柳蚕丝素重链基因的克隆和表达分析
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摘要
蚕丝由丝胶和丝素两部分组成。丝胶主要是由中部丝腺合成,约占重量的25 %,包裹着丝素蛋白。丝素蛋白主要是在后部丝腺合成,是蚕丝中主要的组成部分,约占重量的70%。
丝素重链(Fib-H)作为丝素蛋白中主要的组成部分,与丝素轻链(Fib-L)和P25蛋白以6∶6∶1的摩尔比组成复合体,构成丝素的基本结构单位。研究丝素重链对于提高蚕丝的产量和质量、生产有特殊用途的丝产品、提高蚕的抗病能力和抵抗不良环境的能力以及弄清物种之间的同源性与进化关系等方面具有重要的理论和实际应用价值。
本研究利用反转录PCR的方法首次在柳蚕丝腺中获得了Fib-H基因的5’端序列,片段长2801bp,编码933个氨基酸,包含起始密码子ATG,具有野蚕Fib-H保守结构特征,经过与柞蚕、天蚕、樟蚕、印度柞蚕等昆虫的丝素基因对比,发现有高度的同源性,尤其与同为大蚕蛾科的樟蚕的关系最近。GenBank登录号:GU212855.1。利用半定量PCR对五龄期不同时间Fib-H基因的表达情况进行分析,结果表明:柳蚕幼虫5龄期第1日、第3日、第6日、第9日、第12日的后部丝腺中,Fib-H基因的表达量呈逐渐增加的趋势,与丝腺发育的生理特征相符合。通过对五龄期第1日、第3日、第6日、第9日、第12日中部丝腺、脂肪体等不同组织Fib-H基因的表达情况进行分析,发现Fib-H基因在中部丝腺中也存在一定程度的表达,且变化趋势大致是:低—高—低-高。脂肪体中未检测到该基因表达,表明Fib-H基因的表达具有组织特异性,这与早期的关于Fib-H基因的研究结果基本是一致的。
Silk contains two main parts, sericin and fibroin.Sericin produced in mid-silk glang encapsulates fibroin which takes up 25% of the total weight. Fibroin produced in post-silk gland is the main part of silk which takes up 70% of the total weight.
Fibroin-H as the main part of silk、fibroin-L and P25 with a 6:6:1 molar ratio make up the fundamental structural unit of fibroin.Rearch of fibroin-H has important theoretical and practical value. From theoretical sense, we can ascertain the homology and evolution relationship of different species. From practical possibilities ,we can increase output and quality of silk, produce special silk product,raise silkworm ablility of resistance to disease and the ability to resist bad environment and development new applications.
This experiment cloned Fibroin-H gene sequence of the 5’flanking from Actias selene firstly. This sequence of 2801bp long contains start codon and encodes 933 amino acids with Fibroin-H conservative structural characteristics.We have found that the sequence has a high homology with that of Antheraea pernyi、Antheraea yamamai、Saturnia japonica、Anthereaea mylitta, especially Saturnia japonica.It shows the result is right.The accesssion number is GU212855.1. Through expression analysis of semi-quantitative PCR towards fibroin-H of different time in the fifth instar, we have found expession of fibroin-H from the first day,third day, sixth day, ninth day and twelfth day in the fifth instar of A.selene in post-silk gland increases day by day.It is credible.Through analysis we have detected fibroin-H from the first day,third day, sixth day, ninth day and twelfth day in the fifth instar of A.selene in mid-silk gland and the trend is:low-high-low-high.We have not detected fibroin-H in fat body.It shows expression of fibroin-H has organization specificity which is in accord with previous study.
