家蚕蜕皮触发激素及其受体基因的克隆和表达分析
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摘要
蜕皮是昆虫个体生长所必需的生命过程,是昆虫变态发育的重要过程之一。从形态学角度米说,昆虫蜕皮过程包括旧表皮的脱离和新表皮的形成;从分子生物学角度来看,昆虫蜕皮是多种激素协调调控的结果。尽管已经清楚昆虫蜕皮主要是由保幼激素和蜕皮激素协同控制,但是由于蜕皮的过程相当复杂,蜕皮相关信号通路及关键调节基因仍有待深入研究。家蚕是重要的鳞翅目模式昆虫,其生命周期要经过卵、幼虫、蛹和成虫四个阶段,是研究昆虫蜕皮发育的理想模式之一。在果蝇和烟草天蛾等昆虫蜕皮的激素调控研究中,研究者发现了一个参与蜕皮促发的激素基因,即蜕皮触发激素(Ecdysis triggering hormone,ETH)。基于家蚕基因组数据库,我们对家蚕ETH基因及其受体ETHR进行了克隆和表达分析,期望为阐释家蚕蜕皮的激素调控机制提供更多的数据支撑。
通过生物信息学分析,我们在家蚕基因组精细图预测基因中鉴定获得了与烟草天蛾ETH及其受体ETHR的同源基因,命名为BmETH和BmETHR。根据家蚕预测基因序列设计引物,分别克隆获得了这两个基因的完整ORF序列。其中,BmETH基因ORF全长为303bp,由2个内含子和3个外显子构成,编码100个氨基酸,预测分子量大小为11.49kD,等电点是7.51。SignalP在线信号肽预测发现,BmETH编码的第1到15个氨基酸为信号肽。BmETHR基因ORF全长为810bp,BmETHR基因不含内含子,编码269个氨基酸,预测分子量大小为31.07kD,等电点是9.91蛋白质序列比对分析表明,BmETHR基因与其它昆虫ETHR基因具有保守的跨膜结构域。
采用RT-PCR技术,我们分析了BmETH基因和BmETHR基因在胚胎期、幼虫期、变态期以及在幼虫5龄3天各个组织中的表达情况。结果表明,BmETH在家蚕胚胎发育初期不表达,在胚胎发育后期的表达逐渐升高。在幼虫发育过程中,BmETH基因几乎都是在每个龄期蜕皮前上调表达;在变态发育过程中,BmETH在吐丝前、化蛹前和化蛾前上调表达。BmETH的时期表达特征暗示其与胚胎形态发生及幼虫或蛹期蜕皮紧密相关。在幼虫5龄3天各组织中,BmETH基因仅在气管、丝腺、精巢和中肠等组织中高量表达,这与其他昆虫中ETH基因仅在气管中高量表达的特征有些差异。在不同发育阶段,BmETHR的表达一般随BmETH的表达量变化发生一定程度的变化,提示其与ETH的信号传导相关;但是,BmETHR在胚胎发育早期也有表达,而在幼虫5龄3天的丝腺和中肠组织中低量表达,与ETH的表达并无相关性,暗示其可能不受ETH的调节。另外,我们利用在家蚕中已建立的原位杂交技术,对家蚕BmETH基因在气管中的表达进行了组织定位,结果表明家蚕BmETH基因在气管附着的气门上腺中的Inka细胞中表达。
The ecdysis is necessary for the insect growth and development,and is one of the most important processes during insect metamorphosis.From morphological aspect, insect's ecdysis is a process in which the old epidermis begins to separate and the new epidermis is reconstructed.At the molecular level,the insect ecdysis is regulated cooperatively by a variety of hormones.Although it is clear that the insect ecdysis is mainly controled by juvenile hormone and molting hormone(ecdysone),it is necessary for investigating deeply the signaling pathways and the key genes related to ecdysis. The Bombyx mori is one of the best-characterized models for biochemical,molecular genetic and genomie studies of the order Lepidoptera.It undergos four stages:egg,larva, pupa and adult,and is the ideal model for studying the insect ecdysis.Previous studies in Drosophila melanogaster and Manduca sexta confirmed that a peptide hormone, ecdysis-triggering hormone(ETH) is involved in triggering their ecdysis.We apllied the BLAST approach to search the Bombyx mori genome database and identified the homologs of genes encoding ecdysis triggering hormone and its receptors in M.sexta. We further performed the cloning and expression profiling of these two genes in B. mori.
Based on the predicated orthologs of genes encoding ecdysis triggering hormone and its receptor,we designed the specific primers and cloned their complete open reading frame(ORF),named as BmETH and BmETHR,respectively.The total length of BmETH is 303bp,containing 2 introns and 3 exons,coding 100 amino acids.The predicted molecular weight and isoelectric point of BmETH is 11.49 kD and 7.51, respectively.The 1st to 15th amino acids of the amino acids sequence deduced from BmETH defined as signal peptide region.The BmETHR consists of a total length of 810bp.Bioinformatics analysis showed that BmETHR has no intron and code 269 amino acids.The predicted molecular weight and isoelectric point of BmETHR is 31.07 kD and 9.91,respectively.
