Detection Of Disease Resistance For Cotton Cultivar Transformed With Trichoderma Chitinase Gene In Xinjiang Upland Cotton

Cotton is one of primary economic crops in our country and has an important role in national economy.Xinjiang is located on the northwest of China and its characteristic climate has provided natural conditionand maintenance to cotton growth but in the 1980s, verticillium wilt of cotton began to happen and spreadincreasingly. Elementary investigation in 1990s showed that 40% of cotton growing area had this disease inNorth cotton growing area of Xinjiang and in which there was 8% cotton growing with heavy status, somearea evenly was diseased to 30% which effected fitally the yield and quality of cotton and has become oneobstacle of developing cotton production in Xinjiang Autonomous Region. Therefore, how to breed newcotton materials resistance to disease especiaaly to verticillium wilt of cotton became one major issue to beresolved presently in cotton production and cotton breeding. For this disease there had a short of resistantresource to be applied now. Interestingly transfomating foreign resistant gene into cotton by geneengineering has provided a new path for resistant breeding of cotton.In this experiment,gene Trichoderma chitinase resistance to verticillium wilt of cotton constructed inexpressive vector pART27 was transformed into 4 cultivars and lines of Xinjiang upland cotton throughpollen tube path and transgenic plant were detected by PCR polymerase chain reaction. Test results are asbellows:1.Transformation of gene Trichoderma chitinase Transformation method was ovary injection makeing the gene Trichoderma chitinase get into ovuletransform zygote cell through pollen tube path. The number of injected flowers of acceptor cultivars andlines:line 9, Xinluzhong 15, Xinluzhong 19 and T4 was 581, 2067, 808 and 238, respectively, and the bollnumber was 41, 328, 47 and 39; and the ratio of boll ration was 7%, 15.9%, 5.8%, 16.4% and 12.3%.2.The test of positive reaction of marker genes 1.64kg T0 and T1 seeds were grown in disease plot and got seedling 4012 with percentage of plantlets75.2%. 2-3 leaves plants were sprayed with kanamycin at a consistency of 500ppm, and three times wasproceeded, then got 50 plants with positive reaction of un-colourful results.3.detection of transgenic plants The gene Trichoderma chitinase constructed in the expressive vector pART27 was PCRed and the resultsshowed that the target gene was transformed into the cotton cultivars and lines with NPTâ…¡gene.Extracted liquid by the leaves of transformed plants with the gene Trichoderma chitinase were made andthe results from detection of bacteriostasis showed that the products of Trichoderma chitinase in the leavesof transgenic plants were more than the control plant( non-transgenic plants) and which indicated thatforeign gene was expressed in the plants. The expression status of Trichoderma chitinase gene detected byethyl-enzyme isozyme electrophoresis showed that got special protein band with the same molecularweight vs theoretical estimation. T2 seed of transgenic cotton of upland Xinluzhong 15 and line 9T and theseeds of non-transgenic cotton of upland Xinluzhong 15 and line 9T were grown in disease plot verticilliumwilt of cotton to detect their resistant capacities and at the same time to select the capacities of resistance toverticillium wilt of cotton. Based on the harvest period,we counted the disease index by the result of cuttingstem that the color of vascular bundle and got 23 plants with a high resistant capacities.