The Expression And Regulation Of L-arginine Kinase In Helicoverpa Armigera

Cotton bollworm, Helicoverpa armigera (Hübner) is one of the important agricultural pests in the world. It not only seriously harms the cotton, also endangers the corn, sorghum, soybean and many kinds of crops. In invertebrates, L-arginine kinase(AK) is the important regulation factor in energy metabolism. The related research shows that AK plays an important role in the processes of adaptation environmental factors, immunity and defense of pathogen. Using Bt Cry1Ac susceptible strain (96S) of H.armigera as materials, we cloned and sequenced the AK gene and its promotor, and also related function of AK gene was studied by real time-PCR and RNAi methods. Meanwhile, we also compared the expression difference of AK and related resistant genes between the susceptible strain and the resistant strain (Bt-R,the resistant ratio to Cry1Ac was about 2971). The physiological determinations were measured between two strains. Detail results are as followed:1. The AK gene was cloned from the 96S strain by the informations of homologous genes. We find that it contains two introns and two exons in the middle of AK gene. The protein expression vector with AK gene was constructed, and with the optimized conditions, AK protein was a single soluble bank in SDS-PAGE gel.2. By using genome walking method, the promoter of AK of 96S strain was cloned. We got about 2Kb sequence from the upstream of 5'UTR. We predict the biological functions by the bio-software.3. We used the Real Time-PCR method to study the expression of AK gene in the 96S strain. We found that its expression existed widely and had specificity in different time and tissues. In midgut, there was lots of expression of AK gene. This predicted that maybe in that part it needs more energy. With the increasing of larvae age, AK gene's expression becomes higher. However, in the age of imago, the expression becomes lower.4. By feeding and injection ways, the AK gene in 96S strain of cotton bollworm was successfully interfered. Comparing with the normal growing group, growth and development of interfering group were significantly inhibited. It showed that they appear"suppression"phenomenon. This result is consistent to the physiological determinations and statistical ecology.5. By the RT-PCR and Real-time PCR methods, we compared the the expression of AK in Bt-R strain and 96S, the data indicated that the AK expressions were different in two strains whenever they were in different times and tissues. With the increasing of age, AK expression in Bt-R also becomed higher but the expression was lower than in 96S.6. By the data of measuring the physiological determination, we found that comparing with 96S strain, in Bt-R strain, the concentration of blood lymph protein increased, which indicated that resistant cotton bollworm needs more blood lymph protein to resist bad environment; Bt-R's L-arginine kinase and alkaline phosphatase markedly reduce. In two strains, the enzyme activity of midgut is higher than ones of muscle.This research focused on the molecular analysis of AK gene and AK protein, and also worked on preliminarily research in physiological determination aspect. The data will be beneficial for research in AK of H.armigera. Furthermore, it could provide the theoretical base to basic research of H.armigera.