色素代谢相关基因在家蚕褐色类鹑斑突变品系q-l~b 4龄眠蚕表皮中的表达分析
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摘要
家蚕体表色素主要有黑色素、蝶啶类色素和眼色素,不同家蚕品种的体色和斑纹都是表皮中黑色素、眼色素和蝶啶类色素等相互作用的结果。为了解家蚕褐色类鹑斑突变体(brown quail-like,q-l~b)幼虫表皮色素的代谢情况,利用qRT-PCR技术分别检测q-l~b突变体和其对应的野生型品种0223金黄(0223JH)在4龄入眠后18 h新生表皮中37个色素代谢相关的基因的表达水平。结果表明,相对于野生型品种,大多数与黑色素、蝶啶类色素合成相关的关键基因在q-l~b突变体中表达上调,而与眼色素合成相关的关键基因表达下调,表明黑色素、蝶啶类色素在q-l~b突变体表皮中的含量都增加,而眼色素在q-l~b突变体表皮中的含量相对减少;与几丁质合成及尿酸转运相关的基因在q-l~b突变体表皮中也存在表达差异,其中大部分关键基因表达上调,表明更多的几丁质在q-l~b突变体新生表皮中合成,并伴随更多的尿酸被转运到表皮,与其他色素一起参与表皮构建,形成q-l~b突变体特殊的体色斑纹。此外,部分保幼激素代谢和核受体基因差异表达也可能影响了相关色素的合成及代谢。q-l~b突变体相对于野生型具有更复杂的体色和斑纹,这些基因的差异表达可能与此有关。研究结果为进一步阐述家蚕褐色类鹑斑产生的分子机制打下了基础。
Epidermis pigment of silkworm( Bombyx mori) is mainly composed of melanin,ommochrome and pteridine.All skin color of silkworm larvae is based on the interaction of melanin, ommochromes and pteridines etc. In this study,37 genes in the epidermis of q-l~b mutant 18 hours after 4 th instar molting related to pigmentation were analyzed by quantitative reverse transcription PCR( qRTPCR) with its wild-type strain 0223 JH as the control. Results showed that many key genes involved in biosynthesis of melanin and pteridine were up-regulated in q-l~b ,while key genes related to ommochrome biosynthesis were down-regulated,which indicated that the contents of melanin and pteridine increased but ommochrome con-tent decreased in q-l~b mutant. Key genes in chitin synthesis and genes related to uric acid transportation were differentially expressed in q-l~b mutant as compared with wild-type 0223 JH. Most of the key genes were up-regulated,which implicated that there were more chitin produced in newly grown epidermis of q-l~b mutant,and more uric acids were transported to cuticula,participating epidermis construction with other pigments and forming special body marks in q-l~b mutant. In addition,certain genes related to juvenile hormone metabolism and several nuclear receptor genes were differentially expressed between q-l~b and 0223 JH. These genes might also contribute to the pigmentation in q-l~b mutant. These differentially expressed genes might be responsible for the phenotype of q-l~b . Our research establishes a good foundation for further elucidation on molecular mechanism of pigmentation in the q-l~b mutant.
Epidermis pigment of silkworm( Bombyx mori) is mainly composed of melanin,ommochrome and pteridine.All skin color of silkworm larvae is based on the interaction of melanin, ommochromes and pteridines etc. In this study,37 genes in the epidermis of q-l~b mutant 18 hours after 4 th instar molting related to pigmentation were analyzed by quantitative reverse transcription PCR( qRTPCR) with its wild-type strain 0223 JH as the control. Results showed that many key genes involved in biosynthesis of melanin and pteridine were up-regulated in q-l~b ,while key genes related to ommochrome biosynthesis were down-regulated,which indicated that the contents of melanin and pteridine increased but ommochrome con-tent decreased in q-l~b mutant. Key genes in chitin synthesis and genes related to uric acid transportation were differentially expressed in q-l~b mutant as compared with wild-type 0223 JH. Most of the key genes were up-regulated,which implicated that there were more chitin produced in newly grown epidermis of q-l~b mutant,and more uric acids were transported to cuticula,participating epidermis construction with other pigments and forming special body marks in q-l~b mutant. In addition,certain genes related to juvenile hormone metabolism and several nuclear receptor genes were differentially expressed between q-l~b and 0223 JH. These genes might also contribute to the pigmentation in q-l~b mutant. These differentially expressed genes might be responsible for the phenotype of q-l~b . Our research establishes a good foundation for further elucidation on molecular mechanism of pigmentation in the q-l~b mutant.
