Expression Pattern And Function Study Of Opsin Genes In Helicoverpn Armigera

Helicoverpa armigera (Hubner) are omnivorous, and their reproductive and adaptive capacity are very strong. The outbreak of disaster cotton bollworm has happened all over the world, resulting in economic losses in a wide variety of agricultural crops. Light trap is not only a method to forecast the population dynamics of insect pests, but also an ecological treatment measure to control the behavior of cotton bollworm. Vision plays key roles in the performance of insect behaviors, such as searching for food and potential mates, avoiding predators and unsafe environments. The function of opsin genes and phototactic behavior in cotton bollworm were studied in this study, which was beneficial for revealing the adaptive capacity of nocturnal insects to light, and providing some theoretical basis to control the behavior of cotton bollworm, finally improving the integrated management of H. armigera. This doctoral dissertation contained eight sections.(1)The external structure of compound eye was compared between female and male moths, and the internal structure of compound eye was observed by microexamination. The compound eye was semispherical, and ommatidia lined up tightly. Central ommatidia were hexagonal, whereas marginal ommatidia were not. There were no significances of distance of compound eyes, distance of ocelli, compound eye length, compound eye width, compound eye perimeter, number of ommatidia between female and male moths, but the compound eye of male moth was little bigger than that of female moth. The superposition eye of cotton bollworm contained dioptric apparatus, pigment cell, rhabdom and basement membrance from external to internal.(2)The phototaxis rate to UV (365nm), blue (450nm) and green (505nm) lights of female and male moths with different emergence days was determined by phototactic behavior trial. The phototaxis rate was related with emergence day, but not with type of lights and gender. The phototaxis rates of1-3day old moths were higher than those of0and6day old moths.(3)Total RNA was isolated from the compound eye of H. armigera, and UV, blue and long-wavelength sensitive opsin genes were cloned by RT-PCR and RACE, namded as Ha-UV(GenBank accession number:HQ641391), Ha-BL(GenBank accession number:JX644013) and Ha-LW (GenBank accession number:JX392054).1258bp of Ha-UV,1451bp of Ha-BL and1574bp of Ha-LW encoded opsins of varying lengths:379(Ha-UV),381(Ha-LW) and382(Ha-BL) amino acids.(4)The encoded amino acid sequences were analyzed by online software. The molecular masses of the encoded opsins were predicted to be41.35kDa (Ha-UV, GenBank accession number:ADW20311),43.22kDa (Ha-BL, GenBank accession number:AGH28029), and41.86kDa (Ha-LW, GenBank accession number:AGH28027), and the calculated isoelectric points were8.05(Ha-UV),7.02(Ha-BL), and7.97(Ha-LW). The encoded amino acid sequences contained seven membrance-spanning helical domains, G-protein-coupled receptors and visual pigment (opsin) retinal binding sites. Sequence alignment revealed that Lepidopteran opsins shared significant homology.(5)Whether opsin mRNA levels oscillate in a circadian manner is determined by real-time PCR. Under14L:10D, Ha-UV and Ha-BL levels peaked at ZT1and then decreased, whereas Ha-LW levels tended to decrease during the day and increase at night. The expression pattern of opsin gene was not changed under one day dark condition, illustrating that the opsin gene expression was regulated by biological rhythm. This study also confirmed that two clock genes (Ha-CRY1and Ha-CRY2) performed circadian functions in peripheral tissues (compound eye).(6)Both light exposure and starvation treatment influenced opsin gene expression in H. armigera, while opsin gene expression was not influenced by mating. Six hours light exposure (UV and green light) rather than the scotophase significantly up-regulated Ha-UV. Starvation down-regulated most opsin genes, especially Ha-UV.(7)The antigen was obtained by prokaryotic expression and polypeptide synthesis. The antigen was not obtained by prokaryotic expression. Antibodies of Ha-UV and Ha-LW were generated by polypeptide synthesis, and the quality of antibodies was good by western blot and immunohistochemistry test. Ha-LW protein level was higher at ZT1, ZT5, ZT9and ZT21than those at ZT13and ZT17. Ha-UV protein level was higher at ZT5and ZT9than those at other time points.(8)RNAi was conducted by dsRNA injection. After pupae injection, opsin gene expression was inhibited during pupae stage, however, the inhibitory effect disappeared during adult stage. Opsin gene and opsin expressions after double compound eyes injection were inhibited more effectively than those after one compound eye injection. Significant64.3%reductions of Ha-LW and Ha-UV levels were observed at24h after dsRNA injection, while there were significant36.2%and33.7%reductions of Ha-LW and Ha-UV protein levels at24h after dsRNA injection.