柞蚕新型转基因载体构建和精子介导转化研究
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摘要
柞蚕是我国特有的一种野外饲养的经济昆虫。相比于家蚕,柞蚕由于其种群和生长环境的特殊性使其在抗病害能力、耐气候变化能力等方面具有显著的优势,是不可替代的重要生物资源。因此,开展柞蚕转基因研究,对于了解柞蚕重要基因功能,通过转基因柞蚕育种技术改善柞蚕丝品质,提升柞蚕市场竞争,以及利用柞蚕作为生物反应器,开发柞蚕资源的新用途等都具有十分重要的意义。
本文在前期研究的基础上,对柞蚕转基因载体进行了改造,优化了启动子、转座子和报告基因等重要的转基因元件,构建了新型的柞蚕双元转基因载体；利用荧光标记技术对精子介导途径进行了观察；克隆了人工拼接的蜘蛛拖牵丝基因片段,利用精子介导途径对柞蚕进行转基因研究,获得了报告基因整合和表达的初步证据。主要研究内容和结论如下：
(1)通过染色体步移的方法扩增得到了全长为1927bp的柞蚕肌动蛋白A1启动子,其与家蚕肌动蛋白启动子核心区域同源率为92%,具有典型的真核生物启动子特征。通过对启动子功能区域的删除研究,确定了启动子的表达调控区域。发现A1启动子的F4片段具有较强的正向调控功能,可在转基因研究中作为强启动子调控柞蚕基因的表达,在TATA box上游可能存在抑制元件,对基因表达起到负向调节的作用。
(2)利用Tail-PCR方法加长柞蚕丝素基因5’和3’端序列,并与GFP标记基因融合,构建了新的丝素基因同源表达框Fib-GFP。将上述表达框与A1驱动的红色荧光蛋白基因一同插入到piggyBac转座子两个重复序列之间,得到柞蚕基因转移载体pBac[Al-DsRed+Fib-GFP];利用柞蚕肌动蛋白A1启动子改造辅助转移载体,得到辅助载体pBacAlHelper。将Al-piggyBac表达框插入到包含反向重复序列的转移载体上,构建柞蚕双元基因转移载体pBac[Al-DsRed+Fib-GFP]-Al-piggyBac。通过对Sf9细胞进行转染实验,证明两种转移系统都能够正常工作,而且整合效率比较表明双元表达载体比转移载体与辅助质粒的双质粒载体系统具有更高的整合效率,具有在柞蚕个体转基因研究中应用的潜力。
(3)利用FITC示踪技术研究了柞蚕精子与DNA吸附及转移途径,并建立柞蚕的精子介导转基因体系。将柞蚕精液与FITC标记的外源DNA混合后,荧光显微镜下检测到精子头部有荧光现象,表明柞蚕精子具有摄取外源DNA的能力。将FITC标记的外源DNA导入柞蚕雌蛾体内,在荧光显微镜下观察外源DNA进入雌性生殖系统的运动过程,证实了昆虫通过精子介导实现转基因的途径。利用精子介导将携带GFP基因的载体导入蚕卵,在蚕卵中观察到瞬时表达的绿色荧光,通过RT-PCR和Western blot检测结果证明绿色荧光蛋白基因可经精子介导实现转移和表达。
(4)利用巢式PCR技术从大腹圆蛛Araneus ventricosus基因组中克隆了长度为837bp的蜘蛛拖牵丝蛋白基因(ASP)。将得到的拖牵丝蛋白基因(ASP)分别构建至原核表达载体pGEX-6p-1和真核表达载体pGFP-N2上,成功实现了ASP基因在大肠杆菌和真核细胞中的表达。
(5)将蜘蛛拖牵丝蛋白基因(ASP)克隆至柞蚕双元基因转移载体上,利用精子介导法进行柞蚕转基因实验,经过分子鉴定,证明蛛丝与GFP融合基因在柞蚕丝腺中得到表达。
Antheraea pernyi, common name called Tussah, is an economically important insect and model organisms for basic research. Its transgenic research has very important significance. Compared to the silkworm, the transgenic research of tussah has just initiated, but shown specialty and good research convenience. We focused on tussah research, hoping to make use of molecular biology techniques in transgenic silkworm breeding, to improve the quality of silk, to enhance its market competitiveness, and to improve the utilization of tussah resources.
In this dissertation, tussah transgenic vector was modified by optimizing the promoter, transposons, reporter genes and other important transgenic elements; the mechanism of sperm-mediated pathway was studied by fluorescent labeling; artificial splicing gene fragment of spider draggling silk was cloned, and was transferred into tussah by sperm-mediated pathway. The integration and expression of the reporter gene was verified. The major contents and conclusions are as follows:
(1) Tussah actin promoter Al was amplified by chromosome walking approach, and its total length was1927bp. Compare with silkworm actin promoter, their core region homologous rate was92%. Promoter Al had typical eukaryotic features. Promoter expression regulatory region was idetified through the functional areas deletion. F4fragment of Al promoter showed a strong positive regulatory function, which could be used as strong promoter for transgenic research. Strong suppression components might exist at upsteam of TATA box for the negative regulation of gene expression.
(2) Fibroin gene5'and3'end fragments lengthened by Tail-PCR were fused with GFP marker gene to construct a new fibroin gene homologous expression cassettes Fib-GFP. The expression cassette was inserted into the piggyBac trnnsposon between two repeated sequences to obtain a silkworm gene transfer vector pBac[Al-DsRed+Fib-GFP]; using tussah actin promoter Al transform auxiliary transfer vector to obtain an assistance carrier pBacAlhelper. Connecting the Al-piggyBac expression cassette with the transformation vector containing inverted repeat sequences to obtain a binary silkworm gene transfer vector pBac [Al-DsRed+Fib-GFP]-Al-piggyBac. The optimized silkworm expression vector was used to transfect Sf9cells. The two plasmids vector and binary gene transfer vector were capable of expressing the red fluorescent protein gene. The binary gene transfer vector had higher integration efficiency than two plasmids vector, suggesting a potent silkworm transgenic application.
(3) Tussah Sperm and FITC-labeled exogenous DNA were mixed and laid for a period of time, Fluorescence phenomenon was detected at sperm head by fluorescence microscopy. It showed that tussah sperm has the ability to uptake exogenous DNA. The FITC-labeled exogenous DNA were imported into silkworm moth body through sperm-mediated pathway, and the motion process of exogenous DNA into the female reproductive system was monitored under a fluorescence microscope. The results showed that exogenous DNA could be transfered into the eggs along with sperm movement through sperm-mediated pathway. Then, a vector carried GFP gene was transferred into silkworm eggs by using sperm-mediated pathway. Transient expression of green fluorescence was observed in eggs, RT-PCR and Western blot confirmed its transformation and expression.
(4) The towing silk protein gene (ASP) was cloned by nested PCR technique from the big round belly spider Araneus ventricosus, which was837bp in length. The ASP was constructed into the prokaryotic expression vector pGEX-6p-1and eukaryotic expression vector pGFP-N2, and named as pASG and pASN, respectivly. pASG was induced and expressed in E. coli at16℃,24h, and observed successful expression of the fusion protein GST-ASP; Green fluorescent protein (GFP) expression was observed after pASN was transfected into Sf9insect cells in48h.