引文
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[2] TATEMATSU K,YAMAMOTO K,UCHINO K,et al.Positional cloning of silkworm white egg 2(w-2)locus shows functional conservation and diversification of ABC transporters for pigmentation in insects[J].Genes Cells,2011,16(4):331-342
[3] MENG Y,KATSUMA S,DAIMON T,et al. The silkworm mutant lemon(lemon lethal)is a potential insect model for human sepiapterin reductase deficiency[J]. J Biol Chem,2009,284(17):11698-11705
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[5] LIU C,YAMAMOTO K,CHENG T C,et al.Repression of tyrosine hydroxylase is responsible for the sex-linked chocolate mutation of the silkworm,Bombyx mori[J].Proc Natl Acad Sci USA,2010,107(29):12980-12985
[6] DAI F,QIAO L,TONG X,et al.Mutations of an arylalkylamine-Nacetyltransferase,Bm-iAANAT,are responsible for silkworm melanism mutant[J].J Biol Chem,2010,285(25):19553-19560
[7] ZHAN S,GUO Q,LI M,et al.Disruption of an N-acetyltransferase gene in the silkworm reveals a novel role in pigmentation[J]. Development,2010,137(23):4083-4090
[8] WITTKOPP P,WILLIAMS B,SELEGUE J,et al.Drosophila pigmentation evolution:divergent genotypes underlying convergent phenotypes[J]. Proc Natl Acad Sci USA,2003,100(4):1808-1813
[9] KATO T,SAWADA H,YAMAMOTO T,et al.Pigment pattern formation in the quail mutant of the silkworm,Bombyx mori:parallel increase of pteridine biosynthesis and pigmentation of melanin and ommochromes[J].Pigment Cell Res,2006,19(4):337-345
[10] MENG Y,KATSUMA S,MITA K,et al.Abnormal red body coloration of the silkworm,Bombyx mori,is caused by a mutation in a novel kynureninase[J].Genes Cells,2009,14(2),129-140
[11]赵巧玲,王文波,陈安利,等.家蚕两种新的类鹑斑突变体的遗传分析和SSR标记定位[J].遗传,2014,36(4):369-375
[12] NIE H,LIU C,CHENG T,et al. Transcriptome analysis of integument differentially expressed genes in the pigment mutant(quail)during molting of silkworm,Bombyx mori[J/OL]. PLoS ONE,2014,9(4):e94185[2018-03-25]. https://journals. plos.org/plosone/2017/140409/ncomms0094185/full/ncomms00941-85.html.DOI:10.1371/ncomms0094185
[13] WANG P,QIU Z,XIA D,et al.Transcriptome analysis of the epidermis of the purple quail-like(q-lp)mutant of silkworm,Bombyx mori[J/OL].PLoS ONE,2017,12(4):e0175994[2018-03-25].https://journals. plos. org/plosone/2017/170417/ncomms017599-4/full/ncomms0175994.html.DOI:10.1371/ncomms0175994
[14] 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,2010,12:157-167
[15] WANG P,BI S,WU F,et al.Differentially expressed genes in the head of the 2nd instar pre-molting larvae of the nm2 mutant of the silkworm,Bombyx mori[J/OL]. PLoS ONE,2017,12(7):0180160[2018-03-25]. https://journals. plos. org/plosone/2017/170720/ncomms0180160/full/ncomms0180160. html. DOI:10.1371/ncomms0180160
[16] LIVAK K J,SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2ΔΔCT method[J].Methods,2001,25(4):402-408
[17] WITTKOPP P J,TRUE J R AND CARROLL S B.Reciprocal functions of the Drosophila yellow and ebony proteins in the development and evolution of pigment patterns[J]. Development,2002,129(8):1849-1858
[18] FUTAHASHI R,FUJIWARA H.Melanin-synthesis enzymes coregulate stagespecific larval cuticular markings in the swallowtail butterfly,Papilio xuthus[J]. Dev Genes Evol,2005,215(10):519-529
[19] HAN Q,FANG J,DING H,et al.Identification of Drosophila melanogaster yellow-f and yellow-f2 proteins as dopachrome-conversion enzymes[J].Biochem J,2002,368(1):333-340
[20] XIA A H,ZHOU Q X,YU L L,et al.Identification and analysis of YELLOW protein family genes in the silkworm,Bombyx mori[J/OL].BMC Genomics,2006,7(1):195[2018-03-25]. https://doi.org/10.1186/1471-2164-7-195