丝素重链(Fib-H)作为丝素蛋白中主要的组成部分,与丝素轻链(Fib-L)和P25蛋白以6∶6∶1的摩尔比组成复合体,构成丝素的基本结构单位。研究丝素重链对于提高蚕丝的产量和质量、生产有特殊用途的丝产品、提高蚕的抗病能力和抵抗不良环境的能力以及弄清物种之间的同源性与进化关系等方面具有重要的理论和实际应用价值。
本研究利用反转录PCR的方法首次在柳蚕丝腺中获得了Fib-H基因的5’端序列,片段长2801bp,编码933个氨基酸,包含起始密码子ATG,具有野蚕Fib-H保守结构特征,经过与柞蚕、天蚕、樟蚕、印度柞蚕等昆虫的丝素基因对比,发现有高度的同源性,尤其与同为大蚕蛾科的樟蚕的关系最近。GenBank登录号:GU212855.1。利用半定量PCR对五龄期不同时间Fib-H基因的表达情况进行分析,结果表明:柳蚕幼虫5龄期第1日、第3日、第6日、第9日、第12日的后部丝腺中,Fib-H基因的表达量呈逐渐增加的趋势,与丝腺发育的生理特征相符合。通过对五龄期第1日、第3日、第6日、第9日、第12日中部丝腺、脂肪体等不同组织Fib-H基因的表达情况进行分析,发现Fib-H基因在中部丝腺中也存在一定程度的表达,且变化趋势大致是:低—高—低-高。脂肪体中未检测到该基因表达,表明Fib-H基因的表达具有组织特异性,这与早期的关于Fib-H基因的研究结果基本是一致的。
Silk contains two main parts, sericin and fibroin.Sericin produced in mid-silk glang encapsulates fibroin which takes up 25% of the total weight. Fibroin produced in post-silk gland is the main part of silk which takes up 70% of the total weight.
Fibroin-H as the main part of silk、fibroin-L and P25 with a 6:6:1 molar ratio make up the fundamental structural unit of fibroin.Rearch of fibroin-H has important theoretical and practical value. From theoretical sense, we can ascertain the homology and evolution relationship of different species. From practical possibilities ,we can increase output and quality of silk, produce special silk product,raise silkworm ablility of resistance to disease and the ability to resist bad environment and development new applications.
This experiment cloned Fibroin-H gene sequence of the 5’flanking from Actias selene firstly. This sequence of 2801bp long contains start codon and encodes 933 amino acids with Fibroin-H conservative structural characteristics.We have found that the sequence has a high homology with that of Antheraea pernyi、Antheraea yamamai、Saturnia japonica、Anthereaea mylitta, especially Saturnia japonica.It shows the result is right.The accesssion number is GU212855.1. Through expression analysis of semi-quantitative PCR towards fibroin-H of different time in the fifth instar, we have found expession of fibroin-H from the first day,third day, sixth day, ninth day and twelfth day in the fifth instar of A.selene in post-silk gland increases day by day.It is credible.Through analysis we have detected fibroin-H from the first day,third day, sixth day, ninth day and twelfth day in the fifth instar of A.selene in mid-silk gland and the trend is:low-high-low-high.We have not detected fibroin-H in fat body.It shows expression of fibroin-H has organization specificity which is in accord with previous study.
引文
[1] Yamaguchi K, Kikuchi Y, Takagi T, et al. Primary structure of the silk fibroin light chain determined by cDNA sequencing and peptide analysis[ J ]. J Mol Biol, 1989, 210: 127– 139
[2] Sprague K U. The Bombyx mori silk proteins: characterization of large Polypeptides[ J ]. Biochemistry,1975, 14: 925– 931
[3] Couble P, Moine A, GarelA, et al. Developmental variations of anonfibroin mRNA of Bombyx mori silk gland, encoding for a low-molecular-weight silk protein[ J ]. DevBiol, 1983, 97: 398– 407
[4] Inoue S, Tanaka K, Arisaka F, et al. Silk fibroin of Bombyx mor iis secreted, assembling a high molecularmass elementary unit consisting of H-chain, L-chain, and P25, with a 6∶6∶1 molar ratio[ J ]. J Biol Chem, 2000, 275: 40517– 40528
[5] Tanaka K, Inoue S, Mizuno S. Hydrophobic interaction of P25,containing Asn-linkedoligosaccharidechains, with the H-L complex of silk fibroin produced by Bombyx mori[ J ]. Insect Biochem Mol Biol, 1999, 29: 269– 276
[6] TsujimoroY, SuzukiY. The DNA sequence of Bombyxmori fibroin gene including the 5′flanking, mDNA coding, entire intervening an fibroin protein codingregions[J]. Cel,l 1979, 18: 591-600.
