Functional Analysis Of Puccinellia Tenuiflora Sucrose Non-Fermenting 1-Related Protein Kinase (PutSnRK2)

SnRK2s (Sucrose non-fermenting 1-related protein kinases 2) are a kind of plant-specific serine/threonine protein kinases, which have been cloned in many plants. Plant SnRKs are grouped into three subfamilies (SnRK1,SnRK2, SnRK3). Plant SnRK2s can respond to various stress signals in plants and play an important role in plant stress resistance. In this study, the amino acid sequence, expression characteristics, subcellular localization, purification of recombinant proteins of PutSnRK2 from Puccinellia tenuiflora were characterized to analyze its function. And transgenic plants resistant results were obtained under different stresses to analyze its function in further.First of all, the amino acid sequence of PutSnRK2 was analysed. Bioinformatics analysis results showed that all the family members of plant SnRK2s contain a core domain of Ser/Thr protein kinase. PutSnRK2 showed high similarity with the SnRK2 members which have been reported in Arabidopsis and rice. A phylogenetic tree was constructed using the full-length amino acid sequences of PutSnRK2 and SnRK2 subfamily members of Arabidopsis and rice. The results demonstrated that SnRK2s are highly conserved in plant kingdom, and PutSnRK2 protein had the closest relationship with SnRK2 proteins in rice with the highest homology with the rice SAPK7 in 75% identities.They both belonged to the same subfamily SnRK2b. Next, in order to analyze the function of PutSnRK2, the expression patterns of PutSnRK2 genes were analyzed using RT-PCR under NaHCO3, NaCl, ABA or PEG stresses treatments. The results showed that the expression of PutSnRK2 gene was significantly induced in leaves and roots of the Puccinellia tenuiflora treated by these stresses. The expression of PutSnRK2 was increased in roots and decreased in leaves, which suggested that there existed a relationship between the expression of PutSnRK2 gene and environment stress. The plant protoplasts of transgenic Arabidopsis over-expressing PutSnRK2-GFP were extracted to examine the subcellular localization of the PutSnRK2-GFP protein. The results showed that the PutSnRK2-GFP fusion protein was localized in the cytoplasm.In addition, to analyze the function of PutSnRK2 protein, the PutSnRK2 gene was cloned into the pGEX-6p-3 plasmid and expressed in Escherichia coli BL21 (DE3). GST-PutSnRK2 fusion protein was obtained through induction and purification. These results offered a foundation for analyzing the kinase activity and protein-protein interaction of PutSnRK2 in future. At last, the phenotype of transgenic Arabidopsis over-expressing PutSnRK2 was analyzed.The results showed that transgentic plants overexpressing GFP and PutSnRK2 showed slow growth compared with the wild-type plants under normal condition. However,the overexpressing PutSnRK2 significantly enhanced tolerance to salt and PEG in transgenic plants compared with wild-type plants, which indicated that PutSnRK2 played a key role in plant in response to salt and PEG stress.In conclusion, these results suggested that PutSnRK2 was involved in abiotic stress. This study laid a foundation for further studying the function of PutSnRK2 gene.