The Research Of Germplasm Innovation Of Bt Island Cotton And The Grafted Conduction Of Insect-resistant Protein

Since the Bt gene is taked into cotton, the spread of Bt cotton in China greatly reduced the labor intensity of farmers planting,so it is widely welcomed by farmers.Bt gene (thuringiensis) is a specific genes play an important role in plant insect-resistant (lepidoptera specially) which is studied more in recent years.When insects attack the cotton, insect-resistant gene will be activated and express.Insect-resistant protein are formed by gene transcription, translation, which is high toxicity to the lepidoptera insects. So as to improve the resistance of plants to the lepidoptera insects, and the insect resistance of plants is comprehensive improvement. It is the most ideal genetic engineering in plant insect-resistant by far.In this paper we cloned a Bt gene by cloning method in cotton41, then take it into the island cotton by injection through pollen tube.Through the hybrids in cotton41and island cotton, the grafting with cotton41and island cotton, we want to improve the insect resistance in island cotton in several ways which are transgenosis, introgressive and Protein transfer. Main research results are divided into two parts that are germplasm innovation and the grafted conduction of Insect-resistant protein.The part of germplasm innovation is as follow:1.Designing specific primers p1and p2according to the Bt gene sequences of cotton41for PCR amplification, and get a1824bp long gene.2.Connect the gene which is getted by PCR amplification to the T cloning vector.3.P1and P2respectively containing enzyme digestion sites with sacl and bamhl. Take double digestion with sacl and Bamhl on Bt gene that is connected to cloning vector and pbi121plasmid, then connect ed with T4DNA ligase to construct the plant expression vector.4.1nject the plant vector plasmid into island cotton through pollen tube, Through the resistance of kan, PCR to identify the posotive plants.5.Through the hybrids in cotton41and island cotton, back cross and test cross in F1, to obtain hybrids with Bt gene by the way that is introgressive. And through the resistance of kan, PCR to identify the posotive plants.The part of grafted conduction of Insect-resistant protein is as follow:1. Cotton41and island cotton were grafted as scion and stock mutually.2. Detect the level of Bt protein in island which is grafted to cotton41throuth ELISA techniques, the results show that the Bt toxic protein is transferred from cotton41to island cotton.