Molecular Genetic Mechanism Of Resistance Of Helicoverpa Armigera (H(?)bner) To Transgenic Bt Cotton

The extension of Bt transgenic cotton offers a new method on controlling Helicoverpa armigera which is one of the most important pests of cotton in China. But the resistance risk of H. armigera to transgenic Bt cotton will badly impact the longevity of its application in the cotton production. In this thesis, we used one kind of molecular technique, amplified fragment length polymorphism (AFLP), to map the gene conferring resistance to Bt cotton in H. armigera, decide the sites of the resistance gene. Understanding the resistant mechanism of H. armigera to transgenic Bt cotton based on molecular genetics is an important part of safety evaluation system of transgenic Bt cotton. The results will provide some practical and theoretical value for establishing the resistance management strategies, staying the development of resistance, and improving the progress of the molecular technique.In chapter 1 of this thesis, we summarized the progress of resistance of transgenic crops to pests, resistance of pests to transgenic Bt crops, strategies of resistance management and AFLP, which provide theoretical basis for the successive research.Chapter 2 involves the purification and maintenance of resistant population of H. armigera to transgenic Bt cotton. Selection experiment of purification and maintenance for high resistance to transgenic Bt cotton expressing Cry 1 Ac toxin in population of H. armigera were conducted using a leaf-feeding method. The results showed that the mean survival rate of the neonate feeding on the leaves for 3d were 31.6%~62.1%, and that for 5d were 69.5%~82.3%. By 12 generation of selection, the resistant level of H. armigera toCrylAc was enhanced from 329.7-fold to 2824-fold. The very high level of resistance of//. armigera to Bt toxin, CrylAc, was recovered. It will provide a guarantee for the research work about molecular mechanism of H, armigera to transgenic Bt cotton.The extraction of complete DNA from H. armigera (HUbner) is a key precondition of genetic analysis, which were studied in chapter 3. The analysis of amplified fragment length polymorphism (AFLP) has the potential to become a powerful new DNA fingerprinting technique for studying genetic relationship and genetic diversity in insects. The CTAB based DNA extraction protocol provided DNA suitable for AFLP assay. Its process mainly contained six parts, including digesting of tissue by lysis buffer, precipitation of polysaccharides by CTAB, removal of proteins by chloroform : isoamylalcohol (24:1, V:V), precipitation of DNA by PEG, dissolving DNA by TE buffer and consuming of RNA by RNase A. The ratios of A260/A280 of genomic DNAs gotten by the method were almost between 1.7 and 1.9, and the sizes of products digested by EcoR I / Mse I were mainly 50~1000bp.In chapter 4, we studied the inheritance and AFLP markers of resistance in H. armigera to transgenic Bt cotton. The results of infected diet method showed that the ratio between the survival and the dead in the backcross population (BC) of H. armigera was 1:1 after feeding diet with 40ng/ml CrylAc, a determining dose between resistant colony and heterozygote colony for 5d, which was consistent with that of its resistance genetic model-incompletely recessive autosomal heredity. AFLP is a reliable and powerful approach in detecting polymorphic loci. In this paper, AFLP method was applied to map the resistant gene marker in H. armigera to Bt cotton on the basis of previous studies. The results showed that BtR gene flanked with two AFLP marker, EaaMctaO2 and EaaMctaO3, on the 2nd linkage group in H. armigera, with 10.40 cM and 13.90 cM linkage distance. The AFLP marker, EaaMcta, was collected, cloned, and sequenced, and a band with 681bp length was gotten. The results provide a guidance in understanding, monitoring, and managing resistance to Bt cotton in H. armigera.In chapter 5, the usefulness of EnviroLogix CrylAb/CrylAc plate kits for quantification of the Cry1Ac content was determined with two Bt transgenic cotton varieties, NUCOTN 33B and NUCOTN 99B, and a non-Bt variety, Sumian 12...