Cloning And Sequnce Analysis Of A Protein Kinase Gene GsGSK In Soybean

In this study, the homologous gene cloning strategy was used. The ORF sequence of soybean GmGSK gene registered in the GenBank was used as a reference to design primers. Total RNA was extracted from a wild soybean accession with salt tolerance, and a full-length cDNA was further isolated from wild soybean by RT-PCR. The complete open reading frame sequence was1230bp in length and encoded a peptide of410amino acid residues with molecular weight of46.49kD and an isoelectric point of8.71. Protein sequence analysis showed that it encoded a protein homologous with GSK, containing serine/threonine protein kinase active site, named GsGSK (Genbank Access number:JQ639024).Compared with the nucleotide sequence of the GsGSK gene from cultivated soybean, the GsGSXgene from wild soybean had11SNPs, in which10SNPs were synonymous mutations, and1SNP was non-synonymous mutation, with a homology of99.1%. Compared with the deduced amino.acid sequence encoded by wild, soybean and cultivated soybean, the197th amino acid was tyrosine (Y) for wild soybean and cysteine (C) for cultivated soybean. By aligning the deduced amino acid sequence and GSK genes from other plants, GsGSK showed a high homology with GSK genes.Restriction sites were designed flanking the GsGSK ORF and a plant binary expression vector pCAMBIA3300-GsGSK was constructed. GsGSK gene has been transformed to Arabidopsis by using the agrobacterium infection method. Herbicide resistant plants have been obtained by spraying Basta, and5T1transgenic plants were identified by molecular analysis. We are preparing to harvest the seeds from T1transgenic plants, to test the tolerance of T2transgenic Arabidopsis to stress.The function of the GsGSK gene from wild soybean is still unknown, and further study is required to verify whether it can enhance the stress tolerance of transgenic plants, similarly to that of cultivated soybean.