Creation Of Transgenic Bt Insect Resistant Rice Using A Green Tissue-specific Promoter To Minimize Expression Of Bt Proteins In Seeds

Transgenic insect resistant crops expressing Bt insecticidal proteins have been widely used for crops improvements, for example, maize and cotton. However, as to commercial planting of Bt transgenic rice, one public concern is the Bt protein expressed in the rice seeds. Simultaneously, with the insect-resistant transgenic plants increasingly spread, and lots of Bt toxic proteins transformed alone, the problem about insect-tolerance is becoming more severe. Therefore, to mitigate the concern over the Bt proteins in the transgenic rice and to prevent or delay the insect tolerance we design the experiment using green tissue specific promoter and fusion of two insecticidal genes method. In this paper two transformation vector pGreen-CrylAc-223and pZmUbi-Cry1Ac-223are constructed. Cry1Ac and Cry1Ie genes were fused together. The fusion gene Cry1Ac-223was separately directed by pGreen promoter and ZmUbi promoter. Compared to the toxic protein level, the strength and specificity of the pGreen promoter was investigated. The glyphosate resistance gene G10was used as the selection marker. Transgenic rice lines were obtained by agrobacterium-mediated transformation. In greenhouse, Heliothis armigera (H.armigera) insect-resistant test found the positive lines, into which the insecticidal gene was transformed. Glyphosate resistance bioassay tests showed that the transgenic plants had good effect. According to western blot analysis and comparison of the two promoters, the toxic protein was detected in leaves of transgenic plants containing the specific promoter, but little tested, less than17ng/g. Highly insect-resistant lines with single-copy insertion were selected based on field assessment and southern blot analysis in the T1generation.after analysis of agronomic traits for the single-copy lines, one good line S20was picked. The S20line was studied in detail for breeding new variety and further research resource, including identification of the T-DNA insertion site via Tail-PCR and PCR, the rice leaf folder (Cnaphalocrocis medinalis Guenee) and the striped stem borer(Chilo supperssalis Walker) insects bioassays, and selection homozygous plants by spraying glyphosate.