基于Illumina平台的厚壳贻贝外套膜转录组从头测序
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摘要
为了解贻贝贝壳基质蛋白的分子多样性,采用Illumina高通量测序平台,对厚壳贻贝(Mytilus coruscus)外套膜组织开展转录组文库构建及从头测序研究,总计产出6 970 102 920nt数据,共77 445 588条干净阅读子;经序列拼接与组装,获得厚壳贻贝外套膜转录组单一基因共计106 452个,总长55 222 289nt,平均长度519nt,N50达到754nt;并对上述单一基因进行了生物学功能注释和聚类分析。为进一步挖掘贻贝贝壳基质蛋白相关基因,通过对单一基因的序列注释以及利用已知贝壳基质蛋白序列进行本地搜索,共鉴定出与12种已知贝壳基质蛋白同源的厚壳贻贝外套膜单一基因124条。通过对厚壳贻贝贝壳基质蛋白单一基因进行序列分析和比较,发现厚壳贻贝与其他软体动物,如鲍属、牡蛎属和珠母贝属等在贝壳基质蛋白组成以及序列上具有一定的保守性,暗示着贝壳基质蛋白可能具有共同的起源;同时,首次从贻贝属中发现了3种贝壳棱柱层特异的贝壳基质蛋白单一基因。这为后续贻贝贝壳基质蛋白的大规模鉴定以及对贝壳形成机制的探讨奠定了基础。
Mollusk shell is an exoskeleton made up of organic mineral particles and its function is to protect the inner soft body from predators and environmental damage.Due to its superior mechanical properties,such as stiffness,fracture toughness,and tensile strength,the mollusk shell has been investigated as an excellent biomaterial and as bio-mineralization model over the past decades.Previous studies have revealed that mollusk shell was consist of a mineral phase(calcium carbonate)and an organic matrix(proteins,polysaccharides and lipids).The organic matrix intercalated in a shell is generally assumed to play an important role in crystal nucleation,crystal orientation,crystal size regulation,and crystal polymorphism and contributing to the shell's mechanical properties.Recent advances on ease and availability of high throughput RNA-sequencing have resulted in a sharp increase in transcriptome data for invertebrate bio-minerals.However,for Mytilus,bio-mineralizationmechanisms and shell formation were restricted to obtain by deficiency of transcriptome information.To understand the molecular diversity of matrix proteins from mussel shell and to develop a transcriptome from the adult mantle tissue of M.coruscus,the transcriptome sequencing of M.coruscus mantle was performed by Illumina platform.A total of 77 445 588paired-end reads amounting to 6Gb of sequence data were generated and further assembled de novo with Trinity software.A transcriptome was produced after filtering and quality checking,yielded a final set of 106 425 high quality mantle unigenes for analysis.To obtain an integrated view of the transcriptional events of bio-mineralization related processes in M.coruscus mantle,the unique transcript library was screened for reported shell matrix proteins(SMPs).Taken together,124 unigenes were identified with significant hits(E-value <10-5)to 12 reported bio-mineralization related proteins including all known Mytilus SMPs.The most abundant unigenes out of 124 matched ones were annotated as calponin,followed by fibronectin,perlucin,nacrein,and Shematrin.Most of homologues were fromCrassostrea,Pinctada,Haliotis,and Mytilus,which showed that M.coruscus mantle unigene encoded putative proteins exhibiting sequence similarities with previously characterized SMPs of other Bivalvias,indicating a common originate for these SMPs.In conclusion,a comprehensive transcriptomic resource for the Mytilus SMPs was presented in this paper,providing insight into the protein composition of the shell.The transcriptomic resource can also provide a foundation for further investigations on the formation and bio-mineralization of mussel shell.
