Research Of Rice Gene Targeting And Genetic Transformation System Of Suspension Cells

As the expression product of rice gene Bel,CYP81A6(the cytochrome P450) is responsible for resistance to herbicide bentazon,so the bentazon susceptive rice could be obtained with the knocking out techniques.While the targeting effect of different vectors with various length of homology fragment are distinct.The vector pRB2 with about 2 kb left and 2 kb right homologous fragment and the vector pJL9 with 4 kb left and 4 kb right homologous fragment are constructed in this research,while the pRB2 vector can be used in target gene Bel of ShijinB.Otherwise,Agrobacterim-mediated genetic transformation system of ShijinB suspension cells was successfully constructed in this research.The mainly result as follows:1.Bel targeting vector pRB2 with Bel homology length of 2224 bp on the left and 2394 bp on the right,and the hpt and DT-A as its positive and negative selection marker was contructed,and Bel targeting vector with the about 4kb left and about 4kb right Bel homologous fragment is being constructed.By now,pJL9 vector containing a positive selection marker which flanked by 4373 bp left and 3936 bp right homologous fragment was successfully constructed.2.3 resistant callus were obtained after transformation of 15.425 g rice callus with Bel targeting vector pRB2 and then 28 rice were differentiated from those resistant callus.It was identified using the PCR method that 27 out of 28 differentiated plants are transgenic rice but it was also found that the foreign gene was random inserted in rice genome without homologous recombination.3.Agrobacterim-mediated genetic transformation system of ShijinB suspension cells was established.The callus were induced by the mature seeds of ShijinB,which followed by suspension cultured to obtain dynamic cells.Cells were manipulated as follows:precultured for a week;co-cultured in N6â…¡medium;transferred to N6â… medium including cefotaxime 250 mg/L,carbenicillin 250 mg/L,hygromycin 50 mg/L for screening;transferred onto MSâ… C medium for differentiation.Plantlets coming from resistant callus were transferred to MSâ…¡medium.PCR amplification indicated that the exogenous gene had been introduced into the rice genome.