| 1. Background and SignificanceInsect molting and metamorphosis are important and complex processes of developmental events, these processes are mainly regulated by two hormones: ecdysone (20-hydroxyecdysone,20E) and juvenile hormone (juvenile hormone, JH). 20E provokes larval molting, metamorphosis and eclosion of adult, while JH governs the nature of the developmental transition.During insect molting and metamorphosis, some other physiological processes also changed. For example, some immune related genes are induced by 20E for defending foreign microorganism when insect molting and metamorphosis; during metamorphosis, lepidoptera insect hemocytes counts has obvious changes, the populations of plasmatocytes and granulocytes increased remarkably, and accounted for more than 70% of all cells. Some reports indicate that hemocytes migrate to the tissues and undergo histolysis during larva-pupa metamorphosis. So research on those physiological processes will help us understand the molecular mechanisms of how hormone mediates the growth and development.2. Progress and Pending ProblemsIn mammalian and human, hormone can mediated immunity. However, in insects, whether and how hormone mediate innate immunity is not fully understood. Some reports indicated that 20E can promotes humoral and cellular immunity, and the induction by 20E requires ecdysone receptor (EcR) and its ultraspiracle (USP), JH can inhibite this 20E-induced reponse. However the molecular mechanisms of how hormone promote the innate immunity is not fully understood. Finding of some new genes which not only induced by 20E but also involved in innate immunity will help us understand the molecular mechanisms.The insect embryonic blood cell development is mediated by several transcriptors, such as serpent and U-shaped. When larval blood development, JAK/STAT and Hedgehog (HH) signal pathway also involed in the haematopoiesis. 20E signal pathway play important role during metamophosis, and in this stage the granulocytes amounts increase and constitute more than 70% of all hemocytes, other types of cells amounts also changed. So whether 20E signal pathway involved in the regulate some haematopoiesis related genes will help us understand the mechanism how 20E mediate haematopoiesis.3. Methods and Acquired ResultsIn the current research, we characterized two genes from Helicoverpa armigera. The first gene is specificly expressed during molting and metamorphosis, and named HaBBP (Helicoverpa armigera BTB, BACK, PHR). The second gene main funtions is mediating haematopoiesis in Drosophila, and named HaUSH (H. armigera U-shaped). We investigated the functions of the genes during molting and metamorphosis and provided useful target molecules for understanding the mechanisms of the molting and metamorphosis of insects.(1). H. armigera HaBBP geneThe BBP (BTB, BACK, and PHR) proteins belong to BTB gene family. In addition to the BTB domain, BBP also contain BACK (broad-complex, tramtrack and bric-a-brac) and PHR (PAM, highwire and RPM) domains. The BTB (broad-complex, tramtrack and bric-a-brac) domain is a highly conserved protein-protein interaction motif. The BTB domain-containing proteins mediate a wide variety of biological activities, such as transcription regulation, ion channel assembly, cytoskeletal arrangement and apoptosis. The BACK domain was first identified in BTB proteins and supposedly has important structural or functional roles in E3-mediated protein degradation. The physiological functions of BBP proteins have not been fully investigated.In the current research, we cloned a BBP gene from hemocytes of H. Armigera, named HaBBP. The full length of HaBBP cDNA encodes 535 amino acids, and multi-alignment revealed that HaBBP exhibited a high amino acid sequence homology with corresponding counterparts from other species (70%-91%).We first investigated the expression profile of HaBBP by RT-PCR and western blot, the results showed that HaBBP was expressed in four typical tissues and was much higher in fat body and hemocytes. In fat body and hemocytes, the expression patterns at different developmental stage exhibits that HaBBP is highly expressed during the molting and wandering stages rather than at the feeding stage. In order to examine the effect of hormones on HaBBP expression in the hemocytes,6th instar 0 h larvae were injected with hormone. The results showed that the expression of HaBBP in the hemocytes was upregulated by 20E compared with the control, and methoprene had no evident effect on the expression of HaBBP. To confirm the relationship between HaBBP and 20E signal, the EcR-B1 and USP1 were silenced with RNAi in HaEpi cells. The results showed that the silencing of EcR-B1 and USP1 resulted in significant suppression of HaBBP compared with the control, suggesting that HaBBP is regulated by the 20E signaling pathway, and acts downstream of EcR and USP.To understand the function of HaBBP, we examined its distribution of HaBBP in hemocytes by immunocytochemistry. The results showed that HaBBP only localized in the cytoplasm of granulocytes and plasmatocytes. Considering that granulocytes and plasmatocytes are major hemocytes in innate immunity, we then challenged the larvae with Escherichia. coli. The results showed that HaBBP was upregulated after E. coli stimulation both in the fat body and hemocytes, suggesting that HaBBP is involved in the innate immune response in H. armigera.In conclusion, HaBBP express much higher in fat body and hemocytes during the molting and wandering stages rather than at the feeding stage; 20E treatment could upregulate the expression of HaBBP and knockdown of EcR-B1 and USP1 led to a reduced transcript level of HaBBP; Methoprene had no evident effect on the expression of HaBBP; The HaBBP localized in the cytoplasm of granulocytes and plasmatocytes; Immune stimulation by E. coli caused upregulation of HaBBP in the hemocytes and fat body. Based on these results, HaBBP is regulated by 20E and likely participate in the innate immune response.(2). H. armigera HaUSH geneU-shaped (ush) gene belong to the friend of GATA (FOG) family. So far, all the functions of the FOG family members seem to depend on a GATA factor which is a transcription factor bind to the consensus DNA sequence "GATA". Ush play important role during Drosophila embryo haematopoiesis. However during larvae stage, the function of ush is barely investigated. In the current research, we obtained the whole 3’terminal 1629bp sequence from hemocytes of H, armigera, it encodes 542 amino acids, named HaUSH. Multi-alignment shows that HaUSH has a low amino acid sequence homology with corresponding counterparts from other insect.We investigated the expressional profile by RT-PCR. The results indicated that HaUSH expression was higher in the embryo stage than in the larvae stage. HaUSH expressed in all four tissues, including integument, midgut, fat body, and hemocytes. In the hemocytes, the mRNA level of HaUSH was low at the 5th instar 12 h, and it was slightly increased at the 5th instar 36 h when the larvae underwent molting, then with a subsequent reduce at the 6th feeding stage, HaUSH expression increased and reach the highest at the 6 instar 96 h.20E treatment could upregulate the expression of HaUSH, knockdown of EcR-B1 led to a reduced transcript level of HaBBP. Those results indicate that HaUSH is regulated by 20E and acts downstream of EcR.In sum, HaUSH expression was higher in the embryo stage than in the larvae stage; HaUSH expressed in all four tissues during larvae stage. In hemocytes, HaUSH expression increased during metamorphosis.20E can upregulate HaUSH expression, and silencing of EcR-B1 block the inducing; those results indicate that 20E signal pathway regulates HaUSH.3. The highlight and significance of this researchWe cloned the full length of BBP gene HaBBP cDNA. Using the RT-PCR, western blot, immunocytochemistry and RNAi methods, we investigated HaBBP gene expression profile, subcellular localization and expression profile after E. coli stimulation. The results demonstrated that HaBBP is upregulated by 20E signal pathway, and involved in the innate immune response.We obtained the 3’terminal 1629 bp sequence of HaUSH, using RT-PCR and RNAi methods; we investigated the expressional profile at different developmental stages and tissue distribution of HaBBP, form embyo to prepupa larvae; and analyse the relationship between HaUSH and 20E signal pathway. The results demonstrated that HaUSH is regulated by 20E. the results also indicate that 20E may involved in haematopoiesis through the ush. |
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