Analysis Of GmPKS4 Gene Expression And Identification Of Interaction Protein In Soybean

PKS(PKS,protein kinases),also known as CIPK(CIPK,CBL-interacting protein kinases),encodes a serine/threonine protein kinase and interacts with calcineurin B-like protein(CBL,Calcineurin B-like protein),which exerts a variety signal transduction of plants in stress.In order to preliminarily identify the soybean GmPKS4 gene,the expression of this gene in different tissues of soybean was analyzed;A plant expression vector was constructed to determine the localization of GmPKS4 at the subcellular level;The prokaryotic expression vector was constructed and the GmPKS4 protein was successfully induced.The interacting protein with GmPKS4 was identified by using yeast two-hybrid technique.The overexpression vector was constructed to obtain Arabidopsis thaliana overexpressing GmPKS4 by Floral-Dip.The main experimental results are as follows: 1.The expression of soybean GmPKS4 gene in different tissues was detected by qRT-PCR method.The results showed that the GmPKS4 gene was expressed in roots,stems,leaves and flowers,and the expression level was the highest in leaves.In the embryos of different stages of soybean,the expression level was lower in the early embryos and the highest in the 50 d embryos,then the expression level in the mature soybean grain reduced.2.The plant expression vector pCAMBIA1302-GmPKS4 was constructed and the transient expression of GmPKS4 gene in tobacco mesophyll cells was achieved by injecting tobacco leaves.GmPKS4 was localized on the cell membrane and nucleus by using laser confocal fluorescence microscopy.3.The prokaryotic expression vector pGEX4T-3-GmPKS4 was constructed and transformed into E.coli competent Transetta(DE3).The optimal conditions for induction were 28�C,0.2 mM IPTG for 4 h.Detected by Western blotting at 74 kDa a distinct band indicates that the GmPKS4 protein was expressed successfully,laying the foundation for subsequent purification of the protein.4.The bait expression vector pGBKT7-GmPKS4 and the prey vector pGADT7-GmSNF4 were constructed.Experiments showed that soybean GmPKS4 gene had no toxic to yeast;pGBKT7-GmPKS4 had no self-activation;Yeast two-hybrid experiments showed that GmPKS4 interacts with GmSNF4.5.By Constructing a plant overexpression vector pBASTA-GmPKS4,GmPKS4 was transformed into wild type Arabidopsis thaliana by Floral Dip method,and nine strains of T1 positive plants were obtained.The segregation ratios of the T2 generation positive plants were tested and it was found that three lines were in accordance with the Mendelian separation law,providing a preliminary basis for the phenotypic analysis of the GmPKS4 gene.