Molecular Cloning And Mrna Expression Of Transporter ABCC2Gene In Cotton Bollworm

Helicoverpa armigera (Hubner) is one of the most serious insect pests of cotton in China and other countries in the world. In China, transgenic cotton expressing Bacillus thuringiensis (Bt) toxins was commercialized in1997, and in2012the Bt cotton acreage in China is about four million hectares. The Bt cotton has been widely adopted to control some key lepidopteran pests, especially the cotton bollworm Helicoverpa armigera. Bt cotton has indeed helped to control the damage of cotton bollworm effectively, reduced the need for insecticide sprays, and also enhanced biocontrol services. But evolution of resistance to Bt cotton among target pests is a major threat to the continued success of Bt cotton. There is a major potential risk of the development of resistance to the Bt toxin under the strong selection pressure in the field.The transporter ABCC2has been reported to related to resistance to Cry1Ac in several insects. In this study, the full-length cDNA of the ABCC2gene was cloned from H. armigera by using RT-PCR and RACE techniques. The relative expression of ABCC2gene in the susceptible and Cry1Ac-resistant strains was compared, and the spatio-temporal expression of ABCC2gene was also determined. The results of the study showed that the transporter ABCC2plays no role in the formation of Cry1Ac resistance in the four strains tested.1. Cloning and sequence analysis of HaABCC2gene from H. armigeraThe full-length cDNA of the transporter ABCC2gene (HaABCC2) was cloned from H. armigera. Sequence analysis indicated that the full-length of this target gene was4185bp. HaABCC2has the highest cDNA sequence similarity (93%) with the ABCC2of Heliothis virescens. Comparing the full-length cDNA of HaABCC2between the susceptible and resistant strains of H. armigera, we found the amino acid sequences encoded were identical between strains.2. Temporal and spatial expression of HaABCC2geneThe levels of HaABCC2gene expression in different developmental stages and different tissues of H. armigera were measured by qPCR. From egg to larval stage, the levels of HaABCC2gene expression were on the upward trend. Then the expression level dropped dramatically at pupal stage, and continues to decline to the bottom at adult stage eventually. The levels of HaABCC2gene expression in each stage were respectively5.50(eggs),9.16(1st-2nd),13.85(3rd-4th),26.72(5th),2.83/1.78(female pupae/male pupae) and1.38(male adult) times than that in female adult stage. The levels of HaABCC2gene expression were highest in the foregut; lower in the hindgut, midgut, Malpighian tube, fat body and lowest in the cuticle. The levels of HaABCC2gene expression in all above mentioned tissues and the whole body were respectively977.04,883.32,528.44,59.44,2.83and164.72times than that in the cuticle.3. Expression of HaABCC2gene in susceptible and Bt-resistant strains of H. armigeraThe levels of HaABCC2gene expression in susceptible and Cry1Ac-resistant strains of H. armigera are detected by qPCR. The resistant strains of H. armigera were four representative strains that were resistant to Cry1Ac toxin, including AY423, CYTO, CAD and Lang Fang. The levels of HaABCC2gene expression in the above mentioned4resistant strains were respectively0.68,0.67,1.01and0.67times compared with that of the susceptible strain (SCD).