Function Analyses Of Five Genes Involved In 20E Signal Transduction Pathway And Interaction Among Them

Insect molting and metamorphosis are regulated by two major hormones:the steroid 20-hydroxyecdysone (20E) and the sesquiterpenoid juvenile hormone (JH).20E initiates molting while JH governs the nature of the developmental transition. Upon the binding of 20E to the ecdysone (20E) receptor (EcR), which subsequently binds to its heterodimeric partner ultraspiracle protein (USP) and forms the receptor complex, a molting cascade is initiated by the transcription of a number of transcription factors in the nuclear receptor superfamily including EcR, USP, E74, E75, and hormone receptor 3 (HR3). Subsequently, several late genes in the hormone pathway are upregulated and help mediate the molting process.It has been reported that some chaperone proteins are involved in the maturing of the EcR/USP transcription complex. Two proteins, Hsp90 and Hsc70, have been found as the components of the complex. A chaperone-EcR/USP heterocomplex is required for activation of EcR/USP DNA binding activity. However, little is know about the role of chaperone proteins in the up-or down-regulation of its activity.In the current research, we identified an epidermal cell line from Helicoverpa armigera, which could be used for investigating hormonal regulation on the gene expression in the hormonal signal pathway and performing RNAi to investigate gene function. To probe the role of the Hsc70 in 20E signaling transduction pathway, we have analyzed the role of Hsc70 and its interaction with USP, utilizing molecular cloning, expression analysis, and functional determination of EcRB1, USP1, and Hsc70. The results reveal that Hsc70 plays important roles in regulating the expression of a set of genes involved in the 20E signaling transduction pathway by binding with USP1 and facilitating the expression of EcRB1 and USP1. We also characterized two 20E induced genes (guanylate kinase and adenylate kinase 1) from Helicoverpa armigera. These provided useful target molecules for understanding the signal cascades of 20E. It was also helpful to investigate the mechanisms during the molting and metamorphosis of insects. 1. Identification of an epidermal cell line from Helicoverpa armigera and establishment of a model for investigating hormonal regulation on the gene expression and gene functionAn epidermal cell line from the 5th instar larval integument of Helicoverpa armigera was identified by maker genes. This cell line was established by Shao Honglian in our lab。My own work was the identification of the cell line using several maker genes and conforming that it wasn't contaminated by other tissues. Results showed that two cuticle proteins(Ha-cup1 and Ha-cup4) and Ha-trypsin2 expressed in both HaEpi cell line and epidermis but did not express in haemocytes. In contrast, Ha-cathL expressed only in haemocytes and not in other tissues or the HaEpi cell line. Hmg176 expressed only in the midgut and hexamerin only expressed in the fat body but not in HaEpi cell line and epidermis. These facts indicated that HaEpi cell line was not derived from haemocytes, midgut or fat body but from integument.Futhermore, a model for investigating hormonal regulation on the gene expression and gene function using RNAi was established. Northern blot results showed that HHR3, the maker gene of the 20E signal transduction pathway, was upregulated after being induced by RH-2485. A Dig-labeled HHR3 probe was used to detect 4 isoforms of HHR3 transcripts. Band 1 was dominant and could be induced within 3 h, peaked at 12 h, and declined thereafter. The other bands were notably fainter. Additionally, HHR3 expression could be knocked down using the RNAi method. These characters present us with a model for investigating gene function by RNAi in the cell line.During the 20E induction, nearly all of the examined genes were upregulated by 20E treatment. Ecdysone receptor (EcRb) expression peaked at 3 h,12 h, while ultraspiracle protein (USP1) appeared obviously after 12 h induction with 20E. Ecdysone induced protein E74 (E74a) and E75 (E75b) increased along with the culture over 24 h. Hormone receptor 3 (HHR3) was not detected in the absence of 20E, but rapidly elevated after culturing for 3 h with 20E, peaked at 12 h, and declined gradually thereafter. The ecdysteroid-regulated