Construction Of SSH Library Of Regeneration Related Gene, Cloning And Function Analysis Of GmVSPB Gene In Soybean

In this study, a SSH cDNA library was constructed related with soybean regeneration gene, and a soybean regeneration related gene GmVSPB was cloned. The futher research showed that the GmVSPB play an important role in cotyledons node regeneration and stem elongation in soybean. The main results were showed as followed:1. Soybean DN50was sampled10times by hour after induced by6-BA, which were mixed as material to construct the soybean regeneration related gene cDNA library by SSH. The ESTs were annotated by similarity to orthologs and paralogs detected with Blastn and Blastx. Function analysis showed that genes were annotated by similarity to signal transduction, macromolecules glucose and protein synthesis and metabolism, light and leaf morphogenesis, regulation of apoptosis, the cell defense, cell wall differentiation, and participate in a variety of hormones and cytokinin-mediated signaling pathways, and analysis the relative expression of some genes in the libraby.2. The GmVSPB gene was cloned from the library and ligated with the intermediate expression vector pCAMBIAI121-3300and plant expression vector pCAMBIAI121-3300-GmVSP, then transformed into E.coli and agrobacterium. Arabidopsis thaliana, tobacco, and soybean were used as transgenic receptor by floral dip method, leaf transform, cotyledon nodes, respectively. Identify the overexpressing plants by PPT and PCR.3. The tissue specific expression of the GmVSPB gene showed that the highest amount of gene expression was in cotyledonary node, followed by the stem and root. The expression level of GmVSPB gene began to rise at the4h after treated with6-BA, and reach the peak at the8h.4. Treat the soybean root and lateral root on hormone find that6-BA restrained soybean root and lateral root to growth, the GmVSPB gene is the negative factor to restrain the growth of the main root and lateral root.5. When tobacco treated with6-BA, then the vitro shoot regeneration is impacted on the hormone, the overexpression plants have higher regeneration ability than the wild tobacco.6. The GmVSPB play a role in promoting germination under the treated with6-BA, and overexpression of arabidopsis seed germination have sensitivity on high concentration of cytokinin.