【作者】 郑薇薇；

【导师】 赵小凡；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 昆虫的蜕皮和变态受20-羟基蜕皮酮(20-hydroxyecdysone,20E)和倍半萜类保幼激素(juvenile hormone,JH)的双重作用。20E起始了蜕皮生理过程,而保幼激素则决定着蜕皮的性质。20E通过和蜕皮激素受体(ecdysteroid receptor,EcR)结合,再与超气门蛋白(ultraspiracle protein,USP)结合形成转录复合体,继而起始了蜕皮级联反应,一些早期的蜕皮受体超家族的转录因子开始转录,包括EcR、USP、E74、E75以及激素接受子3(HR3)。随后,激素信号途径中的若干晚期基因被诱导表达,从而介导了蜕皮进程。保幼激素是一种调控昆虫生活史的关键激素,在维持蜕皮期间的幼虫状态和引导昆虫的生殖成熟方面发挥了重要作用。保幼激素可能通过与其可能受体Met相结合来调控幼虫的生长发育。EcR／USP转录复合体中还有伴侣蛋白参与,如热休克蛋白Hsp90和热休克蛋白同源物Hsc70是该异源复合物的组成部分,伴侣蛋白与EcR／USP形成的异源复合物对于EcR／USP的DNA结合活性的发挥是必不可少的。但对伴侣蛋白如何调控EcR／USP的DNA结合活性的分子机制并不清楚。本文鉴定了一个新的棉铃虫表皮细胞系,建立了可被激素诱导和可进行RNA干扰的细胞系模型。在此基础上检测了一系列受20E调控的基因的表达模式。借助细胞系RNAi鉴定了蜕皮激素受体EcRB1和超气门蛋白USP1及热休克蛋白同源物Hsc70在20E信号途径中的功能及作用机理。此外,还鉴定了两个受20E上调的激酶(鸟苷酸激酶和腺苷酸激酶1),为研究20E信号途径提供了有效的分子靶标。1.棉铃虫表皮细胞系鉴定及蜕皮激素诱导模型和RNA干扰模型的建立通过标记基因鉴定了HaEpi为表皮细胞系。棉铃虫表皮细胞系HaEpi由本实验室邵红莲建立,该论文的工作是用各种标记基因鉴定细胞系,证明其没有其它组织污染。研究结果表明,两种表皮蛋白(Ha-cup1和Ha-cup4)与Ha-trypsin2在表皮组织和表皮细胞系中表达,但在血淋巴中无表达。相反,Ha-cathL在血淋巴中特异表达,但在表皮细胞和其它三种组织中无表达。在细胞系上未检测到在中肠组织特异表达的hmg176和在脂肪体中特异表达的hexamerine。这些结果表明该HaEpi系的基因表达模式与表皮组织一致,但不同于血细胞、中肠和脂肪体,说明HaEpi系确属表皮组织来源的表皮细胞系。建立了在HaEpi系上进行20E诱导和进行RNA干扰的方法。Northern blot结果表明,非甾醇类的20E类似物RH-2485处理后的细胞中蜕皮激素信号途径中的标记分子HHR3的表达量上调。HHR3的主带在处理后3小时内就开始表达,12小时后达到最高峰,然后逐渐下降,而其他三条带的表达相对较弱。当我们用HHR3的双链RNA转染细胞沉默HHR3后,RT-PCR结果显示HHR3的表达量显著降低。这些结果表明HaEpi是一个可被20E诱导和可进行RNA干扰的细胞系。这为我们利用RNAi检测基因的功能提供了一个良好的实验模型。利用该可诱导的细胞系模型检测了20E对一系列基因表达的调控。这些基因包括HaEcRB1, HaUSPl, E74A, E75B, HHR3, ecdysteroid-regulated gene (ecdy), carbA2, nuclear transfer factor 2 (NTF2), gamma subunit of guanine nucleotide binding protein(G-pro-γ)和Ha-cup1。结果显示,用1μM 20E处理细胞不同时间段后,几乎所有被检测的基因的表达量都上调。蜕皮激素受体EcRB1的表达量在处理后的3-12 h内最高,而USP1要在处理12h后才有明显表达。其它两个转录因子E74a和E75b的表达在处理3h后也明显上调。HHR3经20E处理后在3h内就开始转录表达并且在12小时后达到高峰,随后逐渐下降。蜕皮激素相关蛋白ecdy和羧肽酶A2(carbA2)的表达量也上调。核转移因子2(NTF2)和G蛋白γ亚基(G-proγ)也可以被20E诱导上调表达。当用20E先处理细胞12h而后再撤掉激素继续培养不同时间段后,EcRB1,USP1和HHR3的表达在撤掉激素6 h后就消失了,其它基因的表达量也相继下降,而表皮蛋白1(cup1)在撤除激素后仍持续表达。这些结果说明20E调控这一组基因的表达,并且EcRB1,USP1和HHR3等上游转录因子的表达依赖于20E的存在。