Cloning Of Epsps Gene And Preliminary Study On Sweet Sorghum Transformation

The gene of5-enolpyruvyl-shikimate-3-phosphate synthase was simply called EPSPS. Plant cell produced the resistance of glyphosate of herbicide mainly through EPSPS active site’s changes, so the gene of glyphosate-resistant was known by us as EPSPS. At present, the glyphosate-resistant soybeans which were widely planted in the United States were developed on the basis of this mechanism. Its exogenous gene what was simply called CP4-EPSPS gene originally came from the gene of EPSP synthase in the CP4strain of soil bacterium.Now sweet sorghum was recognized as the most advantage of the sugar, feed and energy crop. But up to now, the varieties of sweet sorghum with excellent resistant characters were still needed to be reformed. The use of transgenic technology played a great significance in the existing varieties of sweet sorghum for traits’improvement and molecular genetic research in which the performance of herbicide resistance was also a very important aspect. In order to obtain the new varieties of sweet sorghum with high stress resistance, the building of the efficient and stable genetic transformation played a key role, so it is particularly important in the development of the new variety of herbicide-resistant transgenic sweet sorghum.The study showed that the EPSPS gene was cloned from the glyphosate-resistant transgenic soybean. Plant expression vector was constructed and then it was transferred into Agrobacterium tumefaciens for the next step of the genetic transformation. And the mature seeds of BT3which was introduced to be planted in Nanning in Guangxi was treated as explants. The efficient regeneration system was firstly established and the genetic transformation which was mediated by Agrobacterium was discussed. The main research’s results were studied as follows:(1) The full-length of the EPSPS gene was cloned from glyphosate-resistant soybean. It was sequenced to be proved entirely correctly and encoded four hundred and fifty-five amino acids. The plant expression vector PCAMBIA1300was used as vector, UBI as promoter and NOS as end sub. The expression vector of EPSPS was constructed.(2) The efficient and stable tissue culture regeneration system of sweet sorghum BT3was established. The mature seeds were used as explants and firstly treated30s-50s by95%alcohol, then flushed by sterile water at a time. Next, They were treated by0.2%mercuric chloride, and then flushed by sterile water for three or four times. So it could achieve the desired disinfection effect. The optimal culture medium of inducing embryogenic callus was MS+2.0mg/L2,4-D+0.05mg/L6-BA. The effective proliferation culture medium was MS+1.5mg/L2,4-D+0.05mg/L6-BA. The efficient differentiation medium was MS+1.0mg/L6-BA+0.1mg/L NAA. The rooting medium was1/2MS+0.05mg/L IBA or1/2MS+2.0mg/L IBA.(3) The critical concentration of Hygromycin resistance about the embryogenic callus of sweet sorghum was screened out to be75mg/L. The growths of non-transgenic callus couldn’t be suppressed by the Hygromycin concentration of25mg/L. The new callus of individual number was produced in the concentration of Hygromycin medium of50mg/L. All the callus died fast in the concentration of100mg/L, but the survival rate about new callus of one point one one percent was also produced in the concentration of75mg/L. It was in the reasonable range of false positive. The optimum concentration of Carbenicillin about inhibiting Agrobacterium tumefaciens was screened out to be500mg/L. The growths of the callus wouldn’t also be affected. So the screening medium of MS+1.5mg/L2,4-D+0.05mg/L6-BA+75mg/L Hygromycin+500mg/L Carbenicillin was suitable.