[21] MACKAY W J,O'DONNELL J M.A genetic analysis of the pteridine biosynthetic enzyme,guanosine triphosphate cyclohydrolase,in Drosophila melanogaster[J].Genetics,1983,105(1):35-53
[22] FUTAHASHI R,FUJIWARA H.Expression of one isoform of GTP cyclohydrolase I coincides with the larval black markings of the swallowtail butterfly,Papilio xuthus[J]. Insect Biochem Mol Biol,2006,36:63-70
[23] HAYASHI Y. Xanthine dehydrogenase in the silkworm,Bombyx mori[J].Nature,1960,186(4730):1053-1054
[24] MENG Y,KATSUMA S,MITA K,et al.Abnormal red body coloration of the silkworm,Bombyx mori,is caused by a mutation in a novel kynureninase[J].Genes Cells,2009,14(2):129-140
[25] KOMOTO N,SEZUTSU H,YUKUHIRO K,et al.Mutations of the silkworm molybdenum cofactor sulfurase gene,og,cause translucent larval skin[J].Insect Biochem Mol Biol,2003,33:417-427
[26] KIUCHI T,BANNO Y,KATSUMA S,et al.Mutations in an amino acid transporter gene are responsible for sex-linked translucent larval skin of the silkworm,Bombyx mori[J]. Insect Biochem Mol Biol,2011,41:680-687
[27] WANG L,KIUCHI T,FUJII T,et al. Mutation of a novel ABC transporter gene is responsible for the failure to incorporate uric acid in the epidermis of ok mutants of the silkworm,Bombyx mori[J].Insect Biochem Mol Biol,2013,43(7):562-571
[28] KOMOTO N,QUAN G X,SEZUTSU H,et al. A single-base deletion in an ABC transporter gene causes white eyes,white eggs,and translucent larval skin in the silkworm w-3oemutant[J].Insect Biochem Mol Biol,2009,39:152-156
[29] STEINER B,PFISTERWILHELM R,GROSSNIKLAUSBURGIN C,et al.Titres of juvenile hormoneⅠ,ⅡandⅢin Spodoptera littoralis(Noctuidae)from the egg to the pupal moult and their modification by the egg-larval parasitoid Chelonus inanitus(Braconidae)[J].J Insect Physiol,1999,45(4):401-413
[30] OHASHI M,TSUSUAM,KIGUCHI K.Juvenile hormone control of larval colouration in the silkworm,Bombyx mori:characterization and determination of epidermal brown colour induced by the hormone[J].Insect Biochem,1983,13(2):123-127
[31] SAFRANEK L,RIDDIFORD L M.The biology of the black larval mutant of the tobacco hornworm,Manduca sexta[J].J Insect Physiol,1975,21:1931-1938
[32] SHINODA T,ITOYAMA K. Juvenile hormone acid methyltransferase:a key regulatory enzyme for insect metamorphosis[J]. Proc Natl Acad Sci USA,2003,100(21):11986-11991
[33] MERZENDORFER H,ZIMOCH L. Chitin metabolism in insects:structure,function and regulation of chitin synthases and chitinases[J].J Exp Biol,2003,206(24):4393-4412
[34] MERZENDORFER H.Insect chitin synthases:a review[J].J Comp Physiol,2006,176(1):1-15
[35] WILLIS J H,ICONOMIDOU V A,SMITH R F,et al.Cuticular proteins[M].Oxford:Elsevier Pergamon Press,2005:79-109
[36] FUKAMIZO T,KRAMER K J. Mechanism of chitin hydrolysis by the binary chitinase system in insect moulting fluid[J].Insect Biochem,1985,15(2):141-145
[37] EVANS R M AND MANGELSDORF D J.Nuclear receptors,RXR,and the Big Bang[J].Cell,2014,157(1):255-266
[38] YAMADA M,MURATA T,HIROSE S,et al.Temporally restricted expression of transcription factor betaFTZ-F1:significance for embryogenesis,molting and metamorphosis in Drosophila melanogaster[J].Development,2000,127:5083-5092
[39] KING-JONES K,THUMMEL C S.Nuclear receptors—a perspective from Drosophila[J].Nat Rev Genet,2005,6:311-323
[40] YAO Q,ZHANG D,TANG B,et al. Identification of 20-hydroxyecdysone late-response genes in the chitin biosynthesis pathway[J/OL].PLoS ONE,2010,5(11):e14058[2018-03-25]. http://dx.plos.org/10.1371/journal.pone.0014058.pdf