[7] UedaH, Mizuno S, Shimura K. Sequence polymorphisms around the 5′-end of the silkworm fibroin H-chain gene suggesting the occurrence of crossing- over between heteromorphic alleles[J].Gene,1985, 34: 351-355.
[8] MitaK, Ichimura S, ZumaM. Specific codon usage pattern and it implications on the secondary structure ofsilk fibroinmRNA[J]. J MolBiol, 1988, 203: 917-925.
[9] Zhou C Z, Confalinieri F, MedinaN, et al. Fine organization of Bombyx mori fibroin heavy chain gene[ J]. Nucleic Acids Research, 2000, 28(12): 2 413-24 19
[10] Tanaka K.,Mori K.,Mizuno S.Immunological identification of the major disu- lfide-linked light component of silk fibroin.JBiochem(Tokyo),1993(114):1-4
[11] Inoue S.,Tanaka K.,Tanaka H., et al. Assembly of the silk fibroin elementary unit in endoplasmic reticulum and a role of L-chain for protection of alpha1,2 mannose residues in N-linked oligosaccharide chains of fibrohexamerin/P5. Eur-J Biochem,2004(271):356-366
[12] Maekawa H, Suzuki Y. Repeated turn-off and turn-on fibroin gene transcription during silk gland development of Bombyx mori[J]. DevBiol, 1980, 78: 394 - 406
[13] Tsujimoto Y, Hirose S, Tsuda M, et al. Promoter sequence of fibroin gene assigned by invitrtranscription system[J]. Proc Natl Acad Sci USA, 1981, 78: 4838 - 4842
[14] Tsuda M, Suzuki Y. Faithful transcription initiation of fibroin gene in a homologous cell-free system reveals an enhancing effect of 5′flanking sequence far upstream[J]. Cell, 1981, 27: 175 - 182
[15] Takiya S.,Hui C.C.,Suzuki Y.A contribution of the core-promoter and its surrounding regions to the preferential transcription of the fibroin gene in posterior silk gland extracts.Embo J,1990(9):489-496
[16] Tsujimoto Y.,Hirose S.,Tsuda M., et al. Promoter sequence of fibroin gene assigned byin vitro transcription system.Proc Natl Acad Sci U S A,1981(78):4838-4842
[17] Suzuki Y.,Tsuda M.,Takiya S., et al. Tissue-specifictranscription enhancement of the fibroin gene characterized by cell-free systems.Proc NatlAcad Sci U S A,1986(83):9522-9526
[18] Matsuno K.,Hui C.,Takiya S., et al. Transcription signals and protein binding sitesforsericin gene transcription in vitr o.J Biol Chem,1989(264):18707-18713
[19] Nony P.,Prudhomme J.C.,Couble P.Regulation of the P25 gene transcription in the silk gland of Bombyx.Biol Cell,1995(84):43-52
[20] Chevillard M.,Couble P.,Prudhomme J.C.Complete nucleotide sequence of the gene encoding the Bombyx mori silk protein P25 and predicted amino acid sequence of the protein.Nucleic Acids Res,1986(14):6341-6342
[21] Tomita M.,Munetsuna H.,SatoT.,et al.Transgenic silkworms produce recombinanthumantypeIIIprocollagenincocoons.Nat.Biotechnol.,2003(21):52-56
[22] Royer C.,Jalabert A.,Grenier A. et al. Biosynt-hesis and cocoon-export of a re -combinant globular protein in transgenic silkworms.Transgenic Res,2005(14 ):463-472
[23] Inoue S.,Tanaka K.,Tanaka H., et al.Assembly of the silk fibroin elementary unit in endoplasmic reticulum and a role of L-chain for protection of alpha1,2-mannose residues in N-linked oligosaccharide chains of fibrohexamerin/P25.EurJBiochem,2004(271):356-366
[24]汪生鹏,陆长德.家蚕丝素重链启动子克隆片段在家蚕体内和昆虫培养细胞内的渗表达[J].蚕业科学, 2006, 32 4 : 491-496
[25] Kojima K, Kuwana Y, Sezutsu H, et al. A new method for the modification of fibroin heavy chain protein in the transgenic silk-worm[J]. BiosciBiotechBioch, 2007, 71: 2943– 2951
[26]孙霞,朱晨,汪生鹏.家蚕丝素基因表达水平的定量分析[J].蚕业科学,2009,35(1): 30-35
[27] Marshall A.The insects are coming . Nature Biotech, 1998,16: 530~5530
[28] Toshiki T, Chantal T, Corinne T, et al. Germline transformation of the silkworm Bombyx mori Lusing a piggyBAC transposon- drived vector. Nature biotechol, 2000, 18: 81~84.
