Molecular Cloning And Antiviral Function Of P53Gene From Helicoverpa Armigera

Background and significanceLepidoptera is the second big orders of Insecta after Coleoptera. They are very widespread and the majority of them are agroforestry crops pests such as Pierisrapae Linneand, Plutella xylostella and the cotton bollworm{Helicoverpa armigera). The baculovirus is a natural pathogen of Lepidoptera. Because baculovirus is safe for vertebrates, so the biological pest control using baculovirus shows good application prospects. It has important significance to clarify the molecular mechanisms of insect host cells resisting baculovirus infection:enrich the molecular mechanisms of invertebrate antiviral innate immunity; provide theoretical basis for the study and design of new biological control program of pests.Progress and scientific questionThe studies on insect indicate that apoptosis is an important defense mechanism for insect to resist baculovirus infection. However, although the apoptosis signaling pathway has been made more detailed studies in vertebrates, researches on invertebrates especially on Lepidopteran insects are rare. p53is an important molecule involved in human intrinsic apoptosis pathway. In Drosophila melanogaster, p53mutants exhibit pronounced defects in apoptosis induced by a variety of factors such as ionizing radiation and viral infection. However another study shows that UV irradiation or camptothecin can induce normal apoptosis in p53-knockdown Spodopterafrugiperda. On the other hand, p53was involved in the regulation of DNA damage response of mammalian and Drosophila and have the function of promote cell survival. However, whether Lepidopteran p53has the similar effect is unclear. Thus, the cell functions of insect Lepidopteran p53need further verification.Methods and ResultsBased on above scientific questions, we examined the antiviral functions of p53from Helicoverpa armigera in the process of baculovirus-induced apoptosis of vitro epidermal cells by gene cloning, RT-PCR, Q-PCR, overexpression analysis, RNAi, MTT cell viability experiment, virus absolute quantitative and etc.1. Using the cell model of apoptosis of epidermal cell lines in vitro from Helicoverpa armigera induced by Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), we examined the expression patterns of apoptosis-genes caspase-1and caspase-5. The results showed that the expression of caspase-1and caspase-5were up regulated in the general.2、 We cloned the full-length sequence of Helicoverpa p53ORF and sequence analysis showed P53is not conserved in various species. Phylogenetic analysis indicated that Helicoverpa P53has a closer evolutionary relationship with P53s from other lepidopteron.3、 The antibody of Helicoverpa P53was prepared and the expression pattern of P53in epidermis, midgut, fat body was examined. The result showed that P53had high expression level in the epidermal tissue.4、Overexpression analysis showed that P53was a nucleoprotein with pro-apoptotic activity.5、 In order to confirm the regulation function of p53in baculovirus-induced apoptosis, we performed AcMNPV infection experiment in p53-knockdown epidermal cells. The result showed that these epidermal cells underwent apoptosis normally.6. To further study whether Helicoverpa P53had antiviral activity, we performed AcMNPV infection experiment in p53-overexpression epidermal cells. The total viral DNA was extracted at48h and72h postinfection. The result showed that overexpression of p53in HaEpi cells significantly reduced the replication of HaEpi AcMNPV DNA in the cells.7、To further study whether the inhibition of the replication of AcMNPV by overexpessed-P53in HaEpi cells was due to the overexpession of p53induced apoptosis, we performed AcMNPV infection experiment for48h in p53-overexpression epidermal cells and used MTT method to examine the cell survival rate. The result showed that the overexpressed-P53could significantly promote cell survival when the cells were infected by virus.Conclusion and scientific significance1. The vitro studies showed that:Helicoverpa P53was localized in the nucleus with the pro-apoptotic activity.2. While p53has no critical role in the apoptotic regulation in the AcMNPV-infected Helicoverpa epidermal cell line, it has functions in inhibition of viral replication and promotion of cell survival. This paper enriches the study of host and virus interactions in invertebrates and provides a theoretical basis for studing and designing new biological pest control programs.