Bt-pta Gene Rices In Molecular Detection And Identification Of Insect Resistance

Rice is one of the most important food crops and staple food for more than half of the population in the world. For a long time, pest infestation is an important factor in rice production, and the use of transgenic insect-resistant rice cultivating technology can effectively solve this problem.In this study, excellent rice restorer line Fuhui 838 was taken as receptor material,Agrobacterium-mediated transformation method was used, recombinant expression vector p CAMBIA1301-Bt-pta was transferred into transgenic plants, and positive transgenic plants were screened. Through the detection of expression of GUS reporter gene, and detection of PCR, and screening the positive T₀ transgenic rice which containing the purpose genes. Then we harvested the rice and planted them. Through the detection of expression of GUS reporter gene and detection of PCR, detecting the T₁ bivalent genetically modified rice, and then transgenic rice insect-resistance were identified. We harvested the transgenic rice which had a better insect-resistance, and then planted them and gave a detection of PCR, then obtained the homozygous bivalent transgenic insect-resistant rice.The main results obtained in this study are as follows:1. The construction of bivalent plant expression vector: At first, we constructed the intermediate vector, and the basis of the expression vector plasmid p UC57-Bt and p CAMBIA1301 were double digested with HindⅢ and Spe I. The purpose Bt gene and vector backbone p CAMBIA1301 were recycled, then the target gene was connected to the vector backbone by T4 DNA ligase was constructed intermediate vector p CAMBIA1301-Bt. Using the Hind Ⅲ p CU57-pta and intermediate vector p CAMBIA1301-Bt were digested, and recovered the target gene of intermediate vector backbone. Then using the T4 DNA ligase the pta gene was connected to the intermediate vector backbone construct plant expression plasmid p CAMBIA1301-Bt-pta.2. Acquisition and preliminary testing of T₀ transgenic rice: The bivalent plant expression vector p CAMBIA1301-Bt-pta were transfered into rice restorers of Fuhui838 by Agrobacterium-mediated transformation method, after hardening, got a total of105 plant regenerated, and after GUS staining and PCR detection, eventually obtaining three plants T₀ transgenic positive rice.3. The molecular detection and identification of insect resistance of the T₁ transgenic rice: The T₁ transgenic rice were planted which were harvested individually and had a detection of expression of GUS reporter gene, preliminary detected positive transgenic rice. Then we had a detection of PCR, ELISA of Bt and insect-resistance detecting indoor and outdoor, and detected 29 plants insectresistance better bivalent transgenic rice, then had a agronomic traits investigation。4. The acquisition of T₂ homozygous bivalent transgenic rice: The T₁ transgenic rice were harwested individually which had a better insect-resistance, then we planted them and obtained 29 rice lines. We selected ten from each lines randomly and had a detection of PCR. There were 29 strains that containing target genes in all ten plants selected. The obtained 29 strains had target genes and were homozygous, and they can provide restorers materials for grouping with hybrids and obtaining insect-resistant hybrid rice.