[29]朱成钢,金永丰,史锋等用丝心蛋白基因构建转基因家蚕基因打靶载体农业生物技术学报,2002, 10: 171~175.
[30]孟智启,刘静,黄君霆转基因蚕研究进展概述.蚕桑通报1996, 27: 4~6.
[31]李振刚,周丛照,唐恒立等YAC介导的天蚕丝素基因向家蚕的转移——天蚕丝素基因—YAC克隆在家蚕的表达昆虫学报1997, 40 (1): 1~5.
[32] Yamao M, Katayama N, Nakazawa H, et al. Gene targetingin the silkworm by use of a baculovirus. Genes Dev, 1999,20:74~76
[33]苏伦安.野蚕学[M].北京:中国农业出版社,1991,272-273
[34]尹志亮,刘朝良,邵引刚等.柳蚕卵黄原蛋白cDNA的克隆及序列分析[J].激光生物学报,2007,16(5):552-558
[35]钱岑,刘朝良,尹志亮等.柳蚕卵黄原蛋白Vgcdna3’端的克隆及序列分析[J].蚕业科学,2008, 34(1):61-64
[36]董胜张,刘朝良,汪泰初等.绢丝昆虫柳蚕的生物学特性的研究和卵黄原蛋白鉴定[J].蚕业科学, 2003,29(4)439-442
[37]汪泰初,冯纪元,鲍先巡等.绢丝昆虫柳蚕的生物学特性的研究[J].安徽农学通报2005,11(1):68-75
[38]孙孝龙.柳蚕的生物学习性与饲养方法[J].昆虫知识,2007,44(5):751-753
[39] Tamura T.,Thibert C.,Royer C., et al.Germline transformation of the silkworm Bombyxmori L.using apiggy Bactrans poson-derived vector. Nat. Biotech nol 2000(18):81-84
[40] Sheng-Peng WANG, Ting-Qing GUO,Xiu-Yang GUO, et al. Structural Analysis of Fibroin Heavy Chain Signal Peptide of Silkworm Bombyx mori.Acta Biochimica et Biophysica Sinica. 2006, 38(7): 507–513.
[41] Tamura T, et al. The fibroin gene of Antheraea yamamai and Bombyx mori are different in their core regions but reveal astriking sequence similarity in their 5′end and 5′flanking regions [J].Mol.Gen.Genet.1987, 207:189-198.