(5) The Draggling silk protein gene (ASP) was then constructed to binary silkworm gene transfer vector and was transfencted tuaash using sperm-mediated pathway. DsRed fluorescence expression was observed in tussah eggs and blood cells of third instar. Further molecular identifications showed silk protein and GFP gene were expressed in tussah individuals.
本文在前期研究的基础上,对柞蚕转基因载体进行了改造,优化了启动子、转座子和报告基因等重要的转基因元件,构建了新型的柞蚕双元转基因载体；利用荧光标记技术对精子介导途径进行了观察；克隆了人工拼接的蜘蛛拖牵丝基因片段,利用精子介导途径对柞蚕进行转基因研究,获得了报告基因整合和表达的初步证据。主要研究内容和结论如下：
(1)通过染色体步移的方法扩增得到了全长为1927bp的柞蚕肌动蛋白A1启动子,其与家蚕肌动蛋白启动子核心区域同源率为92%,具有典型的真核生物启动子特征。通过对启动子功能区域的删除研究,确定了启动子的表达调控区域。发现A1启动子的F4片段具有较强的正向调控功能,可在转基因研究中作为强启动子调控柞蚕基因的表达,在TATA box上游可能存在抑制元件,对基因表达起到负向调节的作用。
(2)利用Tail-PCR方法加长柞蚕丝素基因5’和3’端序列,并与GFP标记基因融合,构建了新的丝素基因同源表达框Fib-GFP。将上述表达框与A1驱动的红色荧光蛋白基因一同插入到piggyBac转座子两个重复序列之间,得到柞蚕基因转移载体pBac[Al-DsRed+Fib-GFP];利用柞蚕肌动蛋白A1启动子改造辅助转移载体,得到辅助载体pBacAlHelper。将Al-piggyBac表达框插入到包含反向重复序列的转移载体上,构建柞蚕双元基因转移载体pBac[Al-DsRed+Fib-GFP]-Al-piggyBac。通过对Sf9细胞进行转染实验,证明两种转移系统都能够正常工作,而且整合效率比较表明双元表达载体比转移载体与辅助质粒的双质粒载体系统具有更高的整合效率,具有在柞蚕个体转基因研究中应用的潜力。
(3)利用FITC示踪技术研究了柞蚕精子与DNA吸附及转移途径,并建立柞蚕的精子介导转基因体系。将柞蚕精液与FITC标记的外源DNA混合后,荧光显微镜下检测到精子头部有荧光现象,表明柞蚕精子具有摄取外源DNA的能力。将FITC标记的外源DNA导入柞蚕雌蛾体内,在荧光显微镜下观察外源DNA进入雌性生殖系统的运动过程,证实了昆虫通过精子介导实现转基因的途径。利用精子介导将携带GFP基因的载体导入蚕卵,在蚕卵中观察到瞬时表达的绿色荧光,通过RT-PCR和Western blot检测结果证明绿色荧光蛋白基因可经精子介导实现转移和表达。
(4)利用巢式PCR技术从大腹圆蛛Araneus ventricosus基因组中克隆了长度为837bp的蜘蛛拖牵丝蛋白基因(ASP)。将得到的拖牵丝蛋白基因(ASP)分别构建至原核表达载体pGEX-6p-1和真核表达载体pGFP-N2上,成功实现了ASP基因在大肠杆菌和真核细胞中的表达。
(5)将蜘蛛拖牵丝蛋白基因(ASP)克隆至柞蚕双元基因转移载体上,利用精子介导法进行柞蚕转基因实验,经过分子鉴定,证明蛛丝与GFP融合基因在柞蚕丝腺中得到表达。
Antheraea pernyi, common name called Tussah, is an economically important insect and model organisms for basic research. Its transgenic research has very important significance. Compared to the silkworm, the transgenic research of tussah has just initiated, but shown specialty and good research convenience. We focused on tussah research, hoping to make use of molecular biology techniques in transgenic silkworm breeding, to improve the quality of silk, to enhance its market competitiveness, and to improve the utilization of tussah resources.
In this dissertation, tussah transgenic vector was modified by optimizing the promoter, transposons, reporter genes and other important transgenic elements; the mechanism of sperm-mediated pathway was studied by fluorescent labeling; artificial splicing gene fragment of spider draggling silk was cloned, and was transferred into tussah by sperm-mediated pathway. The integration and expression of the reporter gene was verified. The major contents and conclusions are as follows:
(1) Tussah actin promoter Al was amplified by chromosome walking approach, and its total length was1927bp. Compare with silkworm actin promoter, their core region homologous rate was92%. Promoter Al had typical eukaryotic features. Promoter expression regulatory region was idetified through the functional areas deletion. F4fragment of Al promoter showed a strong positive regulatory function, which could be used as strong promoter for transgenic research. Strong suppression components might exist at upsteam of TATA box for the negative regulation of gene expression.
(2) Fibroin gene5'and3'end fragments lengthened by Tail-PCR were fused with GFP marker gene to construct a new fibroin gene homologous expression cassettes Fib-GFP. The expression cassette was inserted into the piggyBac trnnsposon between two repeated sequences to obtain a silkworm gene transfer vector pBac[Al-DsRed+Fib-GFP]; using tussah actin promoter Al transform auxiliary transfer vector to obtain an assistance carrier pBacAlhelper. Connecting the Al-piggyBac expression cassette with the transformation vector containing inverted repeat sequences to obtain a binary silkworm gene transfer vector pBac [Al-DsRed+Fib-GFP]-Al-piggyBac. The optimized silkworm expression vector was used to transfect Sf9cells. The two plasmids vector and binary gene transfer vector were capable of expressing the red fluorescent protein gene. The binary gene transfer vector had higher integration efficiency than two plasmids vector, suggesting a potent silkworm transgenic application.
(3) Tussah Sperm and FITC-labeled exogenous DNA were mixed and laid for a period of time, Fluorescence phenomenon was detected at sperm head by fluorescence microscopy. It showed that tussah sperm has the ability to uptake exogenous DNA. The FITC-labeled exogenous DNA were imported into silkworm moth body through sperm-mediated pathway, and the motion process of exogenous DNA into the female reproductive system was monitored under a fluorescence microscope. The results showed that exogenous DNA could be transfered into the eggs along with sperm movement through sperm-mediated pathway. Then, a vector carried GFP gene was transferred into silkworm eggs by using sperm-mediated pathway. Transient expression of green fluorescence was observed in eggs, RT-PCR and Western blot confirmed its transformation and expression.
(4) The towing silk protein gene (ASP) was cloned by nested PCR technique from the big round belly spider Araneus ventricosus, which was837bp in length. The ASP was constructed into the prokaryotic expression vector pGEX-6p-1and eukaryotic expression vector pGFP-N2, and named as pASG and pASN, respectivly. pASG was induced and expressed in E. coli at16℃,24h, and observed successful expression of the fusion protein GST-ASP; Green fluorescent protein (GFP) expression was observed after pASN was transfected into Sf9insect cells in48h.
(5) The Draggling silk protein gene (ASP) was then constructed to binary silkworm gene transfer vector and was transfencted tuaash using sperm-mediated pathway. DsRed fluorescence expression was observed in tussah eggs and blood cells of third instar. Further molecular identifications showed silk protein and GFP gene were expressed in tussah individuals.
引文
[1]刘彦群,姜德富.柞蚕功能基因研究进展[J].蚕业科学,2008,34(3)：568-574.
[2]向仲怀,杨焕明.家蚕基因组研究对蚕业学科和产业发展的影响[J].世界科技研究与发展,2003,5(6)：1-5.
