Studies On The Resistance Screening And Mechanisms Of Transgenic Cry1Ac Plus CpTI Cotton To Helicoverpa Armigera(Hübner)

The cotton bollworm, Helicoverpa armigera (Hübner), is one of the most serious agricultural pests worldwide. It was characterized with wide harmful scope, great reproductive potential and well environmental adaptability. The transgenic cotton played an important role in the comprehensive prevention of cotton pests. Transgenic cotton expressing the Cry1Ac toxin from Bacillus thuringiensis has been widely planted in China since 1997. However, transgenic cotton shows insecticidal protein endlessly during the whole growing stage, and this will greatly intensify selection pressure for resistance in the H.armigera. The potential risk of resistance will be a limiting factor for the long-term usefulness of transgenic cotton. Resistance selection is a foundation for the study of resistance mechanism. We need to assess the risk of Bt resistance development, investigate susceptibility levels of field populations of H. armigera to Bt cotton and detect frequency of resistance genes at low level in the field, which will provide an accurate information about whether the resistant management strategy is working correctly or not. In present study, we use the seed of CCRI41 and CCRI44 to select the resistant Helicoverpa armigera strains, study the effects of transgenic cotton on enzymes activities in larvae of the H. armigera and estimated frequency of alleles conferring resistance to Cry1Ac in field populations of Helicoverpa armigera.1. Resistance selection of Helicoverpa armigera population to transgenic cotton seedTransgenic Bt+CpTI cotton seed are used as the source of insecticidal protein to select the resistant H. armigera strains which were collect from Anyang, Henan in 2005. The AYBC strain was screening with CCRI41 and the AYBT strain was screening with CCRI44. The control strain which screening with non-transgenic cotton (CCRI49) was called AYCK. The study shows After 14 generations of selection, resistance to Cry1Ac of the selected strains increased to 4.0 fold and 4.6 fold compared with that of the control strain. During the selection, the growing speed of H. armigera was restrained from 11.0 of AYCK to 6.0-7.0 of AYBC and AYBT. The Development rating of H. armigera will increase slowly if screening use a same dosage,and the period of larva growing was prolonged form 18-21d to 31-36d, and the weight of pupa decreased , oviposition number of female, hatchability of egg also decrease sharply. This research provides the foundation for the process of resistant ability and mechanism of H. armigera.2. Effects of transgenic cotton on enzymes activities in larvae of the Helicoverpa armigera.The cotton bollworm (Helicoverpa armigera (Hübner)) larvae were fed on artificial feed which include Bt cotton(CCRI44) and Bt+CpTI cotton(CCRI41) seed powder respectively for 12 generations. Then the activities of three detoxification enzymes (α-naphthyl acetate esterase, acetylcholinesterase and glutathione-S- transferase), two protective enzymes (superoxide dismutase and catalase) and midgut proteases (total protease, trypsin-like enzyme and chymotrypsin-like enzyme) in 5rd instar larvae of the cotton bollworm. The results showed that the activities ofα-naphthylacetate esterase, glutathione-S-transferase were lower than that of the control strian fed on the non-transgenic cotton. Glutathione-S-transferase couldn't be detected in the cotton bollworm which feed Bt+CpTI cotton. There is no significant difference in protective enzymes and total protease among the cotton bollworm. In the Bt-resistant cotton bollworm and (Bt+CpTI)-resisitant cotton bollworm,the activities of active alkaline trypsin-like enzyme were 3.00- and 4.00-fold higher than that of the control, the activities of weak alkaline trypsin-like enzyme and chymotrypsin-like enzyme were greatly lower than that of the control.3. Susceptibility levels of field populations of Helicoverpa armigera to the MVP II WPSusceptibility levels of three field populations of H. armigera collected from Anqiu, Shangdong Province, and Baoding, Hebei Province and Anyang, Henan Province during 2006-2007 were investigated using a surface contamination bioassay. No significant resistance to Cry1Ac was found among these three poplulations.The relative sensitivity ratios is between 1.1to1.7, which indicates there is no significant resistance to CrylAc of the H. armigera field group in these three areas.4. Estimated frequency of alleles conferring resistance to Cry1Ac in field populations of Helicoverpa armigera.Estimated the frequency of alleles conferring resistance to Bt cotton producing the Cry1Ac toxin in field populations of the cotton bollworm Helicoverpa armigera (Hübner) collected from Anqiu, Shangdong Province, and Baoding, Hebei Province and Anyang, Henan Province in 2007. The resistance frequency of Helicoverpa armigera population in Anqiu, Shangdong Province is 1.7×10^-3 and in Baoding, Hebei Province is 2.0×10^-3, and Anyang, Henan Province is 0².9×10^-3. As a result, only a little part of field populations'sensitivity to CrylAc is decreasing, field populations of H. armigera collected from three areas have not evolved prominent resistance to Cry1Ac,the resistance frequency to Cry1Ac is at its normal level. But Bt resistance risk still exists,and the early resistance detection and alarming system should be initiated in China as early as possible.