[42]潘兴亮,曹广力,薛仁宇等家蚕丝素重链启动子驱动DsRed的瞬时分泌表达Chin J Biotech 2009,May 25;25(5):761-766
[43]赵兴海搞好柞蚕综合利用开拓柞蚕丝绸事业新领域辽宁丝绸2009年第1卷
[44]冷春玲TAIL-PCR技术分离柞蚕丝素基因3′端非编码区未知DNA序列[J].华北农学报,2009,24(1):108-111
[45]李文利,金礼吉,范琦等.柞蚕丝素基因5′端部分序列的克隆及结构分析[J].中国农业科学,2002,35 (2):218-221
[46]孟智启,刘静,项友斌.天蚕丝素基因5’末端及其侧翼区域的克隆[J] .蚕业科学, 1997,23 (3):183-184
[47]张俊涛,李文利,张波柞蚕丝蛋白基因3’端不分片段的克隆与表达[J] .辽宁农业科学,2003(2):12-13
[48]周文林,曹锦如,叶爱红等.家蚕丝素蛋白H链基因启动区域的克隆及活性分析[J] .蚕业科学, 2008,34 (1):135-137
[49]李维,刘辉芬,王宇等.家蚕丝心蛋白轻链cDNA和基因调控片段的克隆及序列分析[J].应用与环境生物学报, 2004,10(5):614～617
[50]刘炜彬,贡成良,薛仁宇等家蚕丝蛋白Fhx/P25基因启动子区域的克隆及序列分析[J].动物学研究,2007,28(1):17-23
[51]颜新培,钟伯雄,徐孟奎等家蚕五龄后部丝腺蛋白质构成与茧层量的关系[J].蚕业科学,2003,29(4):344-348
[52]张成岗,贺福初编著.生物信息学方法与实践.科学出版社,2002
[53] Kyte J. Jensen M.Skovgaard, and S.Brunak. Prediction of novel archaeal enzymes from sequence-derived features. Protein Science, 2002,11:2894-2898
[54] Tsujimoto Y, Hirose S, Tsuda M, et al. Promoter sequence of fibroin gene assigned by invitro transcription system [J]. Proc Natl Acad Sci U S A. 1981, 78:4838-4842
[55] Tsuda M, Suzuki Y. Faithful transcription initiation of fibroin gene in a homologous cell-free system reveals an enhancing effect of 5' flanking sequence far upstream [J]. Cell. 1981,27:175-182
[2] Sprague K U. The Bombyx mori silk proteins: characterization of large Polypeptides[ J ]. Biochemistry,1975, 14: 925– 931
[3] Couble P, Moine A, GarelA, et al. Developmental variations of anonfibroin mRNA of Bombyx mori silk gland, encoding for a low-molecular-weight silk protein[ J ]. DevBiol, 1983, 97: 398– 407
[4] Inoue S, Tanaka K, Arisaka F, et al. Silk fibroin of Bombyx mor iis secreted, assembling a high molecularmass elementary unit consisting of H-chain, L-chain, and P25, with a 6∶6∶1 molar ratio[ J ]. J Biol Chem, 2000, 275: 40517– 40528
[5] Tanaka K, Inoue S, Mizuno S. Hydrophobic interaction of P25,containing Asn-linkedoligosaccharidechains, with the H-L complex of silk fibroin produced by Bombyx mori[ J ]. Insect Biochem Mol Biol, 1999, 29: 269– 276
[6] TsujimoroY, SuzukiY. The DNA sequence of Bombyxmori fibroin gene including the 5′flanking, mDNA coding, entire intervening an fibroin protein codingregions[J]. Cel,l 1979, 18: 591-600.
[7] UedaH, Mizuno S, Shimura K. Sequence polymorphisms around the 5′-end of the silkworm fibroin H-chain gene suggesting the occurrence of crossing- over between heteromorphic alleles[J].Gene,1985, 34: 351-355.
[8] MitaK, Ichimura S, ZumaM. Specific codon usage pattern and it implications on the secondary structure ofsilk fibroinmRNA[J]. J MolBiol, 1988, 203: 917-925.
