马氏珠母贝贝壳基质蛋白基因Pm-PNU7的克隆和功能研究
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摘要
【目的】研究马氏珠母贝(Pinctada martensii)贝壳基质蛋白(Shell Matrix Proteins, SMPs)基因(Pm-PNU7)表达与功能。【方法】利用马氏珠母贝基因组和蛋白质组信息分析和RACE技术克隆获得壳基质蛋白新基因Pm-PNU7全长序列,通过原位杂交对其进行定位,RNA干扰检测其对贝壳形成的影响。【结果】Pm-PNU7的cDNA全长为797 bp,编码145个氨基酸,具有特殊的"KGG"重复序列和富含天冬氨酸(Asp)序列"DDDDDDHDD"。qRT-PCR分析表明,Pm-PNU7基因在外套膜边缘区和套膜区显著高表达(P<0.05);ISH检测显示,Pm-PNU7主要定位于边缘区外褶外侧和内侧上皮细胞、中褶内侧上皮细胞以及套膜区外侧上皮细胞。RNA干扰后,Pm-PNU7在外套膜边缘区和套膜区的表达量均显著下调,贝壳棱柱层和珍珠层微观结构均出现不同程度的紊乱。【结论】Pm-PNU7参与了贝壳棱柱层和珍珠层的形成。
【Objective】The expression and function of a novel shell matrix protein Pm-PNU7 in pearl oyster Pinctada martensii were studied according to the genome and proteome analyses.【Method】The full-length sequence of Pm-PNU7 gene of was cloned by RACE technology.Its transcripts was localized by in situ hybridization and the function of the gene in shell formation was studied by RNA interference.【Results】The full-length SMP Pm-PNU7 gene was 797 bp,encoding a novel protein with 145 amino acids with no functional annotation.It contained a special repeating sequence "KGG" and an aspartic acid-rich region "DDDDDDHDD".qRT-PCR analysis results showed that Pm-PNU7 was significantly highly expressed in mantle edge(ME) and mantle pallial(MP)(P<0.05).ISH results showed that Pm-PNU7 mainly distributed in the inner and the outer epidermal cells of the outer fold of ME,the inner epidermal cells of the middle fold of ME,and the outer epidermal cells of MP.After gene knock down of Pm-PNU7 in ME and MP(P<0.05)by RNAi,,the microstructure of both prismatic layers and nacreous layers appeared distorted【Conclusion】Pm-PNU7 could affect the formation of the prismatic and nacreous layers of P.martensii.
【Objective】The expression and function of a novel shell matrix protein Pm-PNU7 in pearl oyster Pinctada martensii were studied according to the genome and proteome analyses.【Method】The full-length sequence of Pm-PNU7 gene of was cloned by RACE technology.Its transcripts was localized by in situ hybridization and the function of the gene in shell formation was studied by RNA interference.【Results】The full-length SMP Pm-PNU7 gene was 797 bp,encoding a novel protein with 145 amino acids with no functional annotation.It contained a special repeating sequence "KGG" and an aspartic acid-rich region "DDDDDDHDD".qRT-PCR analysis results showed that Pm-PNU7 was significantly highly expressed in mantle edge(ME) and mantle pallial(MP)(P<0.05).ISH results showed that Pm-PNU7 mainly distributed in the inner and the outer epidermal cells of the outer fold of ME,the inner epidermal cells of the middle fold of ME,and the outer epidermal cells of MP.After gene knock down of Pm-PNU7 in ME and MP(P<0.05)by RNAi,,the microstructure of both prismatic layers and nacreous layers appeared distorted【Conclusion】Pm-PNU7 could affect the formation of the prismatic and nacreous layers of P.martensii.
引文
[1]闫芳.马氏珠母贝珍珠层形成相关基因的克隆与功能研究[D].湛江:广东海洋大学,2014.
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[14]SUZUKI M,SARUWATARI K,KOGURE T,et al.An acidic matrix protein,Pif,is a key macromolecule for nacre formation[J].Science,2009,325(5946):1388.
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[16]ADDADI L,MORADIAN J,SHAY E,et al.A chemical model for the cooperation of sulfates and carboxylates in calcite crystal nucleation:Relevance to biomineralization[J].Proceedings of the National Academy of Sciences of the United States of America,1987,84(9):2732-2736.
[17]KONG Y,JING G,YAN Z,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[J].Journal of Biological Chemistry,2009,284(16):10841-10854.
[18]TAKAGI R,MIYASHITA T.Prismin:A new matrix protein family in the Japanese pearl oyster(pinctada fucata)involved in prismatic layer formation[J].Zoological Science,2010,27(5):416-426.
[19]YU S,INOUE N,ISHIKAWA T,et al.Pearl microstructure and expression of shell matrix protein genes msi31 and msi60in the pearl sac epithelium of pinctada fucata by in situ hybridization[J].PLoS One,2013,8(1):e52372.
[20]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)[J].Biochemical Journal,2004,382(Pt 1):205.
[21]ZHANG Y,XIE L,MENG Q,et al.A novel matrix protein participating in the nacre framework formation of pearl oyster,Pinctada fucata[J].Comparative Biochemistry&Physiology Part B,2003,135(3):565-573.
[22]师尚丽,焦钰,杜晓东,等.马氏珠母贝外套膜细胞的超微结构观察[J].广东农业科学,2012,39(8):135-137.
[23]GARDNER L D.Spatial analysis of biomineralization associated gene expression from the mantle organ of the pearl oyster Pinctada maxima[J].Bmc Genomics,2011,12(1):455.
[24]YAN Y,YANG D,YANG X,et al.A novel matrix protein,PfY2,functions as a crucial macromolecule during shell formation[J].Scientific Reports,2017,7(1):6021.
