Cloning The Gene Of EPSPS,Constructing Of Expression Vector And Genetic Transform Of Flax

In the experiment,EPSPS gene was cloned by PCR from transgenic herbicide glyphosateresistant oil rape,then it was constructed on the plant expression vectors pEarlygate100andpMDC139by Gateway. Two vectors were transferred into LBA4404by triparantal crossing.Thesystem of oil fax transformation and detection mediated were established，which make thefoundation for cultivating high yield,high-quality and herbicide resistant oil flax in northwest ofChina.The research suggested:1.EPSPS gene was amplified with high-fidelity Q5DNA polymerase by PCR fromtransgenic herbicide glyphosate resistant oil rape genome DNA and was cloned into clonevector pENTRTM/D-TOPO.The sequencing and analysis result showed the identical CDSsequence as the known one in Monsanto company’s patent(US5633435). Then the sequencewas constructed into plant express vectors pEarlygate100and pMDC139, These tworecombinant vectors pEarlygate100and pMDC139with EPSPS gene were confirmed byrestriction enzyme and PCR analysis and the results showed that these recombinant plantvectors were constructed successfully.2.Based on above efficient protocol of plant regeneration, Agrobacterium strainLBA4404containing recombined pEarlygate100-EPSPS and pMDC139-EPSPS were used todevelop the Agrobacterium-mediated flax transformation system. In this experiment we mainlyfocused on selection pressure,and concentration of Ceftriaxone. Results showed that theoptimum concentration of Hygromycin for selection was20mg/L,PPT was mg/l and Glyphosatewas60mg/L. The optimum concentration of Ceftriaxone Sodium which was used for inhibit thegrowth of Agrobacterium was400mg/L respectively.3.The experiment mediated rooting medium of oil flax regenerated seedings by NAA,Activated Carbon and PP333.The result showed the optimal rooting medium for regeneratedseedlings of oil flax was1/2MS+0.01mg/L+0.01mg/L PP333+0.1%ActivatedCarbon。4.Oilflax ‘longya No.10’ were infected by Agrobacterium containing recombinedpMDC139-EPSPS and pEarlygate100-EPSPS plant expression vector.Transformations wereconfirmed by Gus stain and PCR. Obtained positive transgenic plants and established a flaxGenetic transformation detection system.