gene (ecdy),carboxypeptidase (carbA2),nuclear transfer factor 2 (NTF2) and G-protein y subunit (G-proy). Otherwise, withdrawing of 20E after 12 h culture in it, EcRb, USP1 and HHR3 stopped expression at 6 h. Other genes also showed decreased expression along with the incubation time from 6 to 24 h after withdrawal of 20E. The cuticle protein 1 (cup1) did not demonstrate a close relationship to the withdrawing of 20E. This result indicated that the cell line responded well to the 20E analogs, which suggested that a new epidermal cell line which could be induced by ecdysone was successfully developed.In conclusion, HaEpi cell line was indeed derived from integument; it could be used to analyse the hormonal regulation on the gene expression and investigate gene function by RNAi.2. Regulation of USP1, EcRB1 and Hsc70 on the gene expression in the 20E signal transduction pathway in Helicoverpa armigeraThe complete sequences of HaEcRB1,HaUSP1 and HaHsc70 was respectively cloned from Helicoverpa armigera. HaEcRB1 encodes a 545-amino acid protein while HaUSPl and HaHsc70 encode a 414 amino acid protein,a 654-amino acid residue protein respectively. The results from multi-alignment revealed that HaEcRB1, HaUSP1, and HaHsc70 all exhibited a high amino acid sequence homology with corresponding counterparts from other species. Semi-quantitative RT-PCR showed that HaEcRBl and HaUSP1 were obviously upregulated in feeding 5th instar larvae and molting 5th instar larvae in all tested tissues. However, the transcript of HaHsc70 reflected a constitutive expression in all tested tissues and at all three developmental stages. Western blot results revealed that HaHsc70 was upregulated by 20E both in the 20E-injected larvae and in 20E-treated HaEpi cells compared to controls. However, methoprene administered to larvae or HaEpi cells exhibited no obvious effect on the HaHsc70 content.To explore the function of these genes in 20E signal transduction pathway, HaEcRB1,HaUSPl and HaHsc70 were silenced in the HaEpi respectively. Then RT-PCR was used to analyse the expression of several genes, including HaEcRB1, HaUSP1, E74A, E75B, HHR3, ecdy, carbA2, NTF2, G-pro-γ,HaHsc70, Hsp90 and apoptosis inhibitor (apoi). Silencing of HaEcRBl,HaUSP1 and HaHsc70 respectively by RNAi resulted in suppression of a set of 20E induced genes, including the transcription factors E74A, E75B, and HHR3, effector genes ecdy and carbA, and chaperone protein Hsc70, when compared to the control cells treated with GFP dsRNA. However, silencing of them did not result in the suppression of the induction of NTF2, G-pro-γ,Hsp90, and apoi. Interestingly, 20E-induced expression of HaEcRBl and HaUSP1 was significantly suppressed after HaHsc70 was silenced. These results suggest that HaEcRB1,HaUSP1 and HaHsc70 were functioned upstream in the 20E signal transduction pathway.To clarify the mechanism how HaHsc70 is involed in the 20E signal transduction pathway, the subcellular location of HaHsc70 was investigated. It was found to be located in the cytoplasm in control cells. After the cells were incubated with 20E, HaHsc70 was detected not only in the cytoplasm, but also in the nuclei. In contrast, HaUSP1 and HaEcRB1 were predominantly located in the control cells'nuclei. Following induction with 20E, they were notably upregulated in the nucleus. However, when HaHsc70 was knocked down by RNAi, the HaUSP1 and HaEcRB1 signal in the nucleus decreased compared with that in the dsGFP-control cells. These results suggest that HaHsc70 could translocate to nucleus after 20E induction and the expression of HaUSP1 and HaEcRB1 was regulated by HaHsc70.HaHsc70 was capable of binding to HaUSPl in pull-down assays.In sum, HaEcRB1,HaUSP1 and HaHsc70 function upstream in the 20E signal transduction pathway; HaEcRB1 and HaUSP1 mainly locate in nucleus while HaHsc70 locates in cytoplasm; HaHsc70 could translocate to nucleus after 20E induction; Hsc70 participates in the 20E signal transduction pathway via binding to USP1 and mediating the expression of EcRB1, USP1 and then a set of 20E responsive genes.3. Function of guanylate kinase and adenylate kinase 1 in 20E signal transduction pathwaya. Guanylate kinaseA 1,216-bp full length cDNA of HaGK was obtained from the cDNA library of H. armigera. It contains a 606-bp ORF encoding a 202-amino acid residue protein. Analysis of HaGK using the SMART