[2] TATEMATSU K,YAMAMOTO K,UCHINO K,et al.Positional cloning of silkworm white egg 2(w-2)locus shows functional conservation and diversification of ABC transporters for pigmentation in insects[J].Genes Cells,2011,16(4):331-342
[3] MENG Y,KATSUMA S,DAIMON T,et al. The silkworm mutant lemon(lemon lethal)is a potential insect model for human sepiapterin reductase deficiency[J]. J Biol Chem,2009,284(17):11698-11705
[4] FUTAHASHI R,SATO J,MENG Y,et al.yellow and ebony are the responsible genes for the larval color mutants of the silkworm Bombyx mori[J].Genetics,2008,180:1995-2005
[5] LIU C,YAMAMOTO K,CHENG T C,et al.Repression of tyrosine hydroxylase is responsible for the sex-linked chocolate mutation of the silkworm,Bombyx mori[J].Proc Natl Acad Sci USA,2010,107(29):12980-12985
[6] DAI F,QIAO L,TONG X,et al.Mutations of an arylalkylamine-Nacetyltransferase,Bm-iAANAT,are responsible for silkworm melanism mutant[J].J Biol Chem,2010,285(25):19553-19560
[7] ZHAN S,GUO Q,LI M,et al.Disruption of an N-acetyltransferase gene in the silkworm reveals a novel role in pigmentation[J]. Development,2010,137(23):4083-4090
[8] WITTKOPP P,WILLIAMS B,SELEGUE J,et al.Drosophila pigmentation evolution:divergent genotypes underlying convergent phenotypes[J]. Proc Natl Acad Sci USA,2003,100(4):1808-1813
[9] KATO T,SAWADA H,YAMAMOTO T,et al.Pigment pattern formation in the quail mutant of the silkworm,Bombyx mori:parallel increase of pteridine biosynthesis and pigmentation of melanin and ommochromes[J].Pigment Cell Res,2006,19(4):337-345
[10] MENG Y,KATSUMA S,MITA K,et al.Abnormal red body coloration of the silkworm,Bombyx mori,is caused by a mutation in a novel kynureninase[J].Genes Cells,2009,14(2),129-140
[11]赵巧玲,王文波,陈安利,等.家蚕两种新的类鹑斑突变体的遗传分析和SSR标记定位[J].遗传,2014,36(4):369-375
[12] NIE H,LIU C,CHENG T,et al. Transcriptome analysis of integument differentially expressed genes in the pigment mutant(quail)during molting of silkworm,Bombyx mori[J/OL]. PLoS ONE,2014,9(4):e94185[2018-03-25]. https://journals. plos.org/plosone/2017/140409/ncomms0094185/full/ncomms00941-85.html.DOI:10.1371/ncomms0094185
[13] WANG P,QIU Z,XIA D,et al.Transcriptome analysis of the epidermis of the purple quail-like(q-lp)mutant of silkworm,Bombyx mori[J/OL].PLoS ONE,2017,12(4):e0175994[2018-03-25].https://journals. plos. org/plosone/2017/170417/ncomms017599-4/full/ncomms0175994.html.DOI:10.1371/ncomms0175994
[14] 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,2010,12:157-167
[15] WANG P,BI S,WU F,et al.Differentially expressed genes in the head of the 2nd instar pre-molting larvae of the nm2 mutant of the silkworm,Bombyx mori[J/OL]. PLoS ONE,2017,12(7):0180160[2018-03-25]. https://journals. plos. org/plosone/2017/170720/ncomms0180160/full/ncomms0180160. html. DOI:10.1371/ncomms0180160
[16] LIVAK K J,SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2ΔΔCT method[J].Methods,2001,25(4):402-408
[17] WITTKOPP P J,TRUE J R AND CARROLL S B.Reciprocal functions of the Drosophila yellow and ebony proteins in the development and evolution of pigment patterns[J]. Development,2002,129(8):1849-1858
[18] FUTAHASHI R,FUJIWARA H.Melanin-synthesis enzymes coregulate stagespecific larval cuticular markings in the swallowtail butterfly,Papilio xuthus[J]. Dev Genes Evol,2005,215(10):519-529
[19] HAN Q,FANG J,DING H,et al.Identification of Drosophila melanogaster yellow-f and yellow-f2 proteins as dopachrome-conversion enzymes[J].Biochem J,2002,368(1):333-340
[20] XIA A H,ZHOU Q X,YU L L,et al.Identification and analysis of YELLOW protein family genes in the silkworm,Bombyx mori[J/OL].BMC Genomics,2006,7(1):195[2018-03-25]. https://doi.org/10.1186/1471-2164-7-195
