家蚕orcokinin基因的选择性剪接与表达分析
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摘要
Orcokinin(OK)是节肢动物门特有的一种神经肽,该神经肽自从在小龙虾中发现以来,便陆续在节肢动物门其他物种中鉴定到,然而由于其结构的复杂性和功能的多样性,对OK基因的结构与作用机制知之甚少。利用RACE克隆、qRT-PCR等方法对家蚕OK基因(BmOK)的结构与表达进行分析。结果表明BmOK存在选择性剪接,形成2个转录本BmOKA和BmOKB;BmOKA由外显子exon1、exon2、exon3、exon5、exon6、exon7和exon8组成,全长BmOKB由外显子exon1、exon2、exon3和exon4组成。BmOKA由于选择性剪接又有4个亚型,分别为BmOKA1(CDS长度为534 bp)、BmOKA2(CDS长度为564 bp)、BmOKA3(CDS长度为570 bp)和BmOKA4(CDS长度为600 bp),占比为22∶13∶5∶1,4个亚型有相同的5'UTR(62 bp)和3'UTR(188bp); BmOKB只有一种CDS(864 bp),5'UTR序列与BmOKA相同(62 bp),但3'UTR(168 bp)不同。BmOKA主要在家蚕腹神经索表达,在4龄眠后18 h表达量最低; BmOKB在头部和中肠的表达量相对较高,眠期表达量高于其他时期。BmOKA与斜纹夜蛾Orcokinin同源性最高,完整蛋白质前体的同源性为33. 80%。本研究清晰地展示了家蚕BmOK基因的结构与表达模式,为后续分析BmOK基因的功能奠定了理论基础并提供实验依据。
Orcokinin( OK) is a unique neuropeptide of Arthropoda. It has been identified subsequently from many other Arthropoda species after its first finding in Orconectes limosus. However,due to its complexity of structure and diversity of functions,it is rarely known about the structure and expression of OK. In this study,RACE cloning and qRTPCR methods were used for the structure and expression analysis of Bombyx mori OK( BmOK) gene. The results indicated that BmOK gene was alternatively spliced into two transcripts BmOKA and BmOKB. BmOKA consists of exon1,exon2,exon3,exon5,exon6,exon7 and exon8,and BmOKB consists of exon1,exon2,exon3 and exon4.Furthermore,BmOKA contains four subtype transcripts BmOKA1,BmOKA2,BmOKA3 and BmOKA4,their CDS length are 534 bp,564 bp,570 bp and 600 bp respec-tively,and the distribution ratio is 22 ∶ 13 ∶ 5 ∶ 1. The four subtypes have the same 62 bp 5' UTR and 188 bp 3' UTR. There is only one type of BmOKB,with CDS of 864 bp. It has the same 62 bp 5' UTR with BmOKA but different( 168 bp) 3'UTR. The tissue expression profile showed that BmOKA was mainly expressed in ventral nerve cord. The lowest expression level of BmOKA was at 18 hours after forth molting. The expression of BmOKB was relatively higher in head and midgut than in other tissues,and higher in molting period than in other periods. BmOKA has the highest identity with OK from Spodoptera litura. Their complete protein precursors are 33. 80% identical. The above analysis shows clearly the structure and expression pattern of BmOK gene in silkworm,which provides a theoretical basis and experimental reference for subsequent analysis of the function of BmOK gene.
Orcokinin( OK) is a unique neuropeptide of Arthropoda. It has been identified subsequently from many other Arthropoda species after its first finding in Orconectes limosus. However,due to its complexity of structure and diversity of functions,it is rarely known about the structure and expression of OK. In this study,RACE cloning and qRTPCR methods were used for the structure and expression analysis of Bombyx mori OK( BmOK) gene. The results indicated that BmOK gene was alternatively spliced into two transcripts BmOKA and BmOKB. BmOKA consists of exon1,exon2,exon3,exon5,exon6,exon7 and exon8,and BmOKB consists of exon1,exon2,exon3 and exon4.Furthermore,BmOKA contains four subtype transcripts BmOKA1,BmOKA2,BmOKA3 and BmOKA4,their CDS length are 534 bp,564 bp,570 bp and 600 bp respec-tively,and the distribution ratio is 22 ∶ 13 ∶ 5 ∶ 1. The four subtypes have the same 62 bp 5' UTR and 188 bp 3' UTR. There is only one type of BmOKB,with CDS of 864 bp. It has the same 62 bp 5' UTR with BmOKA but different( 168 bp) 3'UTR. The tissue expression profile showed that BmOKA was mainly expressed in ventral nerve cord. The lowest expression level of BmOKA was at 18 hours after forth molting. The expression of BmOKB was relatively higher in head and midgut than in other tissues,and higher in molting period than in other periods. BmOKA has the highest identity with OK from Spodoptera litura. Their complete protein precursors are 33. 80% identical. The above analysis shows clearly the structure and expression pattern of BmOK gene in silkworm,which provides a theoretical basis and experimental reference for subsequent analysis of the function of BmOK gene.