Mollusk shell is an exoskeleton made up of organic mineral particles and its function is to protect the inner soft body from predators and environmental damage.Due to its superior mechanical properties,such as stiffness,fracture toughness,and tensile strength,the mollusk shell has been investigated as an excellent biomaterial and as bio-mineralization model over the past decades.Previous studies have revealed that mollusk shell was consist of a mineral phase(calcium carbonate)and an organic matrix(proteins,polysaccharides and lipids).The organic matrix intercalated in a shell is generally assumed to play an important role in crystal nucleation,crystal orientation,crystal size regulation,and crystal polymorphism and contributing to the shell's mechanical properties.Recent advances on ease and availability of high throughput RNA-sequencing have resulted in a sharp increase in transcriptome data for invertebrate bio-minerals.However,for Mytilus,bio-mineralizationmechanisms and shell formation were restricted to obtain by deficiency of transcriptome information.To understand the molecular diversity of matrix proteins from mussel shell and to develop a transcriptome from the adult mantle tissue of M.coruscus,the transcriptome sequencing of M.coruscus mantle was performed by Illumina platform.A total of 77 445 588paired-end reads amounting to 6Gb of sequence data were generated and further assembled de novo with Trinity software.A transcriptome was produced after filtering and quality checking,yielded a final set of 106 425 high quality mantle unigenes for analysis.To obtain an integrated view of the transcriptional events of bio-mineralization related processes in M.coruscus mantle,the unique transcript library was screened for reported shell matrix proteins(SMPs).Taken together,124 unigenes were identified with significant hits(E-value <10-5)to 12 reported bio-mineralization related proteins including all known Mytilus SMPs.The most abundant unigenes out of 124 matched ones were annotated as calponin,followed by fibronectin,perlucin,nacrein,and Shematrin.Most of homologues were fromCrassostrea,Pinctada,Haliotis,and Mytilus,which showed that M.coruscus mantle unigene encoded putative proteins exhibiting sequence similarities with previously characterized SMPs of other Bivalvias,indicating a common originate for these SMPs.In conclusion,a comprehensive transcriptomic resource for the Mytilus SMPs was presented in this paper,providing insight into the protein composition of the shell.The transcriptomic resource can also provide a foundation for further investigations on the formation and bio-mineralization of mussel shell.
引文
[1]Mann S.Biomineralization Principles and Concepts in Bioinorganic Materials Chemistry.New York,USA:Oxford University Press,2001:24-26.
[2]Freer A,Bridgett S,Jiang J H,et al.Biomineral proteins from Mytilus edulis mantle tissue transcriptome.Marine Biotechnology,2014,16(1):34-45.
[3]Marie B,Marie A,Jackson D J,et al.Proteomic analysis of the organic matrix of the abalone Haliotis asinina calcified shell.Proteome Science,2010,8(1):54-64.
[4]Joubert C,Piquemal D,Marie B,et al.Transcriptome and proteome analysis of Pinctada margaritifera calcifying mantle and shell:Focus on biomineralization.BMC Genomics,2010,11(1):613-625.
[5]Marie B,Trinkler N,Zanella-Cleon I,et al.Proteomic identification of novel proteins from the calcifying shell matrix of the Manila clam Venerupis philippinarum.Marine Biotechnology,2011,13(5):955-962.
[6]Perovic I,Chang E P,Lui M,et al.A nacre protein,n16.3,self-assembles to form protein oligomers that dimensionally limit and organize mineral deposits.Biochemistry,2014,53(16):2739-2748.
[7]Liu L,Hu N,Wang B,et al.A brief utilization report on the Illumina HiSeq 2000sequencer.Mycology,2011,2(3):169-191.
[8]Jackson D J,Wrheide G,Degnan B M.Dynamic expression of ancient and novel molluscan shell genes during ecological transitions.BMCEvolutionBiology,2007,7:160-176.
[9]Jackson D J,McDougall C,Green K,et al.A rapidly evolving secretome builds and patterns a sea shell.BMC Biology,2006,4(1):40-49.
[10]Miyamoto H,Miyashita T,Okushima M,et al.A carbonic anhydrase from the nacreous layer in oyster pearls.Proceedings of the National Academy of Sciences of the USA,1996,93(18):9657-9660.
[11]Huang J,Zhang C,Ma Z J,et al.A novel extracellular EFhand protein involved in the shell formation of pearl oyster.Biochimica et Biophysica Acta:General Subjects,2007,1770(7):1037-1044.
[12]Ferjani I,Fattoum A,Manai M,et al.Two distinct regions of calponin share common binding sites on actin resulting in different modes of calponin-actin interaction.Biochimica et Biophysica Acta:Proteins and Proteomics,2010,1804(9):1760-1767.
[13]Pankov R,Yamada K M.Fibronectin at a glance.Journal of Cell Science,2002,115(20):3861-3863.
[14]Marie B,Le Roy N,Zanella-Cléon I,et al.Molecular evolution of mollusc shell proteins:Insights from proteomic analysis of the edible mussel mytilus.Journal of Molecular Evolution,2011,72(5/6):531-546.
[15]Weiss I M,Kaufmann S,Mann K,et al.Purification and characterization of perlucin and perlustrin,two new proteins from the shell of the mollusk Haliotis laevigata.Biochemical and Biophysical Research Communications,2000,267(1):17-21.