该部分研究得到如下结论：本实验室前期建立的HaEpi系确属表皮组织来源的表皮细胞系；HaEpi系是一个可诱导和可干扰的细胞系；20E可以诱导HaEcRB1等一组基因的上调表达。综上,该细胞系可以用来研究激素的调控效应,并且通过进行RNAi极大地方便了基因功能的研究。2.棉铃虫蜕皮激素受体HaEcRB1、超气门蛋白HaUSPl和热休克蛋白同源物Hsc70在20E信号途径中的功能研究为了研究蜕皮激素信号转导的分子机理,分别克隆得到了棉铃虫蜕皮激素受体HaEcRB1,超气门蛋白HaUSP1及热休克蛋白同系物HaHsc70的全长序列。HaEcRB1全长1871bp,编码545个氨基酸。HaUSPl全长2290bp,编码414个氨基酸。HaHsc70全长2145bp,编码654个氨基酸。DNAman多重序列比对结果表明,HaEcRB1，HaUSP1和HaHsc70分别与同源物种的相似性非常高。HaEcRB1与烟芽夜蛾Heliothis virescens(018473)的相似性达96%；HaUSP1与印度古螟P.interpunctella(AY619987)的相似性为91%；HaHsc70与烟草天蛾M. sexta(AY220911)的相似性高达98%。进一步用半定量RT-PCR研究了HaEcRBl.HaUSP1和HaHsc70在棉铃虫的组织分布和发育阶段表达模式。结果显示,HaEcRB1和HaUSP1的转录子在表皮、中肠、脂肪体和血细胞中都有表达,而在5龄蜕皮和6龄变态时期的表达量明显高于5龄取食时期。HaHsc70在四种组织中也都有表达,但它的转录本水平在各个时期是一致的。说明这三种基因在各种组织中广泛表达,但HaEcRB1和HaUSP1在蜕皮和变态时存在转录水平调控,而HaHsc70属于组成性表达。体外20E诱导细胞和体内注射20E的结果显示,20E可以诱导HaHsc70上调表达,而保幼激素类似物methoprene未影响HaHsc70的表达水平。为了阐明这些基因在蜕皮激素信号转导途径中的功能,我们在表皮细胞系上将HaEcRB1、HaUSP1和HaHsc70分别沉默,然后用RT-PCR检测了HaEcRB1,HaUSP1, E74A,E75B,HHR3,ecdy,carbA2,NTF2,G-pro-γ, HaHsc70,Hsp90和apoptosis inhibitor(apoi)等12个基因的表达情况。结果显示,分别干扰HaUSP1、HaEcRB1和HaHsc70后,一系列基因的表达被抑制,包括转录因子E74A,E75B和HHR3,还有效应基因ecdy以及伴侣蛋白Hsc70,但NTF2、G-pro-γ、Hsp90和apoi的表达量未变化。有趣的是,在沉默HaHsc70后,HaEcRB1和HaUSP1表达明显被抑制了,而分别干扰HaEcRB1、HaUSP1并不互相抑制对方的表达。这些结果说明HaEcRB1、HaUSP1和HaHsc70可能位于20E信号途径的上游发挥作用。为了阐明HaHsc70参与蜕皮激素信号转导的机理,进行了HaHsc70细胞定位研究。免疫细胞化学结果显示,在DMSO处理的对照组细胞中,HaHsc70定位于细胞质中,当用20E处理细胞后,HaHsc70部分迁入核内。然而,在methoprene处理细胞后,未观察到HaHsc70的质核迁移。相反,HaEcRB1和HaUSP1在未处理的细胞中主要位于细胞核中,用20E处理细胞后,HaEcRB1和HaUSP1在细胞核中的表达量明显增加。然而,将HaHsc70干扰后,HaEcRB1和HaUSP1在细胞核中的信号明显减弱。说明20E可使HaHsc70入核表达,并且沉默HaHsc70后影响HaEcRB1和]HaUSP1的表达。体内和体外的pull down结果进一步表明,HaHsc70和HaUSP1之间存在相互作用。因此,HaHsc70可能在20E作用下入核,通过与HaUSP1相结合进而促进了EcR/USP的DNA结合活性,启动20E信号途径中的基因转录,从而起始了20E信号通路。该部分研究得到如下结论：HaEcRB1、HaUSP1和HaHsc70位于20E信号通路的上游；HaEcRB1和HaUSP1主要位于细胞核中,HaHsc70在正常情况下位于细胞质中,20E可使HaHsc70入核；HaHsc70通过与HaUSP1结合来介导20E信号通路中的基因的转录表达。3.鸟苷酸激酶HaGK和腺苷酸激酶1HaAK1在20E信号途径中的功能a.鸟苷酸激酶我们从棉铃虫的cDNA文库中得到了棉铃虫鸟苷酸激酶HaGK。全长1216 bp,编码202个氨基酸。HaGK含有一个鸟苷酸激酶功能域(Gln5-Met191)。