[3]李文利.柞蚕丝素基因转移载体的构建及转基因柞蚕的研究[D].大连理工大学,2003.
[4]XU M., LEWIS R. V. Structure of a protein superfiber:Spider dragline silk [J]. Proc. Natl. Acad. Sci.,USA,1990,87(18):7120-7124.
[5]姜义仁,秦利.柞蚕种质资源研究：回顾与展望[J].中国蚕学会第八届暨国家蚕桑产业技术体系家(柞)蚕遗传育种及良种繁育学术研讨会论文集,2011.
[6]董文娟,王学英,张涛,等.柞蚕滞育蛹抗菌物质产生及其抑菌活性研究[J].安徽农业科学,2007,35(4)：99-991.
[7]范琦.柞蚕核型多角体病毒转移载体的组建及外源基因在柞蚕蛹体内的表达[J].辽宁农业科学,1992,5：002.
[8]LI W, JIN L, AN L. Construction of targeting vector and expression of green fluorescent protein in the silkworm, Antheraea pernyi[J]. DNA and cell biology,2003,22(7):441-446.
[9]SHURONG XU, DANMEI LIU, WENLI LI. Upregulated proteins associated with fibroin synthesis in posterior silk glands of Antheraea pernyi fifth instar larvae[J]. Comp Biochem Physiol Part D Genomics Proteomics.2009,4 (2):105-10.
[10]李维,张义正.黄孢原毛平革菌基因启动子的分离与鉴定[J].生物工程学报,2000,16,(5)：599-602.
[11]刘春,徐汉福,陈玉琳等.家蚕转基因研究及肌动蛋白A3启动子的表达分析[J].蚕学通讯,2007,27(3)：1-6.
[12]向仲怀.蚕丝生物学[M].中国林业出版社,2005.
[13]周文林,曹广力,薛仁宇等.家蚕丝素蛋白轻链基因(fib-L)启动子序列的克隆及其活性分析[J].昆虫学报,2007,50(6)：547-554.
[14]马志明,石晓燕,曹广力等.家蚕核型多角体病毒IE-1基因启动子的克隆和序列分析[J].2001.
[15]赵巧玲,唐顺明,易咏竹等.家蚕CIb1基因启动子活性分析[J].蚕业科学,2006,32(4)：477-481.
[16]MICHAILLE J J, COUBLE P, PRUDHOMME J C, et al. A single gene produces multiple sericin messenger RNAS in the silk gland of Bombyx mori [J]. Biochimie,1986,68(10):1165-1173.
[17]诸戌娴,曹广力,薛仁宇等.家蚕丝胶蛋白基因1(ser-1)启动子的克隆及其活性分析[J].科技通报,2007,23(6)：828-834.
[18]ROBERTSON H M, LAMPE D J. Recent horizontal transfer of a mariner transposable element among and between Diptera and Neuroptera [J]. Molecular biology and evolution,1995,12(5):850-862.
[19]KID WELL M G. Reciprocal differences in female recombination associated with hybrid dysgenesis in Drosophila melanogaster [J].Genet Res,1977,33(1):77-88.
[20]UCHINO K, IMAMURA M, SHIMIZU K, et al. Germ line transformation of the silkworm, Bombyx mori, using the transposable element Minos[J]. Molecular Genetics and Genomics,2007,277(3): 213-220.
[21]LOUKERIS T G, LIVADARAS I, ARCA B, et al. Gene transfer into the medfly, Ceratitis capitata, with a Drosophila hydei transposable element [J]. Science,1995,270(5244):2002-2005.
[22]HANDLER A M. Use of the piggyBac transposon for germ-line transformation of insects[J]. Insect biochemistry and molecular biology,2002,32(10):1211-1220.
[23]FRASER M J. The TTAA-specific family of transposable elements:identification, functional characterization, and utility for transformation of insects[J]. Insect Transgenesis:Methods and Applications,2000:249-268.
[24]ELICK T A, BAUSER C A, FRASER M J. Excision of the piggyBac transposable element in vitro is a precise event that is enhanced by the expression of its encoded transposase[J]. Genetica,1996,98(1): 33-41.
[25]TAMURA T, THIBERT C, ROYER C, et al. GermLine transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector [J]. Nat Biotechnol 2000,18:81-84.
[26]IMAMURA M, NAKAHARA Y, KANDA T, et al. A transgenic silkworm expressing the immune-inducible cecropin B-GFP reporter gene[J]. Insect biochemistry and molecular biology, 2006,36(5):429-434.
[27]ZHONG B, LI J, CHEN J, et al. Comparison of transformation efficiency of piggyBac transposon among three different silkworm Bombyx mori strains[J]. Acta biochimica et biophysica Sinica,2007, 39(2):117-122.
[28]CHIOCCHETTI A, TOLOSANO E, HIRSCH E, et al. Green fluorescent protein as a reporter of gene expression in transgenic mice[J]. Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression,1997,1352(2):193-202.
[29]DAVID H E, DACE A S, DE PMIERAI D L, et al. Construction and ecaluation of transgenic hsp16-GFP-lacZ Caenorhabditiditis elegans strain for environmental monitoring[J]. Environ Toxicol Chem,2003,22:111-118.
[30]ZHANG J Z, GUO X B, XIE S Y, et al. Production of transgenic mice carrying green fluorescence protein gene by alcntiviral vector-mediated approach[J]. Prog Nat Sci,2006,08(16):827-833.
[31]ZHANG Y, PAN D, SUN X, et al. Production of porcine cloned transgenic embryos expiessing green fluorescent protein by somatic cell nuclear transfer[J]. SciSin(c), LifeSci,2006,49(2):164-171.
[32]森肇.在家蚕中的基因整合[J].日本蚕丝学会讲演要旨集,1998,40.
[33]FRADKOV A F, CHEN Y, DING L, et al. Novel fluorescent protein from Discosoma coral and its mutants possesses a unique far-red fluorescence[J]. FEBS letters,2000,479(3):127-130.
[34]YARBROUGH D, WACHTER R M, KALLIO K, et al. Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-A resolution[J]. Proceedings of the National Academy of Sciences,2001,98(2):462-467.
[35]娄桂予,陈敏,陈彬,等HNF1 α对人FXR启动子的调控作用[J].中国生物化学与分子生物学报,2006,22(12)：966-972.
[36]徐春娥,付玲,侯丽华,等.人IFN-β启动子基因报告质粒的构建及SARS-CoV3a和7a蛋白对IFN-β诱生作用的初步研究[J].中国生物工程杂志,2006,26(12)：6-10.
[37]GORDON J W, SCANGOS G A, PLOTKIN D J, et al. Genetic transformation of mouseembryos by microinjection of purified DNA [J]. Proc Natl Acad,1980,77(12):7380-7384.
[38]NINAKI O., MAEKAWA H, GAMO T, et al. Hatchability of silkworm eggs injected with DNA at early embryonic stages[J]. Journal of Sericultural Science of Japan,1985,54.
[39]THOMAS JL.DA ROCHA M.BESSE A,et al.3×P3-EGFP marker facilitates screening of transgenic silkworm Bombyx mori L.from the embryonic stage onwards [J]. Insect Biochem Mol Biol,2002,32:247-253.
[40]UHLIROVA M, ASAHINA M, RIDDIFORD L M, et al. Heat-inducible transgenic expression in the Silkmoth Bombyx mori [J]. Dev. Genes Evol,2002,212(3):145-151.