引文
[1] ONS S,BELL S X,MAESTRO J L. Orcokinins contribute to the regulation of vitellogenin transcription in the cockroach Blattella germanica[J].J Insect Physiol,2015,82:129-133
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[11] CLYNEN E,SCHOOFS L.Peptidomic survey of the locust neuroendocrine system[J].Insect Biochem Mol Biol,2009,39(8):491-507
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[16] STERKEL M,OLIVEIRA P L,URLAUB H,et al. OKB,a novel family of brain-gut neuropeptides from insects[J]. Insect Biochem Mol Biol,2012,42(7):466-473
[17] VEENSTRA J A,IDA T. More Drosophila enteroendocrine peptides:Orcokinin B and the CCHamides 1 and 2[J].Cell Tissue Res,2014,357(3):607-621
[18] BUNGART D,HILBICH C,DIRCKSEN H,et al.Occurrence of analogues of the myotropic neuropeptide orcokinin in the shore crab,Carcinus maenas:evidence for a novel neuropeptide family[J].Peptides,1995,16(1):67-72
[19] SKIEBE P,DREGER M,MESEKE M,et al.Identification of orcokinins in single neurons in the stomatogastric nervous system of the crayfish,Cherax destructor[J]. J Comp Neurol,2002,444(3):245-259
[20] JI C,MIN S C,MIZOGUCHI A,et al.Isoform-specific expression of the neuropeptide orcokinin in Drosophila melanogaster[J]. Peptides,2015,68:50-57
[21] YOSHIAKI T.Recent topics on the regulatory mechanism of ecdysteroidogenesis by the prothoracic glands in insects[J/OL]. Front Endocrinol,2011,2:[2018-03-12]. https://www. frontiersin.org/articles/10.3389/fendo.2011.00107/full
[22]于淼.家蚕PTTH基因转录调控及相关神经肽的发育表达[D].合肥:合肥工业大学,2010
[23] LIVAK K J,SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method[J].Methods,2001,25(4):402-408
[24]陈安利.家蚕“明”死卵突变体l-em基因的定位克隆及功能研究[D].镇江:江苏科技大学,2013
[25] MODREK B,LEE C.A genomic view of alternative splicing[J].Nat Genet,2002,30(1):13-19
[26]陈彬,孙珏,谢曼丽,等.选择性剪接与无义介导的mRNA降解机制研究进展[J].实用医药杂志,2017(11):1039-1043
[27] MATERA A G,WANG Z.A day in the life of spliceosome[J].Nat Rev Mol Cell Biol,2014,15(2):108-121
[28]朱玉贤,李毅,郑晓峰.现代分子生物学[M].北京:高等教育出版社,2007:331-332
[29] MESCE K A,FAHRBACH S E. Integration of endocrine signals that regulate insect ecdysis[J]. Front Neuroendocrinol,2002,23(2):179-199
[30]赵小凡.昆虫蜕皮的分子机理及应用[J].应用昆虫学报,2007,44(3):323-326
[31] YAMANAKA N,REWITZ K F,O’CONNOR M B. Ecdysone control of developmental transitions:lessons from Drosophila research[J].Annu Rev Entomol,2013,58:497-516
[32]王平阳.家蚕新突变体紫色类鹑斑q-lp的定位分析[D].镇江:江苏科技大学,2015
[33] FUTAHASHI R,BANNO Y,FUJIWARA H. Caterpillar color patterns are determined by a two-phase melanin gene prepatterning process:new evidence from tan and laccase2[J]. Evol Dev,2010,12(2):157-167
[2] STANGIER J,HILBICH C,BURDZIK S,et al.Orcokinin:a novel myotropic peptide from the nervous system of the crayfish,Orconectes limosus[J].Peptides,1992,13(5):859-864
[3] YASUDA-KAMATANI Y,YASUDA A. Identification of orcokinin gene-related peptides in the brain of the crayfish Procambarus clarkii by the combination of MALDI-TOF and on-line capillary HPLC/Q-Tof mass spectrometries and molecular cloning[J]. Gen Comp Endocr,2000,118(1):161-172
[4] LI L,PULVER S R,KELLEY W P,et al.Orcokinin peptides in developing and adult crustacean stomatogastric nervous systems and pericardial organs[J].J Comp Neurol,2002,444(3):227-244
[5] SKIEBE P,DREGER M,MESEKE M,et al.Identification of orcokinins in single neurons in the stomatogastric nervous system of the crayfish,Cherax destructor[J]. J Comp Neurol,2002,444(3):245-259
[6] CHRISTIE A E,MCCOOLE M D,HARMON S M,et al.Genomic analyses of the Daphnia pulex peptidome[J]. Gen Comp Endocr,2011,171(2):131-150