[16]Mann K,Weiss I M,AndréS,et al.The amino-acid sequence of the abalone(Haliotis laevigata)nacre protein perlucin.Detection of a functional C-type lectin domain with galactose/mannose specificity.The Federation of European Biochemical Societies Journal,2000,267(16):5257-5264.
[17]Zhang G F,Fang X D,Guo X M,et al.The oyster genome reveals stress adaptation and complexity of shell formation.Nature,2012,490(7418):49-54.
[18]Berman A,Addadi L,Kvick A,et al.Intercalation of sea urchin proteins in calcite:Study of a crystalline composite material.Science,1990,250(4981):664-667.
[19]Miyamoto H,Yano M,Miyashita T.Similarities in the structure of nacrein,the shell-matrix protein,in a bivalve and a gastropod.Journal of Molluscan Studies,2003,69(1):87-89.
[20]Yano M,Nagai K,Morimoto K,et al.Shematrin:A family of glycine-rich structural proteins in the shell of the pearl oyster Pinctada fucata.Comparative Biochemistry and Physiology Part B:Molecular Biology,2006,144(2):254-262.
[21]Kono M,Hayashi N,Samata T.Molecular mechanism of the nacreous layer formation in Pinctada maxima.Biochemical and Biophysical Research Communications,2000,269(1):213-218.
[22]Suzuki M,Saruwatari K,Kogure T,et al.An acidic matrix protein,Pif,is a key macromolecule for nacre formation.Science,2009,325(5946):1388-1390.
[23]Suzuki M,Iwashima A,Tsutsui N,et al.Identification and characterisation of a calcium carbonate-binding protein,blue mussel shell protein(BMSP),from the nacreous layer.ChemBioChem,2011,12(16):2478-2487.
[24]Sudo S,Fujikawa T,Nagakura T,et al.Structures of mollusc shell framework proteins.Nature,1997,387(6633):563-564.
[25]Takeuchi T,Endo K.Biphasic and dually coordinated expression of the genes encoding major shell matrix proteins in the pearl oyster Pinctada fucata.Marine Biotechnology,2006,8(1):52-61.
[26]Suzuki M,Murayama E,Inoue H,et al.Characterization of Prismalin-14,a novel matrix protein from the prismatic layer of the Japanese pearl oyster(Pinctada fucata).Biochemistry Journal,2004,382(1):205-213.
[27]Suzuki M,Nagasawa H.The structure-function relationship analysis of Prismalin-14 from the prismatic layer of the Japanese pearl oyster,Pinctada fucata.The Federation of European Biochemical Societies Journal,2007,274(19):5158-5166.
[28]Kong Y W,Jing G,Yan Z G,et al.Cloning and characterization of Prisilkin-39,a novel matrix protein serving a dual role in the prismatic layer formation from the oyster Pinctada fucata.Journal of Biological Chemistry,2009,284(16):10841-10854.
[29]Jackson D J,McDougall C,Woodcroft B,et al.Parallel evolution of nacre building gene sets in molluscs.Molecular Biology and Evolution,2010,27(3):591-608.
[2]Freer A,Bridgett S,Jiang J H,et al.Biomineral proteins from Mytilus edulis mantle tissue transcriptome.Marine Biotechnology,2014,16(1):34-45.
[3]Marie B,Marie A,Jackson D J,et al.Proteomic analysis of the organic matrix of the abalone Haliotis asinina calcified shell.Proteome Science,2010,8(1):54-64.
[4]Joubert C,Piquemal D,Marie B,et al.Transcriptome and proteome analysis of Pinctada margaritifera calcifying mantle and shell:Focus on biomineralization.BMC Genomics,2010,11(1):613-625.
[5]Marie B,Trinkler N,Zanella-Cleon I,et al.Proteomic identification of novel proteins from the calcifying shell matrix of the Manila clam Venerupis philippinarum.Marine Biotechnology,2011,13(5):955-962.
[6]Perovic I,Chang E P,Lui M,et al.A nacre protein,n16.3,self-assembles to form protein oligomers that dimensionally limit and organize mineral deposits.Biochemistry,2014,53(16):2739-2748.
[7]Liu L,Hu N,Wang B,et al.A brief utilization report on the Illumina HiSeq 2000sequencer.Mycology,2011,2(3):169-191.
[8]Jackson D J,Wrheide G,Degnan B M.Dynamic expression of ancient and novel molluscan shell genes during ecological transitions.BMCEvolutionBiology,2007,7:160-176.
