Target Gene Integration And Expression Of Transgenic Bt Gene Insect - Resistant Early Japonica Rice

Rice is not only an important food and cash crops, but also one of the three crops in Northeast China. More than 65% of the population on rice as the staple food in China, rice production safety relating to people’s livelihood. Nowadays, insect pests pose a serious threat to rice production safety which causing economic losses amounted to 11.5 billion yuan every year. The prevention and treatment of pests mainly dependent on chemical reagents in traditional agricultural production, but long-term heavy use of chemical agents is not only a waste of manpower, material and financial resources, but also cause environmental pollution, pest resurgence and other issues. Therefore, cultivating insect-resistant rice is essential. Currently, transgenic rice technology has become mature and breed a variety of transgenic Bt rice which has the effect of insect-resistant.In this research, univalent Bt insect-resistant rice Kongyu 131 and Songjing 9 as the research objects, using the method of PCR, test strip, real time PCR, ELISA, Southern blot and whole genome resequencing, studied at the level of DNA, mRNA, protein and the correlation of Bt gene transcription and expression, from which to choose the exogenous genetic stability, high level of transcription and expression, low copy number and the insertion point is not destroyed rice endogenous gene insect resistance of the transgenic Bt rice lines. The main results are as follows:1.Use Basta resistance screening, strip testing, PCR and other methods, after T0~T9 generation of screening, obtained early Bt japonica rice Kongyu 131 and Bt super rice Songjing 9 successfully, including 22 strains of HD1(transgenic cry1C* Kongyu 131),10 strains of HD2(transgenic cry2A* Kongyu 131), 45 strains of HD3(transgenic cry1C* Songjing 9) and 26 strains of HD4(transgenic cry2A* Songjing 9).2.Using the method of real time PCR detected the transcription levels of Bt gene in tissues at tillering and heading stage.The results showed that the transcription level of Bt gene is differences even the same organization in the same growing season of different strains in the same strain; in the meanwhile, the same line of different organizations in the same growing season, there are significant differences of Bt gene transcription level, from high to low in turn for leaf > young spike> stem-sheath; In addition, the same line of the same organizations in different growing season, there are significant differences of Bt gene transcription level, from high to low in turn for heading stage > tillering stage.3.Using the method of enzyme-linked immunosorbent assay(ELISA) detected the expression of Bt protein in organizations at tillering stage, heading stage, grain filling stage and mature stage. The results showed that expression of Bt protein is differences even the same organization in the same growing season of different strains in the same strain; in the meanwhile, The same line in the same growing season of Bt protein content in different organizations are significant differences which from high to low in turn for leaf > stem-sheath at tillering stage, leaf > young spike > stem-sheath at heading stage, leaf > stem-sheath> young spike at filling stage, the expression of Bt protein all the above are higher than brown rice at mature stage; In addition, the expression of Bt protein is differences even the same line of the same organizations in different growing season, it is showed that filling stage > heading stage> tillering stage in leaf and stem-sheath of HD1、HD3 lines, filling stage≈heading stage> tillering stage in leaf and stem-sheath of HD2、HD4 lines. The expression of Bt protein in young spike is heading stage≥filling stage> mature stage in different lines.4.Use SPSS software to detect the correlation beween Bt gene transcription level and the expression of Bt protein. The results showed that it is significant positive correlation between the Bt gene transcription level and the expression of Bt protein at 0.01 or 0.05 level in leaf and young spike of HD1,HD2,HD3,HD4 lines.5.Use Southern blot to detect the copy number of selection marker gene and target gene in transgenic Bt rice. The results showed that after continuous passages, the selection marker gene and target gene showed more stable, though the copy number is different, but all between 1 and 2.6. Using the technology of whole genome resequencing to detect the foreign gene’s insertion site in HD1-3、HD2-2、HD3-2 and HD4-3 strains of the transgenic Bt rice. The results showed that the foreign genes were respectively inserted into No.3, No.7, No.8 and No.1 chromosomes.