[41]IMAMURA M, NAKAI J, QUAN GX, et al.Targeted gene expression using the GAL4/UAS system in the silkworm Bombyx mori[J]. Genetics,2003,165:1329-1340.
[42]代红久,徐国江,王铸钢等.利用鳞翅目来源的转座子piggyBac建立高效稳定的转基因家蚕技术[J].科学通报,2005,50(14)：1470-1474.
[43]SANFORD J C, KLEIN T M, WOLF E D, et al. Delivery of substances into cells and tissues using a particle bombardment process [J]. Particulate Science and Technology,1987,5(1):27-37.
[44]KLEIN T M, WOLF E D, WU R, et al. High-velocity microprojectiles for delivering nucleic acids into living cells[J]. Nature,1987,327(6117):70-73.
[45]孟智启,姚山麟,王为民等.基因枪喷射技术在家蚕外源基因转移中的应用[J].浙江农业学报,1990,2：1-6.
[46]陈秀,赵昀,张峰等.新霉素抗性基因在家蚕中的插入和表达[J].生物化学与生物物理学报,1999,31,(1)：90-92.
[47]赵昀,张峰,陈秀等.用绿色荧光蛋白进行转基因蚕研究[J].高技术通讯,1999,6：16-19.
[48]ADACHI T, TOMITA M, SHIMIZU K, et al. Generation of hybrid transgenic silkworms that express Bombyx mori prolyl-hydroxylase alpha-subunits and human collagens in posterior silk glands: production of cocoons that contained collagens with hydroxylated proline residues[J]. Journal of Biotechnology.2006,126(2):205-219.
[49]WONG TK, NEUMANN E. Electric field mediated gene transfer [J].Biochem Biophys Res Commun,1982,107 (2):584.
[50]KENIEHI MOTO, et al. Gene transfer into insect brain nadeellosPeeifieexPerssiom of bombyxin gene[J]. Dev GneeEvol,1999,209:447-450.
[51]张峰,陈秀.能发荧光的转基因家蚕[J].生物化学与生物物理学报：英文版,1999,31(2)：119-123.
[52]赵昀,陈秀,彭卫平等.利用同源重组改变家蚕丝心蛋白重链基因[J].生物化学与生物物理学报.2001,33(1)：112-116.
[53]GUO XY,DONG L, WANG SP, et al. Introduction of foreign genes into silkworm eggs by eleetroPoration And Its aPP lieation in transgenic vector test [J].Acta Bioa Biop Sin,36(5):323-330.
[54]BRACKETT B G, BARANSKA W, SAWICKI W, et al. Uptake of heterologous genome by mammalian spermatozoa and its transfer to ova through fertilization [J]. P Natl Acad Sci USA,1971, 68:353-357.
[55]ROTTMAN OJ, LAMPETER WW. Development of early mouse and rabbit embryos withoutzona pellucida[J]. J Repord Fertil,1981,61(2):303-306.
[56]郭秀洋等.利用精子介导法向蚕卵导入外源基因的研究[J].生物化学与生物物理进展,2001,28(3)：423-425.
[57]王宇,刘辉芬,李维,邱兴林.家蚕转基因方法的初步研究[J].四川动物,2006,3.
[58]张业顺等.家蚕精子介导转基因技术体系的优化[J].蚕业科学,2007,33(2)：201-206.
[59]CHEN YL, REN CZ, GUI MY. Preliminary Study on the artificial insemination of the domesticated silkworm and eri Silkworm [J]. Journal of Xiamen University (Natural Science),1993, S1:9-14.
[60]桂慕燕,陈元霖,刘治纬等.家蚕人工授精与精子超低温保存研究[J].遗传,1997,19(增刊1)：83-85.
[61]代方银,陈智毅,陈元霖等.家蚕白卵突变新系BT924的遗传学研究[J].遗传,2000,22：229-232.
[62]郭秀洋.家蚕遗传操作技术的研究及其应用[D].中国科学院研究生院(上海生命科学研究院),2004.
[63]赵越,李曦,曹广力等.转基因家蚕丝腺组织和转化家蚕培养细胞表达hIGF-I[J]中国科学C辑：生命科学,2009,39：677-684.
[64]GAGNE M, POTHIER F, SIRARD M A. Effect of microinjection time during post-fertilization S-phase on bovine embryonic development[J]. Molecular reproduction and development,1995, 41(2):184-194.
[65]AREZZO F. Sea urchin sperm as a vector offoreign geneticinformation [J]. Cell Biol lnt Rep, 1989,13:391-404.
[66]MILNE C P, ELSCHEN F A, et al. Preliminary Evidence for Honeybee Sperm Mediated DNA Transter [J]. International Symposium on Moleculax Insect Science, Tucson, Afizona, 1989.120-125.
[67]TSAI H J, LAI CH, YANG HS, et al. Sperm as a cartier to introduce an exogenous DNA fragment into the ocyte of Japanese abalone (Halioti. s divorsicolor suportexta) [J]. Transgenic Res, 1997.6:85-95.
[68]KHO H W, ANG L H, et al. Sperm cells as vectors for introducing foreign DNA into zebrafish[J]. Aquaculture,1992,109:1-19
[69]KUZNETSOV A V, KUZNETSOVA I V, et al. Binding of exogenous DNA pRK31acZ by the rab bit spe rmatozoa, its tran sfer in the o eytes, an d expression in the preimplan tation embryos [J]. Russian Journal of Developmental Biology,1995,26:300-309.
[70]PERRY A, WAKAYAMA T, et al. Mammalian transgenesis by intracytoplasmic sperm injection [J]. Science,1999,2114:1180-1183.
[71]NAKANISH A, et al. Gene transfer in the chicken by sperm mediated methods [J]. Mol Report Dev, 1993,36:258-261.
[72]GAGNE M B, POTHIER F, et al. Electroporation of bovine spermatozoa to carry foreign DNA in oocytes [J]. Mol Reprol Del,1991,29:6-15.
[73]SEPERANDIO S, LULLI V, et al.Sperm-mediated DNA transfer in bovine and swine species [J].Anim Biotechnol,1996,7:59-77.
[74]HABROVA V, TAKAC M, et al. Association of Rous sarcoma virus DNA with X.Laevis spermatozoa and its transfer to ova through fertilization[J]. Mol Reprod Dev,1996,44:332-342.
[75]TSAIH J, TSENG F S, et al. Electroporation of sperm to introduce foreign DNA into the genome of loach (Misgurnus anguil-licaudatus) [J]. Can J Fish Aq Sci,1995,52:776-787.
[76]PATIL J G.KHOO H W, et al. Nuclear internalization of foreign DNA by zebrafish spermarozoa and its enhancement by electroporation[J]. J Exp Zool,1996,274:121-129.
[77]CASTRO F O, HERNANDEZ O, et al. Introduction of foreign DNA into Spermatozoa of farm animals [J]. Theriogenology 1991,34:1099-1110.
[78]SIN F Y, BARTLEY A L, et al, Develeopment of a mass gene transfer method in Chinook salmon: optimization of gene transfer by electroporated sperm[J]. Mol Mar Biol Biotechnol,1993 117:57-69
[79]FERNANDEZ MA, MANI SA, RANGARAJAN PN. Sperm-mediated gene transfer into oocytes of the golden hamster:assessment of sperm function[J]. Indian J Exp Biol,1999,37(11):1085
[80]WALL RJ. Sperm-mediated gene transfer:advances in sperm cell research and applications[J]. Transgenic Res,1999,8(4):313
[81]郭冬生,孙亮先,曾志将等.中华蜜蜂精子介导egfp基因转移[J].昆虫学报,2007,50(9)：878-882.