[9]Jackson D J,McDougall C,Green K,et al.A rapidly evolving secretome builds and patterns a sea shell.BMC Biology,2006,4(1):40-49.
[10]Miyamoto H,Miyashita T,Okushima M,et al.A carbonic anhydrase from the nacreous layer in oyster pearls.Proceedings of the National Academy of Sciences of the USA,1996,93(18):9657-9660.
[11]Huang J,Zhang C,Ma Z J,et al.A novel extracellular EFhand protein involved in the shell formation of pearl oyster.Biochimica et Biophysica Acta:General Subjects,2007,1770(7):1037-1044.
[12]Ferjani I,Fattoum A,Manai M,et al.Two distinct regions of calponin share common binding sites on actin resulting in different modes of calponin-actin interaction.Biochimica et Biophysica Acta:Proteins and Proteomics,2010,1804(9):1760-1767.
[13]Pankov R,Yamada K M.Fibronectin at a glance.Journal of Cell Science,2002,115(20):3861-3863.
[14]Marie B,Le Roy N,Zanella-Cléon I,et al.Molecular evolution of mollusc shell proteins:Insights from proteomic analysis of the edible mussel mytilus.Journal of Molecular Evolution,2011,72(5/6):531-546.
[15]Weiss I M,Kaufmann S,Mann K,et al.Purification and characterization of perlucin and perlustrin,two new proteins from the shell of the mollusk Haliotis laevigata.Biochemical and Biophysical Research Communications,2000,267(1):17-21.
[16]Mann K,Weiss I M,AndréS,et al.The amino-acid sequence of the abalone(Haliotis laevigata)nacre protein perlucin.Detection of a functional C-type lectin domain with galactose/mannose specificity.The Federation of European Biochemical Societies Journal,2000,267(16):5257-5264.
[17]Zhang G F,Fang X D,Guo X M,et al.The oyster genome reveals stress adaptation and complexity of shell formation.Nature,2012,490(7418):49-54.
[18]Berman A,Addadi L,Kvick A,et al.Intercalation of sea urchin proteins in calcite:Study of a crystalline composite material.Science,1990,250(4981):664-667.
[19]Miyamoto H,Yano M,Miyashita T.Similarities in the structure of nacrein,the shell-matrix protein,in a bivalve and a gastropod.Journal of Molluscan Studies,2003,69(1):87-89.
[20]Yano M,Nagai K,Morimoto K,et al.Shematrin:A family of glycine-rich structural proteins in the shell of the pearl oyster Pinctada fucata.Comparative Biochemistry and Physiology Part B:Molecular Biology,2006,144(2):254-262.
[21]Kono M,Hayashi N,Samata T.Molecular mechanism of the nacreous layer formation in Pinctada maxima.Biochemical and Biophysical Research Communications,2000,269(1):213-218.
[22]Suzuki M,Saruwatari K,Kogure T,et al.An acidic matrix protein,Pif,is a key macromolecule for nacre formation.Science,2009,325(5946):1388-1390.
[23]Suzuki M,Iwashima A,Tsutsui N,et al.Identification and characterisation of a calcium carbonate-binding protein,blue mussel shell protein(BMSP),from the nacreous layer.ChemBioChem,2011,12(16):2478-2487.
[24]Sudo S,Fujikawa T,Nagakura T,et al.Structures of mollusc shell framework proteins.Nature,1997,387(6633):563-564.
[25]Takeuchi T,Endo K.Biphasic and dually coordinated expression of the genes encoding major shell matrix proteins in the pearl oyster Pinctada fucata.Marine Biotechnology,2006,8(1):52-61.
[26]Suzuki M,Murayama E,Inoue H,et al.Characterization of Prismalin-14,a novel matrix protein from the prismatic layer of the Japanese pearl oyster(Pinctada fucata).Biochemistry Journal,2004,382(1):205-213.
[27]Suzuki M,Nagasawa H.The structure-function relationship analysis of Prismalin-14 from the prismatic layer of the Japanese pearl oyster,Pinctada fucata.The Federation of European Biochemical Societies Journal,2007,274(19):5158-5166.
[28]Kong Y W,Jing G,Yan Z G,et al.Cloning and characterization of Prisilkin-39,a novel matrix protein serving a dual role in the prismatic layer formation from the oyster Pinctada fucata.Journal of Biological Chemistry,2009,284(16):10841-10854.
[29]Jackson D J,McDougall C,Woodcroft B,et al.Parallel evolution of nacre building gene sets in molluscs.Molecular Biology and Evolution,2010,27(3):591-608.