We further investigated the gene expression of BmETH and BmETHR at the different stage of silkworm development by RT-PCR method.During embryogenesis, BmETH was not expressed at the early stage,but was highly detected at the later stage. The expression of BmETH was upregulated not ony before the ecdysis of every larval instar,but also before spinning,pupation,and eclosion of silkworm individuals.These stage-specific expression patterns indicated that BmETH is closely associated with the molting during silkworm embryogenesis,larval growth and metamorphosis.In addition,BmETH was prevalently expressed in trachea,silk gland,testis,and midgut. Interestingly,the expression of BmETHR changed following the change of the expression of BmETH at different stages.However,BmETHR was also detected at transcriptional level at the early stage of silkworm embryogenesis,indicating that the transcription of BmETHR may not be regulated by BmETH.Moreover,using in situ hybridization system,we located the tissue expression of BmETH gene.The result showed that BmETH was expressed in the Inka cell,which placed at the stegma upper-glands of the epidermis.
通过生物信息学分析,我们在家蚕基因组精细图预测基因中鉴定获得了与烟草天蛾ETH及其受体ETHR的同源基因,命名为BmETH和BmETHR。根据家蚕预测基因序列设计引物,分别克隆获得了这两个基因的完整ORF序列。其中,BmETH基因ORF全长为303bp,由2个内含子和3个外显子构成,编码100个氨基酸,预测分子量大小为11.49kD,等电点是7.51。SignalP在线信号肽预测发现,BmETH编码的第1到15个氨基酸为信号肽。BmETHR基因ORF全长为810bp,BmETHR基因不含内含子,编码269个氨基酸,预测分子量大小为31.07kD,等电点是9.91蛋白质序列比对分析表明,BmETHR基因与其它昆虫ETHR基因具有保守的跨膜结构域。
采用RT-PCR技术,我们分析了BmETH基因和BmETHR基因在胚胎期、幼虫期、变态期以及在幼虫5龄3天各个组织中的表达情况。结果表明,BmETH在家蚕胚胎发育初期不表达,在胚胎发育后期的表达逐渐升高。在幼虫发育过程中,BmETH基因几乎都是在每个龄期蜕皮前上调表达;在变态发育过程中,BmETH在吐丝前、化蛹前和化蛾前上调表达。BmETH的时期表达特征暗示其与胚胎形态发生及幼虫或蛹期蜕皮紧密相关。在幼虫5龄3天各组织中,BmETH基因仅在气管、丝腺、精巢和中肠等组织中高量表达,这与其他昆虫中ETH基因仅在气管中高量表达的特征有些差异。在不同发育阶段,BmETHR的表达一般随BmETH的表达量变化发生一定程度的变化,提示其与ETH的信号传导相关;但是,BmETHR在胚胎发育早期也有表达,而在幼虫5龄3天的丝腺和中肠组织中低量表达,与ETH的表达并无相关性,暗示其可能不受ETH的调节。另外,我们利用在家蚕中已建立的原位杂交技术,对家蚕BmETH基因在气管中的表达进行了组织定位,结果表明家蚕BmETH基因在气管附着的气门上腺中的Inka细胞中表达。
The ecdysis is necessary for the insect growth and development,and is one of the most important processes during insect metamorphosis.From morphological aspect, insect's ecdysis is a process in which the old epidermis begins to separate and the new epidermis is reconstructed.At the molecular level,the insect ecdysis is regulated cooperatively by a variety of hormones.Although it is clear that the insect ecdysis is mainly controled by juvenile hormone and molting hormone(ecdysone),it is necessary for investigating deeply the signaling pathways and the key genes related to ecdysis. The Bombyx mori is one of the best-characterized models for biochemical,molecular genetic and genomie studies of the order Lepidoptera.It undergos four stages:egg,larva, pupa and adult,and is the ideal model for studying the insect ecdysis.Previous studies in Drosophila melanogaster and Manduca sexta confirmed that a peptide hormone, ecdysis-triggering hormone(ETH) is involved in triggering their ecdysis.We apllied the BLAST approach to search the Bombyx mori genome database and identified the homologs of genes encoding ecdysis triggering hormone and its receptors in M.sexta. We further performed the cloning and expression profiling of these two genes in B. mori.
Based on the predicated orthologs of genes encoding ecdysis triggering hormone and its receptor,we designed the specific primers and cloned their complete open reading frame(ORF),named as BmETH and BmETHR,respectively.The total length of BmETH is 303bp,containing 2 introns and 3 exons,coding 100 amino acids.The predicted molecular weight and isoelectric point of BmETH is 11.49 kD and 7.51, respectively.The 1st to 15th amino acids of the amino acids sequence deduced from BmETH defined as signal peptide region.The BmETHR consists of a total length of 810bp.Bioinformatics analysis showed that BmETHR has no intron and code 269 amino acids.The predicted molecular weight and isoelectric point of BmETHR is 31.07 kD and 9.91,respectively.