[82]SU G M, LIU FY, QIN L, et al. The application of artificial insemination technology insistant hybridization between Antheraea pernyi and Dictyoploca japonica [J]. Acta Sericologica Sinica, 2011,37:544-548.
[83]郭秀洋.外源基因在家蚕汇总的插入与表达研究[D].中国农业科学院,2000.
[84]左正宏等.家蚕精子介导报告基因gfp和egfp转移技术的研究[J].厦门大学学报,2006,45(4)：580-584.
[85]GANDOLFIF. Sperm-mediated transgenesis[J]. Theriogenology,2000,53(1):127
[86]SPADAFORA C. Sperm cells and foreign DNA:a controversial relation [J]. Bioessays,1998,20: 955-964.
[87]LAVITRANO M, CAMAIONI A, FAZIO VM, et al. Sperm cells as vectors for introducing foreign DNA into eggs.genetic transformation of mice [J]. Cell,1989,57(5):717-723.
[88]BRINSTER R L, SANDGREN E P, BEHRINGER R R, et al. No simple solution for making transgenic mice [J].Cell,1989,59:239-241.
[89]SMITH K, SPADAFORA C. Sperm-mediated gene transfer:Applications and implications [J]. Bioessays,2005,27:551-562
[90]PHILLIPS D M. Insect sperm:their structure and morphogenesis[J]. The Journal of cell biology,1970, 44(2):243.
[91]FAWCETT D W, ANDERSON W A, PHILLIPS D M. Morphogenetic factors influencing the shape of the sperm head [J]. Developmental biology,1971,26(2):220-251.
[92]BACCETTIB. Insect sperm cells [J]. Advances in insect physiology,1972,9:315-397.
[93]LEVIATAN R, FRIEDLANDER M. The eupyrene-apyrene dichotomous spermatogenesis of Lepidoptera:I. The relationship with postembryonic development and the role of the decline in juvenile hormone titer toward pupation [J]. Developmental Biology,1979,68(2):515-524.
[94]FRIEDLANDER M, JANS P, BENZ G. Precocious reprogramming of eupyrene-apyrene spermatogenesis commitment induced by allatectomy of the penultimate larval instar of the moth Actias selene [J]. Journal of Insect Physiology,1981,27(4):267-269.
[95]FRIEDLANDER M, BENZ G. Control of spermatogenesis resumption in post-diapausing larvae of the codling moth [J]. Journal of Insect Physiology,1982,28(4):349-355.
[96]詹光杰,奚耕思.昆虫的精子[J].生物学教学,2007,32(5)：4-5.
[97]卢中建,王荫长,尤子平.小地老虎真核和无核精子的超微结构[J].南京农业大学学报,1993,16(3)：38-43.
[98]刘绪生.棉铃虫精子行为研究[D].南京农业大学,2001.
[99]孔海龙,梁醒财,罗佑珍.柑橘凤蝶生殖系统解剖学研究[J].云南农业大学学报,2006,21(4)：459-462.
[100]卢筝.小地老虎生殖系统的研究[J].昆虫学报,1982,25(3)：268-274.
[101]叶恭银,吴生泉.天蚕雄性内生殖器官的结构及其发育观察[J].浙江,农业学报,1996,8(6)：368-371.
[102]OMURA S. Studies on the reproductive system of the male of Bombys mori. Post-testicular organs and postiesticular behavious of the spermatozoa [J]. Fac Agr Hokkaido Imp Univ,1938,40: 129-170.
[103]OSANAI M, KASUGA H, AIGAKI T. The spermatophore and its structural changes with time in the bursa copulatrix of the silkworm, Bombyx mori[J]. Journal of morphology,1987,193(1):1-11.
[104]卢中建,王荫长,尤子平.小地老虎精子发生和形成的定量分析[J].南京农业大学学报,1992,16(3)：39-44.
[105]KATSUNO S. The Relation of the Behavioral and Morphological Changes of the Spermatozoa in the Male Reproductive Organ to Fertilization of Antheraea yamamai:(Lepidoptera Saturniidae) [J]. Journal of the Faculty of Agriculture, Hokkaido University,1983,61(2):272-278.
[106]小林腾,米川昌志,横山纪子,中恒雅,田中一行[J].应动昆,1991,36：231-237.
[107]叶恭银,龚和.保幼激素和蜕皮激素对天蚕卵巢发育的影响[J].浙江农业大学学报,1999,25(3)：276-280.
[108]MINORU OSANAI,梁振中.从家蚕蛾精液中分离出有核精束和无核精子[J].中国蚕业,1992,1.
[109]须贝悦治.家蚕人工生殖及其利用——Ⅳ：精子和卵细胞的成熟[J].广东蚕业,1992,2.
[110]KAWAMURA N, YAMASHIKI N, SAITOH H, et al. Peristaltic squeezing of sperm bundles at the late stage of spermatogenesis in the silkworm, Bombyx mori[J]. Journal of Morphology,2000, 246(2):53-58.
[111]张波等.柞蚕精子形态的显微观察[J].北方蚕业,2008,29(2)：7-8
[112]张波,王林美,叶博等.柞蚕蛹精巢内精子形成过程的显微观察[J].北方蚕业,2010,31(2)：13-16.
[113]HIGHTOWER R C, MEAGHER R B. The molecular evolution of actin [J]. Genetics,1986,114: 315-322.
[114]MOUNIER N, GOUY M, MOUCHIROUD D, et al. Insect muscle actins differ distinctly from invertebrate and vertebrate cyto-plasmic actins [J]. J Mol Evol,1992,34:406-415.
[115]BUNGER M K, MORAN S M, GLOVER E, et al. Resistance to 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity and abnormal liver development in mice carrying a mutation in the nuclear localization sequence of the aryl hydrocarbon receptor[J]. J Biol Chem,2003,278:17767-17774.
[116]KITADE Y, YAMAZAKI S, SAGA N. A method for extraction of high molecular weight DNA from the macroalga porphyra yezoensis (Rhodophyta) [J]. Journal of Phycology,1996,32(3):496-498.
[117]MOUNIER N, PRUDHOMME J C. Isolation of actin genes in Bombyx mori:the coding sequence of a cytoplasmic actin gene expressed in the silk gland is interrupted by a single intron in an unusual position [J]. Biochimie,1986,68(9):1053-1061.
[118]MANGE A, PRUDHOMME J C. Comparison of Bombyx mori and Helicoverpa armigera cytoplasmic actin genes provides clues to the evolution of actin genes in insects [J]. Mol Biol Evol, 1999,16:165-172.
[119]MANGE A, JULIEN E, PRUDHOMME J C, et al. A Strong Inhibitory Element Down-regulates SRE-stimulated Transcription of the A3 Cytoplasmic Actin Gene of Bombyx mori [J]. J Mol Biol, 1997,265:266-274.
[120]ZHU SL, GUO HL, Hu ZY, et al. Characterization of the promoter elements of Bombyx mori bidensovirus nonstructural gene 1 [J]. Curr Microbiol,2012,65:643-648.
