Molecular Characterization Of Helicoverpa Armigera Atg8 And Its Effects On The Growth And Development Of Insects

Autophagy is an important process which turnovers intracellular substances evolutionarily in Eukaryotes, it has an very important biological function on the growth and develepment of organisms. Low levels of autophagic activity has a protective effect on the cells, the cells can reduce mortality; the high level of autophagy activity is related to cell survival. The occurrence and regulation mechanism of autophagy is complex, which involves a number of autophagy-related genes. Yeast Atg8 and mammalian microtubule-associated protein light chain 3 (LC3) are landmark proteins essential for autophagy. Here the lepidopteran Atg8, a homolog of LC3, is characterized. In the initial stage of autophagy, Atg8 protein is gathered in autophagosome membranes firstly, involving in the formation of autophagosome membrane. In this process, C-terminal part of the Atg8 proteins is cut by a cysteine protease Atg4, forming Atg8-PE. The ratio of Atg8-PE/Atg8 can show the level of autophagy.The amino acid sequences of Atg8 among insects are highly conserved. Firstly, we designed degenerate primers to amplify the sequence of specific fragment of Atg8 in Helicoverpa armigera. Then we used the RACE technique to get the full-length of Atg8. Atg8 proteins which were important landmark proteins fused with tags are commonly used to detect autophagy. The expression patterns of Lepidopteran insect Atg8 are relatively well documented. However, the influence of protein tags on characterization of Atg8 is still not very clear. Thus, a series of plasmids expressing Atg8 fusion proteins were constructed using H. armigera Atg8 gene in the present study. To determine the localization of Atg8 in S1-HP cells, the distribution of endogenous Atg8, HA-Atg8 and fluorescent protein tagged Atg8 were observed. The results showed that most of Atg8 fusion protein was distributed punctatly in the cytoplasm. Interestingly, when the plasmid pEGFP-Nl-IE2-mCherry-Atg8 or the plasmid pEGFP-C1-IE2-EGFP-Atg8 was expressed in S1-HP cells, the green fluorescence or the red fluorescence was localized in both cytoplasm and nucleoplasm, indicating that the subcellular location of the Atg8 was greatly affected by the fluorescent protein tags. Western Blot showed that the Cleavage of EGFP-Atg8 differed from that of mCherry-Atg8. Both cleaved and non-cleaved Atg8 fusion proteins could enter nucleus. When the plasmid pEGFP-C1-IE2-EGFP-Atg8 was expressed in S1-HP cells, the result of Western Blot demonstrated that the size of EGFP-Atg8 fusion protein with different distribution in S1-HP cells was identical. The different localization of the Atg8 proteins were associated with its expression levels, In conclusion, most of Atg8 fusion proteins were distributed punctatly in the cytoplasm when the vector was lower; the fusion proteins were transported into the nucleus from the cytoplasm when the vector was higher. By constructing a series of mutation vectors, we discovered the key amino acid which affected the function and localization of Atg8 proteins. We also fund that HA tag may influence the processing of Atg8 proteins using Western Blot technique.RNAi was applied to verify the function of the Atg8, one of the autophagy related gene, during the growth and development of H. Armigera. Meanwhile, we employed Western Blot to test the expression of Atg8 protein in different organs and tissues of the fifth instar, the results indicated that the fat body and midgut had the highest expression. In this study, each H. Armigera, aging the fifth instar, was selected to be injected with 4μg Atg8-dsRNA and got its midgut after 48h. The results of real-time fluorescence quantitative technique (qPCR) and Western Blot technique showed that Atg8 transcript levels decreased by 50%. Moreover, the weight、eggs and egg hatchability of the experimental group and the control group’s were collected. Compared with the control group, the fecundity and egg hatchability of experimental group approximately decreased by 20%.