Study On Integration Characteristic And Expression Stabilization Of Foreign Gene In Transgenic Birch (Betula Platyphylla)

This paper took the insect-resistant transgenic birch (Betula platyphylla) grown in greenhouse for four years as study materials. The genomic DNA and RNA of leaves were extracted and the integration and the expression characteristic of the extraneous genes in the insect-resistant transgenic birch were analysed by methods of multiplex PCR (MPCR), reverse primer PCR (RP-PCR), inverse PCR (IPCR), RT-PCR, and insect-feeding experiment of the transgenic plants respectively. Results are as follows:1. The condition of the multiplex-PCR method fast examination for extraneous gene integration in transgenic birch was conformed. Simultaneously the improved mPCR method provided a rapid and reliable technique for detecting multiple genes in transgenic birch.2. The condition of reverse primer PCR (RP-PCR) and direct primer PCR method in transgenic birch was conformed. The result of reverse primer PCR (RP-PCR) and direct primer PCR associating with Southern blotting could determine the copy number of T-DNA integrated into the host genome. Then the transgenic birch was classified according to the T-DNA copy number.3. Improved inverse PCR (iPCR) method was utilized and 12 T-DNA flanking sequences (included right border and left border) of the transgenic birches were cloned. All sequences were analyzed. The results primarily revealed integrated characteristic of the flanking sequence: AT in rich ( more than about 60%) and incomplete integration of T-DNA such as deletion and filler DNA et al;The result proved an improved reverse PCR method applied to T-DNA flanking sequence analysis of the transgenic birch was feasibility and rationality.4. Utilizing the insect-feeding experiment associated with half quota RT-PCR method, how strong the target gene expression and insect-resistance were showed. Also, compared with the early study, the insect-resistant stabilization of transgenic birch was evaluated. In the end, the insect-resistant plants were selected, and the relation between target gene's expression and the anti- insect function was established.