The Function Of Adenylate Kinase2(AK2) In The Growth And Development Of Helicoverpa Armigera

Background and research purposeThe cotton bollworm (Helicoverpa armigera) is holometabolous insect belong to Lepidoptera noctuidae. As it is easy to keep and the growth cycle is short, it became an important experimental animal to investigate insect growth and development. The cotton bollworm is also an important agricultural pest which is harmful to the cotton, corn and other crops. To find the important genes that regulate the growth and development of H.armigera is important for the control of cotton bollworm.The biological functions require energy security as well as insect development. The adenylate kinase is a phosphotransferase (AK) contributes to catalyze a reversible reaction of2ADP(GDP)?) ATP(GTP)+AMP when Mg2+existing and plays important roles in the growth and development of organisms. The dynamics of AK-catalyzed phosphotransfer regulates multiple intracellular and extracellular energy-dependent and nucleotide signaling processes, including excitation-contraction coupling, hormone secretion, energetics of cell cycle, cell and ciliary motility, nuclear transport, DNA synthesis and repair, and developmental programming. AK2belongs to AK family with the phosphotransferase activity. Here, the purpose of our research is to investigate the role of AK2linking to energy metabolism in the growth and development of inset. In this study, we found the important genes that regulate the growth and development of H. armigera providing theoretical foundation and target genes for pest control.Research progress and scientific questionIt is reported that AK2is involved in cell proliferation in vertebrates. Knockdown of zebra fish AK2caused leukocyte development defects indicating the importance of AK2in leukocyte differentiation. In human, mutations in the AK2gene cause a profound haematopoietic defect indicating AK2is required for the proliferation and differentiation of cells. However, there are also reports indicating AK2may be involved in cell apoptosis. The precise role of AK2regulating cell functions is unclear. In insect, a recent study reveals that homozygous AK2(-/-) larvae of Drosophila melanogaster ceased growth and died before reaching the third instar larval stage indicating AK2is essential for insect development. However, the molecular mechanism of AK2in regulating insect development is unclear.Methods and ResultsBased on above scientific questions, we examined the function and possible molecular mechanism of AK2by gene cloning, recombinant protein expression, polyclonal antibody preparation, Western blot, immunocytochemistry, RNAi and RT-PCR etc.We cloned the full length sequence of Helicoverpa AK2, and sequence analysis showed AK2is highly conserved in various animals. To investigate the role of AK2in insect development, we first examined the expression profiles of AK2in the tissues during larval development. The results of Western blot showed that AK2expresses in various tissues including epidermis, midgut, fat body and haemocytes reflecting a constitutive expression. These results implied that the AK2plays essential roles in the tissues during insect larval development. To determine the location of AK2in the cells, immunocytochemistry was performed in the Helicoverpa epidermal cell line (HaEpi). Result suggested that AK2locates in the mitochondria.To analyze the function of AK2in cell proliferation, HaEpi was incubated with AK2recombinant protein expressed in E. coli in MTT assay. Result showed that AK2promotes cell proliferation. In order to investigate the roles of AK2in vivo in insect development, we knocked down AK2by RNAi. Results showed that knockdown of AK2in larvae resulted in the larval growth arrest, body weight decreased, developmental stage delayed and decrease of circulating hemocytes. To demonstrate the molecular reason that the slow down growth and delayed development of the larvae after knockdown of AK2, we examined the transcription of some genes that are involved in development, including the molting hormone nuclear receptor EcRB1, transcription factors USP1and Broad, and protein kinase AKT. The results of semi-quantitative RT-PCR showed that knockdown of AK2suppressed genes transcription.Conclusion and significanceTaken as a whole, we identified that AK2is an essential gene for the growth and development of insect. AK2, located in mitochondria, is involved in the growth and development of insect through promoting cell proliferation and regulating gene transcription. Loss of AK2caused profound growth defects providing a good target gene for pest control in agriculture.