[21] MACKAY W J,O'DONNELL J M.A genetic analysis of the pteridine biosynthetic enzyme,guanosine triphosphate cyclohydrolase,in Drosophila melanogaster[J].Genetics,1983,105(1):35-53
[22] FUTAHASHI R,FUJIWARA H.Expression of one isoform of GTP cyclohydrolase I coincides with the larval black markings of the swallowtail butterfly,Papilio xuthus[J]. Insect Biochem Mol Biol,2006,36:63-70
[23] HAYASHI Y. Xanthine dehydrogenase in the silkworm,Bombyx mori[J].Nature,1960,186(4730):1053-1054
[24] MENG Y,KATSUMA S,MITA K,et al.Abnormal red body coloration of the silkworm,Bombyx mori,is caused by a mutation in a novel kynureninase[J].Genes Cells,2009,14(2):129-140
[25] KOMOTO N,SEZUTSU H,YUKUHIRO K,et al.Mutations of the silkworm molybdenum cofactor sulfurase gene,og,cause translucent larval skin[J].Insect Biochem Mol Biol,2003,33:417-427
[26] KIUCHI T,BANNO Y,KATSUMA S,et al.Mutations in an amino acid transporter gene are responsible for sex-linked translucent larval skin of the silkworm,Bombyx mori[J]. Insect Biochem Mol Biol,2011,41:680-687
[27] WANG L,KIUCHI T,FUJII T,et al. Mutation of a novel ABC transporter gene is responsible for the failure to incorporate uric acid in the epidermis of ok mutants of the silkworm,Bombyx mori[J].Insect Biochem Mol Biol,2013,43(7):562-571
[28] KOMOTO N,QUAN G X,SEZUTSU H,et al. A single-base deletion in an ABC transporter gene causes white eyes,white eggs,and translucent larval skin in the silkworm w-3oemutant[J].Insect Biochem Mol Biol,2009,39:152-156
[29] STEINER B,PFISTERWILHELM R,GROSSNIKLAUSBURGIN C,et al.Titres of juvenile hormoneⅠ,ⅡandⅢin Spodoptera littoralis(Noctuidae)from the egg to the pupal moult and their modification by the egg-larval parasitoid Chelonus inanitus(Braconidae)[J].J Insect Physiol,1999,45(4):401-413
[30] OHASHI M,TSUSUAM,KIGUCHI K.Juvenile hormone control of larval colouration in the silkworm,Bombyx mori:characterization and determination of epidermal brown colour induced by the hormone[J].Insect Biochem,1983,13(2):123-127
[31] SAFRANEK L,RIDDIFORD L M.The biology of the black larval mutant of the tobacco hornworm,Manduca sexta[J].J Insect Physiol,1975,21:1931-1938
[32] SHINODA T,ITOYAMA K. Juvenile hormone acid methyltransferase:a key regulatory enzyme for insect metamorphosis[J]. Proc Natl Acad Sci USA,2003,100(21):11986-11991
[33] MERZENDORFER H,ZIMOCH L. Chitin metabolism in insects:structure,function and regulation of chitin synthases and chitinases[J].J Exp Biol,2003,206(24):4393-4412
[34] MERZENDORFER H.Insect chitin synthases:a review[J].J Comp Physiol,2006,176(1):1-15
[35] WILLIS J H,ICONOMIDOU V A,SMITH R F,et al.Cuticular proteins[M].Oxford:Elsevier Pergamon Press,2005:79-109
[36] FUKAMIZO T,KRAMER K J. Mechanism of chitin hydrolysis by the binary chitinase system in insect moulting fluid[J].Insect Biochem,1985,15(2):141-145
[37] EVANS R M AND MANGELSDORF D J.Nuclear receptors,RXR,and the Big Bang[J].Cell,2014,157(1):255-266
[38] YAMADA M,MURATA T,HIROSE S,et al.Temporally restricted expression of transcription factor betaFTZ-F1:significance for embryogenesis,molting and metamorphosis in Drosophila melanogaster[J].Development,2000,127:5083-5092
[39] KING-JONES K,THUMMEL C S.Nuclear receptors—a perspective from Drosophila[J].Nat Rev Genet,2005,6:311-323
[40] YAO Q,ZHANG D,TANG B,et al. Identification of 20-hydroxyecdysone late-response genes in the chitin biosynthesis pathway[J/OL].PLoS ONE,2010,5(11):e14058[2018-03-25]. http://dx.plos.org/10.1371/journal.pone.0014058.pdf