Function Researches Of Prodeath-S/TK And CKS1in The Development Of Helicoverpa Armigera

Background, scientific questions and significance of researchesInsect development is regulated by insulin,20E and JH. Insulin primarily regulates cell proliferation and growth, while20E and JH coordinately regulate molting and metamorphosis. During the development process of holometabolous insect, when JH exists, high titer of20E can initiate the larval molting. When JH is absent, high titer of20E will initiate the metamorphosis. Insulin mainly promotes larval growth, and therefore the prothoracic gland (PG) growth, which produces high level of20E to initiate metamorphosis. The cross talk of three hormones orchestrates insect growth, molting and metamorphosis.Tissue remodeling occurs during insect metamorphosis, that is the larval tissues degradation by programmed cell death (PCD), and the adult tissues formation by cell proliferation. Insect tissue remodeling presents good model to study the hormonal regulation on PCD and cell proliferation. Protein kinase participate in various important physiological processes via promote substrates phosphoralation, a set of protein kinase were reported to promote programmed cell death, CKS1as a key factor in regulating cell cycle, can interact with CDKs and Cyclins, co-regulate cell proliferation. While the functions of protein kinase and CKS1in insect development and metamorphosis are still obscure, and in which way insulin,20E and JH regulate the expression of these genes are still unclear. Deeply study the functions and hormone regulation of CKS1and Prodeath S/TK can help to understand the molecular mechanisms of insect growth and metamorphosis, provide more valuable information for further study of insect physiology and pest management.Results and conclusionThe Lepidoptera, cotton bollworm (Helicoverpa armigera) and HaEpi (Helicoverpa epidermis cell line) were used to study the functions amd molecular mechanisms of CKS1and Prodeath S/TK in insect development and tissues remolding by various molecular methods. The results are as follows:1. Prodeath S/TK takes part in programmed cell death (PCD) during metamorphosisProdeath S/TK is highly expressed at the molting and metamorphosis stages both in transcription level and in translation level It can be upregulation by20E. Knockdown of it by RNAi can suppress the expression of genes related to PCD in20E signal pathway, block the old tissues degradation and metamorphosis. Immunocytochemistry showed that Prodeath S/TK is mainly localized in cytoplasm to play its function.20E can not shift its subcellular localization. Overexpression analysis suggests CKS1can promote programmed cell death. Deletion mutations indicate S_TKc is its function domain. The N-and C-terminal determine its subcellular localization. The prodeath S/TK takes part in20E signal pathway in cytoplasm to promote cell death by20E regulation.2. CKS1takes part in insulin signal pathway to promote cell proliferationCKS1is highly expressed at the growth stage of insect development and is decreased at metamorphosis stage. Knockdown of CKS1in vivo by RNAi can suppress the expression of genes related to development in insulin signal pathway, block insect development and form small pupae. Both insulin and20E can upregulate the expression level of CKS1, respectively, while high titer of20E can block insulin-induced expression of CKS1. Overexpression of CKS1in HaEpi cell line can promote cell proliferation, with the subcellular localization mainly in the nucleus, and N45as its activity site. Knockdown of CKS1in vivo can block cell proliferation in midgut, affect insect growth, repress larvae reach to critical body weight, therefore block the initiation of metamorphosis. During larval growth stage, CKS1takes part in insulin signal pathway to promote cell proliferation. When larvae reach to critical body weight, high titer of20E overlaps insulin to block the expression of CKS1, then stops cell proliferation and initiate metamorphosis.Innovations and significationsThis work identified a novel protein kinase for the first time and named it as Prodeath S/TK. The prodeath S/TK participates in20E signal pathway to regulate old tissues degradation via PCD at metamorphosis stage. This work also demonstrates the function CKS1in insect development, which determines larval growth or metamorphosis by hormone regulation. These data can help to deeply understand the interaction of insulin and20E, thereby to understand the insect development and pest management.