[25]WANG X,HARIMOTO K,FUJI R,et al.Pinctada fucata mantle gene 4(PFMG4)from pearl oyster mantle enhances osteoblast differentiation[J].Journal of the Agricultural Chemical Society of Japan,2015,79(4):558-565.
[26]JIAN L,XU G,XIE J,et al.Dual Roles of the lysine-rich matrix protein(KRMP)-3 in shell Formation of pearl oyster,Pinctada fucata[J].PLoS One,2015,10(7):e0131868.
[2]欧阳健明.生物矿化的基质调控及其仿生应用[M].北京:化学工业出版社,2006.
[3]CARIOLOU M A,MORSE D E.Purification and characterization of calcium-binding conchiolin shell peptides from the mollusc,Haliotis rufescens,as a function of development[J].Journal of Comparative Physiology B,1988,157(6):717-729.
[4]ADDADI L,WEINER S.Biomineralization:A pavement of pearl[J].Nature,1997,389(6654):912-915.
[5]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[J].Comparative Biochemistry&Physiology Part B,2006,144(2):254-262.
[6]ZHANG C,XIE L P,HUANG J,et al.A novel matrix protein family participating in the prismatic layer framework formation of pearl oyster,Pinctada fucata[J].Biochemical&Biophysical Research Communications,2006,344(3):735-740.
[7]ZHANG Y,XIE L,MENG Q,et al.A novel matrix protein participating in the nacre framework formation of pearl oyster,Pinctada fucata[J].Comparative Biochemistry&Physiology Part B Biochemistry&Molecular Biology,2003,135(3):565-573.
[8]GOTLIV B A,KESSLER N,SUNEREL J L,et al.Asprich:A novel aspartic acid-rich protein family from the prismatic shell matrix of the bivalve Atrina rigida[J].Chembiochem,1997,6(2):304-314.
[9]TSUKAMOTO D,SARASHINA I,ENDO K.Structure and expression of an unusually acidic matrix protein of pearl oyster shells[J].Biochemical&Biophysical Research Communications,2004,320(4):1175-1180.
[10]SARASHINA I,ENDO K.The complete primary structure of molluscan shell protein 1(MSP-1),an acidic glycoprotein in the shell matrix of the scallop Patinopecten yessoensis[J].Marine Biotechnology,2001,3(4):362.
[11]SARASHINA I,ENDO K.Primary structure of a soluble matrix protein of scallop shell;implications for calcium carbonate biomineralization[J].American Mineralogist,1998,83(11):1510-1515.
[12]DU X D,FAN G Y,JIAO Y,et al.The pearl oyster Pinctada fucata martensii genome and multi-omic analyses provide insights into biomineralization[J].Gigascience,2017,6(8):1-12.
[13]ZHANG G,FANG X,GUO X,et al.The oyster genome reveals stress adaptation and complexity of shell formation[J].Nature,2012,490(7418):49-54.
[14]SUZUKI M,SARUWATARI K,KOGURE T,et al.An acidic matrix protein,Pif,is a key macromolecule for nacre formation[J].Science,2009,325(5946):1388.
[15]MARIE B,JOUBERT C,TAYALéA,et al.Different secretory repertoires control the biomineralization processes of prism and nacre deposition of the pearl oyster shell[J].Proceedings of the National Academy of Sciences of the United States of America,2012,109(51):20986-20991.
[16]ADDADI L,MORADIAN J,SHAY E,et al.A chemical model for the cooperation of sulfates and carboxylates in calcite crystal nucleation:Relevance to biomineralization[J].Proceedings of the National Academy of Sciences of the United States of America,1987,84(9):2732-2736.
[17]KONG Y,JING G,YAN Z,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[J].Journal of Biological Chemistry,2009,284(16):10841-10854.
[18]TAKAGI R,MIYASHITA T.Prismin:A new matrix protein family in the Japanese pearl oyster(pinctada fucata)involved in prismatic layer formation[J].Zoological Science,2010,27(5):416-426.
[19]YU S,INOUE N,ISHIKAWA T,et al.Pearl microstructure and expression of shell matrix protein genes msi31 and msi60in the pearl sac epithelium of pinctada fucata by in situ hybridization[J].PLoS One,2013,8(1):e52372.
[20]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)[J].Biochemical Journal,2004,382(Pt 1):205.
[21]ZHANG Y,XIE L,MENG Q,et al.A novel matrix protein participating in the nacre framework formation of pearl oyster,Pinctada fucata[J].Comparative Biochemistry&Physiology Part B,2003,135(3):565-573.
[22]师尚丽,焦钰,杜晓东,等.马氏珠母贝外套膜细胞的超微结构观察[J].广东农业科学,2012,39(8):135-137.
[23]GARDNER L D.Spatial analysis of biomineralization associated gene expression from the mantle organ of the pearl oyster Pinctada maxima[J].Bmc Genomics,2011,12(1):455.
[24]YAN Y,YANG D,YANG X,et al.A novel matrix protein,PfY2,functions as a crucial macromolecule during shell formation[J].Scientific Reports,2017,7(1):6021.
[25]WANG X,HARIMOTO K,FUJI R,et al.Pinctada fucata mantle gene 4(PFMG4)from pearl oyster mantle enhances osteoblast differentiation[J].Journal of the Agricultural Chemical Society of Japan,2015,79(4):558-565.
[26]JIAN L,XU G,XIE J,et al.Dual Roles of the lysine-rich matrix protein(KRMP)-3 in shell Formation of pearl oyster,Pinctada fucata[J].PLoS One,2015,10(7):e0131868.