[121]张小辉,祁艳霞.真核生物启动子TATA-box, GC-box和CAAT-box的分析[J].安徽农业科学,2008,36(4)：1380-1381.
[122]LIN YS, GREEN MR. Similarities between prokaryotic and eukaryotic cyclic AMP-responsive promoter elements [J]. Nature,1989,340:656-659.
[123]TIMMERS H T, MEYERS R E, SHARP P A. Composition of transcription factor B-TFIID [J]. Proceedings of the National Academy of Sciences,1992,89(17):8140-8144.
[124]GARCIA J A, OU S H, WU F, et al. Cloning and chromosomal mapping of a human immunodeficiency virus 1 TATA element modulatory factor [J]. Proceedings of the National Academy of Sciences,1992,89(20):9372-9376.
[125]KAGEYAMA R, PASTAN I. Molecular cloning and characterization of a human DNA binding factor that represses transcription [J]. Cell,1989,59:815-825.
[126]LEMAIRE P, VESQUE C, SCHMITT J, et al. The serum-inducible mouse gene Krox-24 encodes a sequence-specific transcriptional activator [J]. Molecular and cellular biology, 1990,10(7):3456-3467.
[127]DING H, BENOTMANE A M, SUSKE G, et al. Functional interactions between Spl or Sp3 and the helicase-like transcription factor mediate basal expression from the human plasminogen activator inhibitor-1 gene [J]. Journal of Biological Chemistry,1999,274(28):19573-19580.
[128]MCVEY D, STRAUSS M, Gluzman Y. Properties of the DNA-binding domain of the simian virus 40 large T antigen[J]. Molecular and cellular biology,1989,9(12):5525-5536.
[129]WU F K, GARCIA J A, HARRICH D, et al. Purification of the human immunodeficiency virus typel enhancer and TAR binding proteins EBP-1 and UBP-1 [J]. The EMBO journal,1988, 7(7):2117-2129.
[130]MOSER M, IMHOF A, PSCHERER A., et al. Cloning and characterization of a second AP-2 transcription factor:AP-2 beta[J]. Development,1995,121(9):2779-2788.
[131]PEDONE P. V., GHIRLANDO R., CLORE G. M., et al. The single Cys2-His2 finger domain of the GAGA protein flanked by basic residues is sufficient for high-affinity specific DNA binding[J]. Proceedings of the National Academy of Sciences,1996,93(7):2822-2826.
[132]FATYOL K, ILLES K, SZALAY A. An alternative intronic promoter of the Bombyx A3 cytoplasmic actin gene exhibits a high level of transcriptional activity in mammalian cells [J]. Mol Gen Genet 1999,261:337-345.
[133]MOHUN T, TAYLOR M, GARRETT N, et al.The CArG promoter sequence is necessary for muscle-specific transcription of the cardiac actin gene in Xenopus embryos [J]. The EMBO Journal, 1989,8(4):1153-1161.
[134]CASES I, DE LORENZO V. Promoters in the environment:transcriptional regulation in its natural context [J]. Nat RevMicrobiol,2005,3(5):105-118.
[135]LIU YQ, LI Y, LI X, et al. The origin and dispersal of the domesticated Chinese oak silkworm, Antheraea pernyi, in China:A reconstruction based on ancient texts[J]. Journal of Insect Science, 2010,10:1-10.
[136]吴雪峰.家蚕转基因载体pBacA3E, pBacA4R在昆虫培养细胞中的表达研究[D].西南大学,2006.
[137]HOCHI S, NINOMIYA T, MIZUNO A, et al. Fate of exogenous DNA carried into mouse eggs by spermatozoa [J]. Anim Biotechnol,1990,1:21-31.
[138]LAVITRANO M, BUSNELLI M, CERRITO M G, et al. Sperm-mediated gene transfer [J]. Reprod Fert Develop,2005,18:19-23.
[139]GARCIA-VAZQUEZ FA, RUIZ S, GRULLON LA., et al. Factors affecting porcine sperm mediated gene transfer [J]. Res Vet Sci,2011,91:446-453.
[140]TAMURA T, KANDA Y, TAKIYA S, et al. Transient expression of chimeric CAT genes injected into early embryos of the domesticated silk worm Bombyx mori [J]. Jpn J Genet,1990,65:401-410.
[141]SHAMILA Y, MATHAVAN S. Sperm-mediated gene transfer in the silkworm Bombyx mori [J]. Arch Insect Biochem,1998,37:168-177.
[142]ZHAO Y, LI X, CAO G, et al. Expression of hIGF-I in the silk glands of transgenic silkworms and in transformed silkworm cells [J].Sci China Ser C,2009,52:1131-1139.
[143]CONG L, CAO G, REN YX, et al. Reducing blood glucose level in TIDM mice by orally administering the silk glands of transgenic hIGF-I silkworms [J]. Biochem Biophys Res Commun, 2011,410:721-725.
[144]XUE R, CHEN H, CUI L, et al. Expression of hGM-CSF in silk glands of transgenic silkworms using gene targeting vector [J]. Transgenic Res,2012,21:101-111.
[145]CAO GL, XUE RY, SHENG WD, et al. Research on hGM-CSF transgenetic silkworm with piggyback transposon [J]. Acta Sericologica Sinica,2006,32:324-327.
[146]LIU JF, SU Q, AN LJ, et al. Transfer of a minimal linear marker-free and vector-free smGFP cassette into soybean via ovary-drip transformation [J]. Biotechnol Lett,2009,31:295-303.
[147]CHANG KT, IKEDA A, HAYASHI K, et al. Possible mechanisms for the testis-mediated gene transfer as a new method for producing transgenic animals [J]. J Reprod Dev,1999,45:37-42.
[148]赵万源,陈德明.用大体放射自显影术研究家蚕精液在雌虫生殖系统中的分布和转移[J].昆虫学报,1966,1：006.
[149]SUZUKI N, OKUDA T, SHINBO H. Sperm precedence and sperm movement under different copulation intervals in the silkworm, Bombyx mori [J].Insect Physiol,1996,42:199-204.
[150]胡巧玲,李晓东,沈家骢.仿生结构材料的研究进展[J].材料研究学报,2003,17(4)：337-344.
[151]ARCIDIACONO S, MELLO C, KAPLAN D, et al. Purification and characterization of recombinant spider silk expressed in Escherichia coli [J]. Appl Microbiol Biotechnol,1998,49(1):31-38.
[152]FAHNESTOCK S R, AND BEDZYK L A. Production of synthetic spider dragline silk protein in Pichia pastoris.Appl [J]. Microbiol Biotechnol,1997,47(1):33-39.
[153]KANE J F. Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coil [J]. Current Opinion in Biotechnology,1995,6(5):494-500.
[154]马鹤雯,张立树等.外源丙氨酸提高蜘蛛牵引丝蛋白天然基因在原核系统中的表达[J].中国生物工程杂志,2007,27(1)：47-51.
[155]LAZARIS A, ARCIDIACONO S, HUANG Y, et al. Spider silk fibers spun from soluble recombinant silk produced in mammalian cells [J].Science,2002,295(5554):472-476.
[156]任洪林,柳增善,潘风光等.大腹圆蛛主壶腹腺cDNA文库构建和丝蛋白基因筛选[J].生物技术,2002,12(5)：1-3
[157]王宝玲.柞蚕种卵孵化率的影响因子研究[J].现代农业科技,2012,13：183.