[9] Zhou C Z, Confalinieri F, MedinaN, et al. Fine organization of Bombyx mori fibroin heavy chain gene[ J]. Nucleic Acids Research, 2000, 28(12): 2 413-24 19
[10] Tanaka K.,Mori K.,Mizuno S.Immunological identification of the major disu- lfide-linked light component of silk fibroin.JBiochem(Tokyo),1993(114):1-4
[11] Inoue S.,Tanaka K.,Tanaka H., et al. Assembly of the silk fibroin elementary unit in endoplasmic reticulum and a role of L-chain for protection of alpha1,2 mannose residues in N-linked oligosaccharide chains of fibrohexamerin/P5. Eur-J Biochem,2004(271):356-366
[12] Maekawa H, Suzuki Y. Repeated turn-off and turn-on fibroin gene transcription during silk gland development of Bombyx mori[J]. DevBiol, 1980, 78: 394 - 406
[13] Tsujimoto Y, Hirose S, Tsuda M, et al. Promoter sequence of fibroin gene assigned by invitrtranscription system[J]. Proc Natl Acad Sci USA, 1981, 78: 4838 - 4842
[14] Tsuda M, Suzuki Y. Faithful transcription initiation of fibroin gene in a homologous cell-free system reveals an enhancing effect of 5′flanking sequence far upstream[J]. Cell, 1981, 27: 175 - 182
[15] Takiya S.,Hui C.C.,Suzuki Y.A contribution of the core-promoter and its surrounding regions to the preferential transcription of the fibroin gene in posterior silk gland extracts.Embo J,1990(9):489-496
[16] Tsujimoto Y.,Hirose S.,Tsuda M., et al. Promoter sequence of fibroin gene assigned byin vitro transcription system.Proc Natl Acad Sci U S A,1981(78):4838-4842
[17] Suzuki Y.,Tsuda M.,Takiya S., et al. Tissue-specifictranscription enhancement of the fibroin gene characterized by cell-free systems.Proc NatlAcad Sci U S A,1986(83):9522-9526
[18] Matsuno K.,Hui C.,Takiya S., et al. Transcription signals and protein binding sitesforsericin gene transcription in vitr o.J Biol Chem,1989(264):18707-18713
[19] Nony P.,Prudhomme J.C.,Couble P.Regulation of the P25 gene transcription in the silk gland of Bombyx.Biol Cell,1995(84):43-52
[20] Chevillard M.,Couble P.,Prudhomme J.C.Complete nucleotide sequence of the gene encoding the Bombyx mori silk protein P25 and predicted amino acid sequence of the protein.Nucleic Acids Res,1986(14):6341-6342
[21] Tomita M.,Munetsuna H.,SatoT.,et al.Transgenic silkworms produce recombinanthumantypeIIIprocollagenincocoons.Nat.Biotechnol.,2003(21):52-56
[22] Royer C.,Jalabert A.,Grenier A. et al. Biosynt-hesis and cocoon-export of a re -combinant globular protein in transgenic silkworms.Transgenic Res,2005(14 ):463-472
[23] Inoue S.,Tanaka K.,Tanaka H., et al.Assembly of the silk fibroin elementary unit in endoplasmic reticulum and a role of L-chain for protection of alpha1,2-mannose residues in N-linked oligosaccharide chains of fibrohexamerin/P25.EurJBiochem,2004(271):356-366
[24]汪生鹏,陆长德.家蚕丝素重链启动子克隆片段在家蚕体内和昆虫培养细胞内的渗表达[J].蚕业科学, 2006, 32 4 : 491-496
[25] Kojima K, Kuwana Y, Sezutsu H, et al. A new method for the modification of fibroin heavy chain protein in the transgenic silk-worm[J]. BiosciBiotechBioch, 2007, 71: 2943– 2951
[26]孙霞,朱晨,汪生鹏.家蚕丝素基因表达水平的定量分析[J].蚕业科学,2009,35(1): 30-35
[27] Marshall A.The insects are coming . Nature Biotech, 1998,16: 530~5530
[28] Toshiki T, Chantal T, Corinne T, et al. Germline transformation of the silkworm Bombyx mori Lusing a piggyBAC transposon- drived vector. Nature biotechol, 2000, 18: 81~84.
[29]朱成钢,金永丰,史锋等用丝心蛋白基因构建转基因家蚕基因打靶载体农业生物技术学报,2002, 10: 171~175.
[30]孟智启,刘静,黄君霆转基因蚕研究进展概述.蚕桑通报1996, 27: 4~6.
[31]李振刚,周丛照,唐恒立等YAC介导的天蚕丝素基因向家蚕的转移——天蚕丝素基因—YAC克隆在家蚕的表达昆虫学报1997, 40 (1): 1~5.
