Cloning And Functional Study Of GmSOT1 Gene From Soybean

Soybean [Glycine max (L.)] which is an important economic and food crops blongs to leguminosae, providing a wide range of plant proteins for human being and important raw material for industrial production. Glucosinolates is a kind of secondary metabolites with sulfur and nitrogen, which exist in the crucifer. Its hydrolysis product proliferation and insect-resistant can inhibit bacteria, fungi, and so on. Sulphotransferase is one of the key enzymes in the synthesis pathway of core structure. Thus it has an important position.This study extracted RNA from roots of soybean through the method of reverse genetics, and reversed transcription into cDNA. we have gotten the CDS (coding sequences) of GmSOT1gene, being composed of 1014 bp nucleotide and encoding a length of 337 amino acid residues of protein. Then soybean GmSOTl gene was amplificated by PCR (polymerase chain reaction), and connected to the polyclonal loci of plant binary expression vector pCAMBIA2301, named pCAMBIA2301-35s:GmSOT1. And then, the GmSOT1 gene was integrated to soybean by Agrobacterium-mediated genetic transformation. We can saw the transgenic plants which were overexpressed of the GmSOTl gene and knock-out were smaller than the controls. Above all, the knock-out plantlets of GmSOT1gene were smaller and darker than the controls. There was abvious whitening at the both ends of seeds.To study the expression location of GmSOT1genes in plant cells, this study constructed a plasmid vector that was fused GFP before C-terminus of GmSOT1 gene. The fluorescence was observed that gene was expressed in cell membranes and cytoplasms under the fluorescence microscope through the transient expression of tobacco leaves.To reform rooting system of transgenic soybean, we made focus on rooting initial explants, concentration of IBA and rooting pre-treatment of soybean resistant shoots. The results show that the rate of inducing adventitious roots with hypocotyls was higher than inducing adventitious roots without hypocotyls. The survival rate of resistant soybean plantlets was 74.88% and the roots were robust when the IBA is 1.0 mg/L. The rooting perior was reduced to 19.21 d and the rooting frequency of Soybean adventitious roots induction added to 98.42% after being pre-treated as pre-treatment Ⅲ, namely:cultured in 1/8 MSB5 medium for 5 d, then transferred to 1/4 MSB5 medium for 5 d, finally transfered into 1/2 MSB5+1.0 mg/L IBA to induced adventitious roots. And then, the genetic transformation efficiency of soybean was increased to 23.6%.In-depth study of GmSOTl gene not only helps to conprehend the molecular mechanism and physiological function of sulfotransferase in synthesis of glucosinolate, but also is expected to create disease resistance soybean germplasm which was increased capacity of nitrogen fixation. The function research of GmSOTl gene is keeping on.