[2]向仲怀,杨焕明.家蚕基因组研究对蚕业学科和产业发展的影响[J].世界科技研究与发展,2003,5(6)：1-5.
[3]李文利.柞蚕丝素基因转移载体的构建及转基因柞蚕的研究[D].大连理工大学,2003.
[4]XU M., LEWIS R. V. Structure of a protein superfiber:Spider dragline silk [J]. Proc. Natl. Acad. Sci.,USA,1990,87(18):7120-7124.
[5]姜义仁,秦利.柞蚕种质资源研究：回顾与展望[J].中国蚕学会第八届暨国家蚕桑产业技术体系家(柞)蚕遗传育种及良种繁育学术研讨会论文集,2011.
[6]董文娟,王学英,张涛,等.柞蚕滞育蛹抗菌物质产生及其抑菌活性研究[J].安徽农业科学,2007,35(4)：99-991.
[7]范琦.柞蚕核型多角体病毒转移载体的组建及外源基因在柞蚕蛹体内的表达[J].辽宁农业科学,1992,5：002.
[8]LI W, JIN L, AN L. Construction of targeting vector and expression of green fluorescent protein in the silkworm, Antheraea pernyi[J]. DNA and cell biology,2003,22(7):441-446.
[9]SHURONG XU, DANMEI LIU, WENLI LI. Upregulated proteins associated with fibroin synthesis in posterior silk glands of Antheraea pernyi fifth instar larvae[J]. Comp Biochem Physiol Part D Genomics Proteomics.2009,4 (2):105-10.
[10]李维,张义正.黄孢原毛平革菌基因启动子的分离与鉴定[J].生物工程学报,2000,16,(5)：599-602.
[11]刘春,徐汉福,陈玉琳等.家蚕转基因研究及肌动蛋白A3启动子的表达分析[J].蚕学通讯,2007,27(3)：1-6.
[12]向仲怀.蚕丝生物学[M].中国林业出版社,2005.
[13]周文林,曹广力,薛仁宇等.家蚕丝素蛋白轻链基因(fib-L)启动子序列的克隆及其活性分析[J].昆虫学报,2007,50(6)：547-554.
[14]马志明,石晓燕,曹广力等.家蚕核型多角体病毒IE-1基因启动子的克隆和序列分析[J].2001.
[15]赵巧玲,唐顺明,易咏竹等.家蚕CIb1基因启动子活性分析[J].蚕业科学,2006,32(4)：477-481.
[16]MICHAILLE J J, COUBLE P, PRUDHOMME J C, et al. A single gene produces multiple sericin messenger RNAS in the silk gland of Bombyx mori [J]. Biochimie,1986,68(10):1165-1173.
[17]诸戌娴,曹广力,薛仁宇等.家蚕丝胶蛋白基因1(ser-1)启动子的克隆及其活性分析[J].科技通报,2007,23(6)：828-834.
[18]ROBERTSON H M, LAMPE D J. Recent horizontal transfer of a mariner transposable element among and between Diptera and Neuroptera [J]. Molecular biology and evolution,1995,12(5):850-862.
[19]KID WELL M G. Reciprocal differences in female recombination associated with hybrid dysgenesis in Drosophila melanogaster [J].Genet Res,1977,33(1):77-88.
[20]UCHINO K, IMAMURA M, SHIMIZU K, et al. Germ line transformation of the silkworm, Bombyx mori, using the transposable element Minos[J]. Molecular Genetics and Genomics,2007,277(3): 213-220.
[21]LOUKERIS T G, LIVADARAS I, ARCA B, et al. Gene transfer into the medfly, Ceratitis capitata, with a Drosophila hydei transposable element [J]. Science,1995,270(5244):2002-2005.
[22]HANDLER A M. Use of the piggyBac transposon for germ-line transformation of insects[J]. Insect biochemistry and molecular biology,2002,32(10):1211-1220.
[23]FRASER M J. The TTAA-specific family of transposable elements:identification, functional characterization, and utility for transformation of insects[J]. Insect Transgenesis:Methods and Applications,2000:249-268.
[24]ELICK T A, BAUSER C A, FRASER M J. Excision of the piggyBac transposable element in vitro is a precise event that is enhanced by the expression of its encoded transposase[J]. Genetica,1996,98(1): 33-41.
[25]TAMURA T, THIBERT C, ROYER C, et al. GermLine transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector [J]. Nat Biotechnol 2000,18:81-84.
[26]IMAMURA M, NAKAHARA Y, KANDA T, et al. A transgenic silkworm expressing the immune-inducible cecropin B-GFP reporter gene[J]. Insect biochemistry and molecular biology, 2006,36(5):429-434.
[27]ZHONG B, LI J, CHEN J, et al. Comparison of transformation efficiency of piggyBac transposon among three different silkworm Bombyx mori strains[J]. Acta biochimica et biophysica Sinica,2007, 39(2):117-122.
[28]CHIOCCHETTI A, TOLOSANO E, HIRSCH E, et al. Green fluorescent protein as a reporter of gene expression in transgenic mice[J]. Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression,1997,1352(2):193-202.
[29]DAVID H E, DACE A S, DE PMIERAI D L, et al. Construction and ecaluation of transgenic hsp16-GFP-lacZ Caenorhabditiditis elegans strain for environmental monitoring[J]. Environ Toxicol Chem,2003,22:111-118.
[30]ZHANG J Z, GUO X B, XIE S Y, et al. Production of transgenic mice carrying green fluorescence protein gene by alcntiviral vector-mediated approach[J]. Prog Nat Sci,2006,08(16):827-833.
[31]ZHANG Y, PAN D, SUN X, et al. Production of porcine cloned transgenic embryos expiessing green fluorescent protein by somatic cell nuclear transfer[J]. SciSin(c), LifeSci,2006,49(2):164-171.
[32]森肇.在家蚕中的基因整合[J].日本蚕丝学会讲演要旨集,1998,40.
[33]FRADKOV A F, CHEN Y, DING L, et al. Novel fluorescent protein from Discosoma coral and its mutants possesses a unique far-red fluorescence[J]. FEBS letters,2000,479(3):127-130.
[34]YARBROUGH D, WACHTER R M, KALLIO K, et al. Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-A resolution[J]. Proceedings of the National Academy of Sciences,2001,98(2):462-467.
[35]娄桂予,陈敏,陈彬,等HNF1 α对人FXR启动子的调控作用[J].中国生物化学与分子生物学报,2006,22(12)：966-972.
[36]徐春娥,付玲,侯丽华,等.人IFN-β启动子基因报告质粒的构建及SARS-CoV3a和7a蛋白对IFN-β诱生作用的初步研究[J].中国生物工程杂志,2006,26(12)：6-10.
[37]GORDON J W, SCANGOS G A, PLOTKIN D J, et al. Genetic transformation of mouseembryos by microinjection of purified DNA [J]. Proc Natl Acad,1980,77(12):7380-7384.
[38]NINAKI O., MAEKAWA H, GAMO T, et al. Hatchability of silkworm eggs injected with DNA at early embryonic stages[J]. Journal of Sericultural Science of Japan,1985,54.
[39]THOMAS JL.DA ROCHA M.BESSE A,et al.3×P3-EGFP marker facilitates screening of transgenic silkworm Bombyx mori L.from the embryonic stage onwards [J]. Insect Biochem Mol Biol,2002,32:247-253.
