a relationship between the expression of PutSnRK2 gene and enrironment stress.The experiment used pYES2 as expression vector, and pYES2-PutSnRK2 was transformed into yeast (Saccharomyces cerevisiae) stain INVScl. The result showed that the growth of cells expressing PutSnRK2 was better than control under Na2CO3, NaHC03 and H2O2 stress, especially under NaCl and Mannitol. These results suggested that the expression of PutSnRK2 in yeast increased the cells' tolerance to various environmental stress. Additionally, in order to understand the sublocation of PutSnRK2, we expressed the GFP-PutSnRK2 fusion protein in yeast. The results showed that the GFP-PutSnRK2 fusion protein located in the nucleus and the cytoplasm.The gene was cloned into pBI121, and introduced into A.thaliana by Agrobacterium tumefaciens-mediated transformation using the vacuum infiltration method. The 16 independent and stable expression lines of over-expressing PutSnRK2-GFP gene were confirmed by PCR and Northern blot analysis. Salt tolerance of the three independent transgenic lines were examined, and the transgenic plants grew better than wild-type plants. These results indicated that PutSnRK2 played an important role in plant response to salt.We chose the transgenic lines with high expression, in order to identify the expression of PutSnRK2 interacted genes. The result showed that PutSnRK2 can regulate the expression of AREB downstream genes, and may be have interact with some AREB upstream genes to regulate the expression of AREB.