We further investigated the gene expression of BmETH and BmETHR at the different stage of silkworm development by RT-PCR method.During embryogenesis, BmETH was not expressed at the early stage,but was highly detected at the later stage. The expression of BmETH was upregulated not ony before the ecdysis of every larval instar,but also before spinning,pupation,and eclosion of silkworm individuals.These stage-specific expression patterns indicated that BmETH is closely associated with the molting during silkworm embryogenesis,larval growth and metamorphosis.In addition,BmETH was prevalently expressed in trachea,silk gland,testis,and midgut. Interestingly,the expression of BmETHR changed following the change of the expression of BmETH at different stages.However,BmETHR was also detected at transcriptional level at the early stage of silkworm embryogenesis,indicating that the transcription of BmETHR may not be regulated by BmETH.Moreover,using in situ hybridization system,we located the tissue expression of BmETH gene.The result showed that BmETH was expressed in the Inka cell,which placed at the stegma upper-glands of the epidermis.
引文
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[2]Sullivan,A.A.,Thummel,C.S.Temporal profiles of nuclear receptor gene expression reveal coordinate transcriptional responses during Drosophila development[J].Mol Endocrinol,2003,17(11):2125-2137.
[3]Kingan,T.G.,Adams,M.E.Ecdysteroids regulate secretory competence in Inka cells[J].J Exp Biol,2000,203(Pt 19):3011-3018.
[4]Takaki,K.,Sakurai,S.Regulation of prothoracic gland ecdysteroidogenic activity leading to pupal metamorphosis[J].Insect Biochem Mol Biol,2003,33(12):1189-1199.
[5]Gauthier,S.A.,Hewes,R.S.Transcriptional regulation of neuropeptide and peptide hormone expression by the Drosophila dimmed and cryptocephal genes[J].J Exp Biol,2006,209(Pt 10):1803-1815.
[6]Zitnan,D.,Kingan,T.G,Hermesman,J.L.Identification of ecdysis-triggering hormone from an epitracheal endocrine system[J].Science,1996,271(5245):88-91.
[7]Zilberstein,Y.,Ewer,J.,Ayali,A.Neuromodulation of the locust frontal ganglion during the moult:a novel role for insect ecdysis peptides[J].J Exp Biol,2006,209(Pt 15):2911-2919.
[8]Morton,D.B.,Truman,J.W.Steroid regulation of the peptide-mediated increase in cyclic GMP in the nervous system of the hawkmoth,Manduca sexta[J].J Comp Physiol[A],1985,157(4):423-432.
[9]Park,Y,Zitnan,D.,Gill,S.S.Molecular cloning and biological activity of ecdysis-triggering hormones in Drosophila melanogaster[J].FEBS Lett,1999,463(1-2):133-138.
[10]Zitnan,D.,Adams,M.E.Excitatory and inhibitory roles of central ganglia in initiation of the insect ecdysis bMHavioural sequence[J].J Exp Biol,2000,203(Pt 8):1329-1340.
[11]Mesce,K.A.,Fahrbach,S.E.Integration of endocrine signals that regulate insect ecdysis[J].Front Neuroendocrinol,2002,23(2):179-199.
[12]Siew,Y.C,Gilbert,L.I.Effects of moulting hormone and juvenile hormone on insect endocrine gland activity[J].J Insect Physiol,1971,17(11):2095-2104.
[13]Bergot,B.J.,Schooley,D.A.Juvenile hormone titer determinations in the southwestern corn borer,Diatraea grandiosella,by electron capture-gas chromatography[J].Life Sci,1976,18(8):811-820.
[14]O'Brien,M.A.,Taghert,P.H.A peritracheal neuropeptide system in insects:release of myomodulin-like peptides at ecdysis[J].J Exp Biol,1998,201(Pt 2):193-209.
[15]Arakane,Y,Li,B.,Muthukrishnan,S..Functional analysis of four neuropeptides,MH,ETH,CCAP and bursicon,and their receptors in adult ecdysis bMHavior of the red flour beetle,Tribolium castaneum[J].Mech Dev,2008,125(11-12):984-995.
[16]Bowser,P.R.,Tobe,S.S.Bioinformatic analysis of neuropeptide and receptor expression profiles during midgut metamorphosis in Drosophila melanogaster[J].Peptides,2006,27(3):583-589.
[17]Davis,M.M.,O'Keefe,S.L.,Primrose,D.A.A neuropeptide hormone cascade controls the precise onset of post-eclosion cuticular tanning in Drosophila melanogaster[J].Development,2007,134(24):4395-4404.
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