[32] Yamao M, Katayama N, Nakazawa H, et al. Gene targetingin the silkworm by use of a baculovirus. Genes Dev, 1999,20:74~76
[33]苏伦安.野蚕学[M].北京:中国农业出版社,1991,272-273
[34]尹志亮,刘朝良,邵引刚等.柳蚕卵黄原蛋白cDNA的克隆及序列分析[J].激光生物学报,2007,16(5):552-558
[35]钱岑,刘朝良,尹志亮等.柳蚕卵黄原蛋白Vgcdna3’端的克隆及序列分析[J].蚕业科学,2008, 34(1):61-64
[36]董胜张,刘朝良,汪泰初等.绢丝昆虫柳蚕的生物学特性的研究和卵黄原蛋白鉴定[J].蚕业科学, 2003,29(4)439-442
[37]汪泰初,冯纪元,鲍先巡等.绢丝昆虫柳蚕的生物学特性的研究[J].安徽农学通报2005,11(1):68-75
[38]孙孝龙.柳蚕的生物学习性与饲养方法[J].昆虫知识,2007,44(5):751-753
[39] Tamura T.,Thibert C.,Royer C., et al.Germline transformation of the silkworm Bombyxmori L.using apiggy Bactrans poson-derived vector. Nat. Biotech nol 2000(18):81-84
[40] Sheng-Peng WANG, Ting-Qing GUO,Xiu-Yang GUO, et al. Structural Analysis of Fibroin Heavy Chain Signal Peptide of Silkworm Bombyx mori.Acta Biochimica et Biophysica Sinica. 2006, 38(7): 507–513.
[41] Tamura T, et al. The fibroin gene of Antheraea yamamai and Bombyx mori are different in their core regions but reveal astriking sequence similarity in their 5′end and 5′flanking regions [J].Mol.Gen.Genet.1987, 207:189-198.
[42]潘兴亮,曹广力,薛仁宇等家蚕丝素重链启动子驱动DsRed的瞬时分泌表达Chin J Biotech 2009,May 25;25(5):761-766
[43]赵兴海搞好柞蚕综合利用开拓柞蚕丝绸事业新领域辽宁丝绸2009年第1卷
[44]冷春玲TAIL-PCR技术分离柞蚕丝素基因3′端非编码区未知DNA序列[J].华北农学报,2009,24(1):108-111
[45]李文利,金礼吉,范琦等.柞蚕丝素基因5′端部分序列的克隆及结构分析[J].中国农业科学,2002,35 (2):218-221
[46]孟智启,刘静,项友斌.天蚕丝素基因5’末端及其侧翼区域的克隆[J] .蚕业科学, 1997,23 (3):183-184
[47]张俊涛,李文利,张波柞蚕丝蛋白基因3’端不分片段的克隆与表达[J] .辽宁农业科学,2003(2):12-13
[48]周文林,曹锦如,叶爱红等.家蚕丝素蛋白H链基因启动区域的克隆及活性分析[J] .蚕业科学, 2008,34 (1):135-137
[49]李维,刘辉芬,王宇等.家蚕丝心蛋白轻链cDNA和基因调控片段的克隆及序列分析[J].应用与环境生物学报, 2004,10(5):614～617
[50]刘炜彬,贡成良,薛仁宇等家蚕丝蛋白Fhx/P25基因启动子区域的克隆及序列分析[J].动物学研究,2007,28(1):17-23
[51]颜新培,钟伯雄,徐孟奎等家蚕五龄后部丝腺蛋白质构成与茧层量的关系[J].蚕业科学,2003,29(4):344-348
[52]张成岗,贺福初编著.生物信息学方法与实践.科学出版社,2002
[53] Kyte J. Jensen M.Skovgaard, and S.Brunak. Prediction of novel archaeal enzymes from sequence-derived features. Protein Science, 2002,11:2894-2898
[54] Tsujimoto Y, Hirose S, Tsuda M, et al. Promoter sequence of fibroin gene assigned by invitro transcription system [J]. Proc Natl Acad Sci U S A. 1981, 78:4838-4842
[55] Tsuda M, Suzuki Y. Faithful transcription initiation of fibroin gene in a homologous cell-free system reveals an enhancing effect of 5' flanking sequence far upstream [J]. Cell. 1981,27:175-182