Insect The Gene Cry2a ~ * Early Japonica Transformation

As the biggest area of japonica rice in China, Heilongjiang province held the balance for agricultural production of our country. Since 1998, Chilo suppressalis was first found in the field of Wuchang city of Harbin region, it had became more serious year after year and spread widely which significantly reduced the yield of rice. The transgenic technique could be an effective way to solve the pest problems.In this study, Bt gene cry2A^* under the regulation of ubi promoter with the selective gene bar was transferred into typical varieties of japonica rice in Heilongjiang province via Agrobacterium-mediated transformation, in order to improve their stem borer resistance. The main results were as follows:1. Enzyme digestion analysis and PCR detection indicated that cry2A^* gene could be transferred into the rice of Heilongjiang via Agrobacterium-mediated transformation.2. The japonica rice transgenic system of Agrobacterium-mediated transformation had been established by optimizing many factors involving in transformation effect, which were callus induction, callus subculture, callus preculture, infection time, co-culture, using washing solution with osmotic potential, adding 15mg/L PPT in screening medium, pre-regeneration, adding 250mg/L CN in screening medium, rooting, acclimation and transfer.3. Cry2A* gene was transferred into the genome of Heilongjiang rice varieties such as Kongyu131, Sonjing9 and Sonjing6 with the optimized japonica rice transgenic system of Agrobacterium-mediated transformation.4. PCR was carried out to detect the integration of cry2A^* gene in T₀ regeneration plants, showing that the ratio of positive plants were 98.2% in Kongyu131, 100% in both Sonjing9 and Sonjing6.5. Resistance to PPT analyzing in T₁ regeneration plants of Kongyu131 suggested that most of transgenic lines follow Mendal'inheritance law 3:1, indicating that transgenic gene was integrated in one locus of plant genome. RT-PCR assay indicated that the cry2A^* gene expressed in the transgenic plants.