Cloning And Functional Analysis Of An Abiotic Stress Inducible Protein Kinase Gene GmGSK In Soybean

High salt, drought, low temperature and other abiotic stresses are important environmental factors to affect crop yield. Plants reduce their own damage through long-term evolution to resist the external environment stress resistance. Protein kinase may be reversible through the phosphorylation to participate in stress signal transduction, thereby further respond to external stresses on plants. Protein kinase played a very important role in the stress-tolerant pathway in plants.We used wheat TaGSKl gene sequence as a query. A full-length cDNA of the GSK-3-like gene was obtained by in silico cloning from Soybean database of GenBank and was further isolated from G. max by RT-PCR. The complete cDNA open reading frame sequence is 1230 bp in length and encoded 410 amino acid residues with a theoretical molecular weight of 46.5 kD and an isoelectric point of 8.64. Protein sequence analysis showed that it encoded a protein homologous with GSK, containing serine/threonine protein kinase active site, named GmGSK (Genbank Access number:FJ460228).Southern blot analysis revealed that it had at least two copies in G. max genome. Northern blot analysis indicated that GmGSK expressed in all tested tissues, with highest expression in root. Semi-quantitative RT-PCR analysis revealed that GmGSK gene could be induced by NaCl, NaHCO3, drought, ABA and cold, which suggested that GmGSK might be involved in soybean abiotic stress signal transduction process or tolerance to abiotic stress.We used pCAMBIA1302 to construct GmGSK:GFP vector and GmGSK:GFP fusion protein was introduced into tobacco by Agroinfiltration. The subcellular localization of GmGSK indicated that it was distributed in the cell membrane and cytoplasmic. GmGSK was transformed into Escherichia coli. (BL21) and successfully expressed with a 46.5kD detected. And Saccharomyces cerevisiae (INVSc1) transformed with GmGSK increased their tolerance obviously to NaCl, NaHCO3 and drought stresses than wild type.Finally, in order to further validate GmGSK biological functions in plants, a recombinant vector pCAMBIA3300-GmGSK was construted and transferred into tobacco for over-expression analysis. The results showed that the growth, relative biomass and rooting rate of transgenic tobaccos were significantly improved than wild type. Transgenic tobacco was treated with NaCl solution and results showed that transgenic tobacco could increase their salt tolerance significantly compared with wild type tobacco. Physiological analysis showed that transgenic tobacco increase the soluble sugar content significantly faster than wild type tobacco and extravasation relative conductivity growth rate was significantly slower than wild type tobacco under salt stress, which help the transgenic tobacco to improve salt tolerance.These results suggested that soybean GmGSK gene played an important role to resist abiotic stress. Over-expressed GmGSK gene could significantly improve the salt tolerance of yeast and tobacco, which indicated that GmGSK gene could be a promising gene in stress tolerance improvement in soybean and other plants in the future.