software showed that HaGK contained a GuKc domain (Gln5-Met191). HaGK had the highest similarity to guanylate kinase from Bombyx. mori (84%identity). In addition, the identities of HaGK to the guanylate kinase from Aedes aegypti, Acyrthosiphon pisum and Tribolium castaneum were 67%,61%, and 60%, respectively.To explore its roles in 20E signal pathway, qRT-PCR results showed that HaGK was obviously upregulated in 5th molting larvae in epidermis, midgut and fat body compared to those at 5th feeding larvae. Furthermore, the gene was detected at all developmental stages and it reached the peaks at the 5th instar 36 h with head capsule slippage and 6th instar 72 h (6-72h, metamorphic molting). This expression pattern suggests that HaGK could be closely related to molting and metamorphosis and might be regulated by ecdysone.To validate this hypothesis, cells were treated by hormones and the expression pattern was detected. HaGK was upregulated by 20E in 20E-treated HaEpi cells compared to controls. Its expression began to increase within 1 h after induction, peaked at 3 h, and then declined. In contrast, the expression of HaGK decreased after treatment with methoprene as compared to the control group. Notably, it was also upregulated by 20E and methoprene together, but the upregulation appeared only after treatment after 3 h.To determine its subcellular location, immunocytochemistry was performed in the HaEpi. HaGK was mainly detected in the cytoplasm in the control group.20E and methoprene had no effect on the location of HaGK.RNAi experiments revealed that the expression of HaGK was largely decreased after HaEcRB1 and HaUSP1 was silenced, showing that HaGK mightbe located downstream in 20E pathway.In summary, HaGK was obviously upregulated in 5th molting and 6th metamorphic molting larvae; its expression could be upregulated by 20E and suppressed by methoprene; HaGK might act downstream EcRB1 and USP1 in 20E signal pathway. These results suggest that HaGK participate in insect molting and metamorphosis in addition to function in energy metabolism.b. Adenylate kinase 1A 1,125-bp full length cDNA for HaAKl was obtained from the cDNA library of H. armigera. It contains a 897-bp ORF encoding a 299-amino acid residue protein. Analysis of HaAK1 using the SMART software showed that HaAK1 contained an AK domain (Ile48-Tyr205). HaAK1 had the highest similarity to adenylate kinase from A.aegypti (78% identity). In addition, the identities of HaAK1 to the adenylate kinase from C. elegans and D. rerio were 63%,60%, respectively.To determine its tissue distribution, qRT-PCR was used. Results showed that HaAK1 were obviously upregulated in 5th molting larvae in epidermis, midgut and fat body compared to those at 5th feeding larvae. However, a rather weak signal was observed in the haemocyte. To investigate the expression pattern of HaAKl in the epidermis during different developmental stages, the cDNAs of the epidermis from the 5th instar 24 h (5-24) to the 1st day pupae (p0) were determined by qRT-PCR. The gene was detected at all developmental stages and it reached the peaks at the 5th instar 36 h with head capsule slippage and 6th instar 72 h (6-72h, metamorphic molting). This expression pattern suggests that HaAK1 could be closely related to molting and metamorphosis and might be regulated by ecdysone.To confirm this, HaEpi cells were treated by 20E and methoprene. The results revealed that HaAK1 was upregulated by 20E in 20E-treated HaEpi cells compared to controls. Its expression peaked at 3 h, and then declined, In contrast, the expression of HaAK1 decreased after treatment with methoprene as compared to the control group. Notably, it was also upregulated by 20E and methoprene together, but the upregulation appeared only after treatment after 12 h.Immunocytochemistry was performed in the HaEpi to determine its subcellular location. HaAK1 was mainly detected in the cytoplasm in the control group.20E and methoprene had no effect on the location of HaGK.RNAi experiments revealed that silence of HaEcRB1 had no effect on the expression of HaAK1, suggesting that HaAK1 could not be directly regulated by EcRB1 but maybe other isoforms of EcRB1.In brief, HaAK1 was upregulated in 5th molting larvae; its expression could be upregulated by 20E in vitro. These results suggest that HaAK1 might participate in insect molting process.