HaGK除与家蚕的鸟苷酸激酶具有最高的相似性(84%)外,与埃及伊蚊鸟苷酸激酶有67%的相似性,与豌豆蚜鸟苷酸激酶有61%的相似性,与赤拟古盗鸟苷酸激酶有60%的相似性。为了研究该基因在20E信号途径中的功能,用qRT-PCR的方法检测了5龄取食幼虫、5龄蜕皮幼虫及6龄变态幼虫的mRNA表达模式。结果发现HaGK的转录子在三个时期的幼虫的表皮、中肠和脂肪体中都有表达,而血细胞中的表达量极低。此外,HaGK在蜕皮期和变态期的表达水平明显高于取食期。HaGK的转录本在5龄24 h开始就有表达,在5龄36 h(头壳爆裂期)时达到峰值,然后在6龄0 h(刚蜕完皮)开始下降。有趣的是,HaGK的表达在6龄72 h又达到第二次高峰,随后逐渐降低,到6龄96 h表达水平极低。当幼虫进入预蛹期时,HaGK的mRNA又开始表达,直到新蛹形成一直维持在一定的水平。这样的表达模式说明HaGK与蜕皮和变态密切相关,极有可能受蜕皮激素调控。为了验证该假设,用激素处理表皮细胞不同时间段后检测其表达模式。结果表明,HaGK可以被20E明显的上调。它的表达量在20E诱导后的1h内就开始上升，在3 h时达到最高峰,然后逐渐下降。相反,methoprene处理细胞后HaGK的表达量和对照组相比明显下降。当同时用两种激素处理细胞时,HaGK的表达量较对照组也增加了,但却是在激素处理3h后才开始上升,说明methoprene延迟了20E的作用。为了确定HaGK在细胞内的分布及激素对其定位的影响,取棉铃虫的表皮细胞做免疫细胞化学,以不添加抗HaGK血清的兔前血清作为阴性对照,结果显示,在DMSO处理的细胞中,HaGK主要位于细胞质。当用20E处理细胞1 h或6 h时,HaGK仍然主要集中于胞质中,表明该激素未引起HaGK的亚细胞定位变化。当用methoprene处理细胞后,得到了与20E诱导类似的结果,HaGK主要定位于胞质中。RNAi实验结果表明,在分别干扰掉20E受体复合体的EcRB1和USP1后,在20E作用下HaGK未能被上调,说明HaGK在20E信号途径中可能位于EcRB1和USP1的下游。该部分研究得到如下结论：HaGK在蜕皮与变态时期高表达；20E可使HaGK的表达量上调,而methoprene则抑制其表达；HaGK可能在20E信号途径中位于EcRB1和USP1的下游来发挥作用。这些结果表明,鸟苷酸激酶除了在核苷酸代谢方面发挥作用外,可能还参与了昆虫蜕皮和变态的生理过程。b.腺苷酸激酶1我们从棉铃虫的cDNA文库中得到了棉铃虫腺苷酸激酶1HaAK1。全长1125 bp,编码299个氨基酸。HaAK1含有一个腺苷酸激酶功能域(Ile48-Tyr205)。HaAK1与埃及伊蚊腺苷酸激酶有52%的相似性,与果蝇腺苷酸激酶1有44%的相似性,与线虫腺苷酸激酶1有50%的相似性。为了鉴定HaAK1存在于哪个组织,用qRT-PCR的方法检测了5龄取食幼虫、5龄蜕皮幼虫及6龄变态幼虫的mRNA。结果发现HaAK1的转录子在三个时期的幼虫的表皮,中肠和脂肪体中都有表达,而血细胞中基本检测不到。此外,HaAK1基因在蜕皮期的表达水平明显高于取食期。HaAK1的转录本在5龄24 h开始就有表达,在5龄36 h(头壳爆裂期)有一个表达高峰。说明HaAK1与蜕皮密切相关,极有可能受蜕皮激素调控。我们用激素处理表皮细胞不同时间段后检测其表达模式。结果表明,HaAK1的表达量在被20E处理6 h后明显的上调。相反,在methoprene处理细胞后的6 h内HaAK1的表达量无明显变化,但在12 h后开始显著下调。值得注意的是,当同时用两种激素处理细胞时,HaAK1的表达量较对照组也增加了,但主要是在激素处理12 h后显著上升,说明methoprene延迟了20E的作用。我们用免疫细胞化学检测了HaAK1在细胞内的分布及激素对其定位的影响。结果显示,在对照组中,HaAK1主要位于细胞质。当用20E处理细胞时,HaAK1的定位未发生变化。当用methoprene处理细胞后,得到了与20E诱导类似的结果,HaAK1主要定位于胞质中。在表皮细胞系上干扰掉20E受体复合体组分之一的EcRB1后,检测发现20E作用后HaAK1的表达量未受影响,说明EcRB1并未直接调控HaAK1,可能存在EcR的其它异构体来调控HaAK1。该部分研究得到如下结论：HaAK1在蜕皮时期高表达；体外20E可以促进HaAK1的表达；HaAK1主要位于细胞质。这些结果表明,腺苷酸激酶1可能参与了昆虫蜕皮的生理过程。更多还原

【Abstract】 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.更多还原