Primary Study On The Transformation Of Nicotiana Benthamiana With CP Gene Cloned From Grapevine Leafroll Associated Virus-2

The disease caused by Grapevine leafroll-associated virus(GLRaVs) is widely distributed in China and causes great losses in quality and yield to infected grapes.In recent years,the strategy using coat protein of virus to obtain resistant crops has been proved to be a new efficient measure for the control of this virus disease.In this paper, we constructed the plant expression vector of grapevine leafroll associated virus 2 (GLRaV-2) coat protein(CP) gene,transformed Agrobecterium tumefaciens EHA105 containing the recombinant plasmid into Nicotiana benthamiana via the leaf explants, and obtained the transgenic plants of GLRaV-2 CP.The main results obtained are as follows:1.The plant expression vector of GLRaV-2 CP gene sense strand was constructed. Synthesized primer CP2-F/CP2-R,which contained a Xba I site at 5'terminal region and a Sma I site at 3'terminal region,then amplified GLRaV-2 CP gene by PCR using the primer.The PCR product was ligated to the plant expression vector pCAMBIA1301S,and transformed the plasmid into Escherichia colia DH10B cell. Selected positive clones by PCR and double-digested with Xba I and Sma I,and the plant expression vector of GLRaV-2 CP gene sense strand was constructed successfully.2.The GLRaV-2 CP was transformed into Nicotiana benthamiana successfully. The Nicotiana benthamiana,leaf explants were transformed with Agrobecterium tumefaciens EHA105 containing the plasmid pCAMBIA1301S,plasmid of GLRaV-2 CP sense strand,and plasmid of GLRaV-2 CP antisense strand obtained by our laboratory before,respectively.Through co-culture,select culture and rooting culture, 119 complete Nicotiana benthamiana plants were obtained,including 58 transgenic GLRaV-2 CP sense,35 transgenic GLRaV-2 CP antisense and 26 transgenic pCAMBIA1301S.The rate of differentiation was more than 80 persent.Extracted the total DNA from the Nicotiana benthamiana transformed with plasmids by CTAB method,identified them by PCR using specific primers and the positive detection rate was more than 80 percent.3.Constructing the propagation,rooting and transplanting system of Transgenic tobacco's.Propagating the GLRaV-2 CP,plasmid pCAMBIA1301S and GLRaV-2 RdRp transgenic tobacco plants and each of the leaf explants can differentiate almost 50 buds.Culturing the buds on three different kinds of the rooting medium,the results showed that the medium MS+IBA 0.1 mg/L was the best,the rooting rate was 100 percent.Then transplanted the rooting plants into the greenhouse,and the survival rate of transplant was more than 70 percent.