Studies On Obtaining, Identification And Exogenic Expression Of Transgenic Cotton Plants

Cotton has a long growth period and wide range of insect pest, with the damage of 10-16% every year, and even up to 30% in disaster year. The long term use of chemical pesticides to control insect pest not only increases the production cost of cotton, but also causes a series problems, such as the enhanced resistance of pest to pesticide, environment pollution and the ecological unbalance. By transformation, the cotton's ability of anti-insect could be raised, so the cost of insect pest is decreased and environment is protected. In currently cotton genetic engineering, a few resistant exogenic genes were used such as Bacllus thuringiensis {Bt) gene, Protease Inhibitor {PI) gene , Amylase Inhibitor (AI) gene, lectin gene, lipoxygenase gene, scorpion toxin gene, spider toxin gene, and so on. Among them, Bt, PI and lectin gene are mostly used. In this study, Bt and gna (galanthus nivals agglutinin) were transformed into cotton to increase the resistance to insect and aphid.Pollen-tube pathway transformation method was used to transform cotton variety SN with pBSGS1M carrying Bt and gna about 20 hours after pollination. The seeds were planted in greenhouse, then, 12 transgenic plants were obtained by kanamycin selection. Both PCR and Southern blotting analyses showed that Bt and gna had been integrated into cotton genome. The result of insect test showed that corrected death rate of the tested bollworm larva (Helicoverpa armigera) was over 80% within 5 days, and the left larva's growth and development was significantly suppressed. The test of aphid {Myzus persicae) growth suppression showed that there was the stronger anti-aphid activity in all transgenic plants, averagely, it could inhibit 60% density of aphid, the highest was 70%. All of facts indicated that we have obtained transgenic cottons resistant to bollworm larva and aphis byusing of the modified anti-insect genes.It showed, by the sandwich ELISA, that Bt protein had been expressed in all transgenic cotton to some extent. There were spatio-temporal changes of the content of toxin protein along with the developmental process of transgenic cotton. The toxin protein in adult leaf of square stage reached 2923.7ng/g FW. Since there was a signal peptide in the expression carrier, Bt protein could be introduced into the cell gap. Using the method developed in this test, the intercellular washing fluids was extracted. It was found that the toxic protein in intercellular washing fluids was 53.57ng/g FW by ELISA, so concluded that the expressed toxic protein had been secreted into the intercellular. Therefore, the orientation in the cotton cell of Bt protein would be explored.