[7] PASCUAL N,CASTRESANA J,VALERO M L,et al.Orcokinins in insects and other invertebrates[J].Insect Biochem Mol Biol,2004,34(11):1141-1146
[8] HOFER S,DIRCKSEN H,TOLLB CK P,et al.Novel insect orcokinins:characterization and neuronal distribution in the brains of selected dicondylian insects[J].J Comp Neurol,2005,490(1):57-71
[9] LIU F,BAGGERMAN G,D'HERTOG W,et al.In silico identification of new secretory peptide genes in Drosophila melanogaster[J].Mol Cell Proteomics,2006,5(3):510-522
[10] HOFER S,HOMBERG U.Evidence for a role of orcokinin-related peptides in the circadian clock controlling locomotor activity of the cockroach Leucophaea maderae[J]. J Exp Biol,2006,209(14):2794-2803
[11] CLYNEN E,SCHOOFS L.Peptidomic survey of the locust neuroendocrine system[J].Insect Biochem Mol Biol,2009,39(8):491-507
[12] ONS S,RICHTER F,URLAUB H,et al. The neuropeptidome of Rhodnius prolixus brain[J].Proteomics,2009,9(3):788-792
[13] ONS S,STERKEL M,DIAMBRA L,et al. Neuropeptide precursor gene discovery in the chagas disease vector Rhodnius prolixus[J].Insect Mol Biol,2011,20(1):29-44
[14] YAMANAKA N,ROLLER L,ZITNAN D,et al.Bombyx orcokinins are brain-gut peptides involved in the neuronal regulation of ecdysteroidogenesis[J].J Comp Neurol,2011,519(2):238-246
[15] JIANG H,HONG G K,PARK Y. Alternatively spliced orcokinin isoforms and their functions in Tribolium castaneum[J].Insect Biochem Mol Biol,2015,65:1-9
[16] STERKEL M,OLIVEIRA P L,URLAUB H,et al. OKB,a novel family of brain-gut neuropeptides from insects[J]. Insect Biochem Mol Biol,2012,42(7):466-473
[17] VEENSTRA J A,IDA T. More Drosophila enteroendocrine peptides:Orcokinin B and the CCHamides 1 and 2[J].Cell Tissue Res,2014,357(3):607-621
[18] BUNGART D,HILBICH C,DIRCKSEN H,et al.Occurrence of analogues of the myotropic neuropeptide orcokinin in the shore crab,Carcinus maenas:evidence for a novel neuropeptide family[J].Peptides,1995,16(1):67-72
[19] SKIEBE P,DREGER M,MESEKE M,et al.Identification of orcokinins in single neurons in the stomatogastric nervous system of the crayfish,Cherax destructor[J]. J Comp Neurol,2002,444(3):245-259
[20] JI C,MIN S C,MIZOGUCHI A,et al.Isoform-specific expression of the neuropeptide orcokinin in Drosophila melanogaster[J]. Peptides,2015,68:50-57
[21] YOSHIAKI T.Recent topics on the regulatory mechanism of ecdysteroidogenesis by the prothoracic glands in insects[J/OL]. Front Endocrinol,2011,2:[2018-03-12]. https://www. frontiersin.org/articles/10.3389/fendo.2011.00107/full
[22]于淼.家蚕PTTH基因转录调控及相关神经肽的发育表达[D].合肥:合肥工业大学,2010
[23] LIVAK K J,SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method[J].Methods,2001,25(4):402-408
[24]陈安利.家蚕“明”死卵突变体l-em基因的定位克隆及功能研究[D].镇江:江苏科技大学,2013
[25] MODREK B,LEE C.A genomic view of alternative splicing[J].Nat Genet,2002,30(1):13-19
[26]陈彬,孙珏,谢曼丽,等.选择性剪接与无义介导的mRNA降解机制研究进展[J].实用医药杂志,2017(11):1039-1043
[27] MATERA A G,WANG Z.A day in the life of spliceosome[J].Nat Rev Mol Cell Biol,2014,15(2):108-121
[28]朱玉贤,李毅,郑晓峰.现代分子生物学[M].北京:高等教育出版社,2007:331-332
[29] MESCE K A,FAHRBACH S E. Integration of endocrine signals that regulate insect ecdysis[J]. Front Neuroendocrinol,2002,23(2):179-199
[30]赵小凡.昆虫蜕皮的分子机理及应用[J].应用昆虫学报,2007,44(3):323-326
[31] YAMANAKA N,REWITZ K F,O’CONNOR M B. Ecdysone control of developmental transitions:lessons from Drosophila research[J].Annu Rev Entomol,2013,58:497-516
[32]王平阳.家蚕新突变体紫色类鹑斑q-lp的定位分析[D].镇江:江苏科技大学,2015
[33] FUTAHASHI R,BANNO Y,FUJIWARA H. Caterpillar color patterns are determined by a two-phase melanin gene prepatterning process:new evidence from tan and laccase2[J]. Evol Dev,2010,12(2):157-167