[40]UHLIROVA M, ASAHINA M, RIDDIFORD L M, et al. Heat-inducible transgenic expression in the Silkmoth Bombyx mori [J]. Dev. Genes Evol,2002,212(3):145-151.
[41]IMAMURA M, NAKAI J, QUAN GX, et al.Targeted gene expression using the GAL4/UAS system in the silkworm Bombyx mori[J]. Genetics,2003,165:1329-1340.
[42]代红久,徐国江,王铸钢等.利用鳞翅目来源的转座子piggyBac建立高效稳定的转基因家蚕技术[J].科学通报,2005,50(14)：1470-1474.
[43]SANFORD J C, KLEIN T M, WOLF E D, et al. Delivery of substances into cells and tissues using a particle bombardment process [J]. Particulate Science and Technology,1987,5(1):27-37.
[44]KLEIN T M, WOLF E D, WU R, et al. High-velocity microprojectiles for delivering nucleic acids into living cells[J]. Nature,1987,327(6117):70-73.
[45]孟智启,姚山麟,王为民等.基因枪喷射技术在家蚕外源基因转移中的应用[J].浙江农业学报,1990,2：1-6.
[46]陈秀,赵昀,张峰等.新霉素抗性基因在家蚕中的插入和表达[J].生物化学与生物物理学报,1999,31,(1)：90-92.
[47]赵昀,张峰,陈秀等.用绿色荧光蛋白进行转基因蚕研究[J].高技术通讯,1999,6：16-19.
[48]ADACHI T, TOMITA M, SHIMIZU K, et al. Generation of hybrid transgenic silkworms that express Bombyx mori prolyl-hydroxylase alpha-subunits and human collagens in posterior silk glands: production of cocoons that contained collagens with hydroxylated proline residues[J]. Journal of Biotechnology.2006,126(2):205-219.
[49]WONG TK, NEUMANN E. Electric field mediated gene transfer [J].Biochem Biophys Res Commun,1982,107 (2):584.
[50]KENIEHI MOTO, et al. Gene transfer into insect brain nadeellosPeeifieexPerssiom of bombyxin gene[J]. Dev GneeEvol,1999,209:447-450.
[51]张峰,陈秀.能发荧光的转基因家蚕[J].生物化学与生物物理学报：英文版,1999,31(2)：119-123.
[52]赵昀,陈秀,彭卫平等.利用同源重组改变家蚕丝心蛋白重链基因[J].生物化学与生物物理学报.2001,33(1)：112-116.
[53]GUO XY,DONG L, WANG SP, et al. Introduction of foreign genes into silkworm eggs by eleetroPoration And Its aPP lieation in transgenic vector test [J].Acta Bioa Biop Sin,36(5):323-330.
[54]BRACKETT B G, BARANSKA W, SAWICKI W, et al. Uptake of heterologous genome by mammalian spermatozoa and its transfer to ova through fertilization [J]. P Natl Acad Sci USA,1971, 68:353-357.
[55]ROTTMAN OJ, LAMPETER WW. Development of early mouse and rabbit embryos withoutzona pellucida[J]. J Repord Fertil,1981,61(2):303-306.
[56]郭秀洋等.利用精子介导法向蚕卵导入外源基因的研究[J].生物化学与生物物理进展,2001,28(3)：423-425.
[57]王宇,刘辉芬,李维,邱兴林.家蚕转基因方法的初步研究[J].四川动物,2006,3.
[58]张业顺等.家蚕精子介导转基因技术体系的优化[J].蚕业科学,2007,33(2)：201-206.
[59]CHEN YL, REN CZ, GUI MY. Preliminary Study on the artificial insemination of the domesticated silkworm and eri Silkworm [J]. Journal of Xiamen University (Natural Science),1993, S1:9-14.
[60]桂慕燕,陈元霖,刘治纬等.家蚕人工授精与精子超低温保存研究[J].遗传,1997,19(增刊1)：83-85.
[61]代方银,陈智毅,陈元霖等.家蚕白卵突变新系BT924的遗传学研究[J].遗传,2000,22：229-232.
[62]郭秀洋.家蚕遗传操作技术的研究及其应用[D].中国科学院研究生院(上海生命科学研究院),2004.
[63]赵越,李曦,曹广力等.转基因家蚕丝腺组织和转化家蚕培养细胞表达hIGF-I[J]中国科学C辑：生命科学,2009,39：677-684.
[64]GAGNE M, POTHIER F, SIRARD M A. Effect of microinjection time during post-fertilization S-phase on bovine embryonic development[J]. Molecular reproduction and development,1995, 41(2):184-194.
[65]AREZZO F. Sea urchin sperm as a vector offoreign geneticinformation [J]. Cell Biol lnt Rep, 1989,13:391-404.
[66]MILNE C P, ELSCHEN F A, et al. Preliminary Evidence for Honeybee Sperm Mediated DNA Transter [J]. International Symposium on Moleculax Insect Science, Tucson, Afizona, 1989.120-125.
[67]TSAI H J, LAI CH, YANG HS, et al. Sperm as a cartier to introduce an exogenous DNA fragment into the ocyte of Japanese abalone (Halioti. s divorsicolor suportexta) [J]. Transgenic Res, 1997.6:85-95.
[68]KHO H W, ANG L H, et al. Sperm cells as vectors for introducing foreign DNA into zebrafish[J]. Aquaculture,1992,109:1-19
[69]KUZNETSOV A V, KUZNETSOVA I V, et al. Binding of exogenous DNA pRK31acZ by the rab bit spe rmatozoa, its tran sfer in the o eytes, an d expression in the preimplan tation embryos [J]. Russian Journal of Developmental Biology,1995,26:300-309.
[70]PERRY A, WAKAYAMA T, et al. Mammalian transgenesis by intracytoplasmic sperm injection [J]. Science,1999,2114:1180-1183.
[71]NAKANISH A, et al. Gene transfer in the chicken by sperm mediated methods [J]. Mol Report Dev, 1993,36:258-261.
[72]GAGNE M B, POTHIER F, et al. Electroporation of bovine spermatozoa to carry foreign DNA in oocytes [J]. Mol Reprol Del,1991,29:6-15.
[73]SEPERANDIO S, LULLI V, et al.Sperm-mediated DNA transfer in bovine and swine species [J].Anim Biotechnol,1996,7:59-77.
[74]HABROVA V, TAKAC M, et al. Association of Rous sarcoma virus DNA with X.Laevis spermatozoa and its transfer to ova through fertilization[J]. Mol Reprod Dev,1996,44:332-342.
[75]TSAIH J, TSENG F S, et al. Electroporation of sperm to introduce foreign DNA into the genome of loach (Misgurnus anguil-licaudatus) [J]. Can J Fish Aq Sci,1995,52:776-787.
[76]PATIL J G.KHOO H W, et al. Nuclear internalization of foreign DNA by zebrafish spermarozoa and its enhancement by electroporation[J]. J Exp Zool,1996,274:121-129.
[77]CASTRO F O, HERNANDEZ O, et al. Introduction of foreign DNA into Spermatozoa of farm animals [J]. Theriogenology 1991,34:1099-1110.
[78]SIN F Y, BARTLEY A L, et al, Develeopment of a mass gene transfer method in Chinook salmon: optimization of gene transfer by electroporated sperm[J]. Mol Mar Biol Biotechnol,1993 117:57-69
[79]FERNANDEZ MA, MANI SA, RANGARAJAN PN. Sperm-mediated gene transfer into oocytes of the golden hamster:assessment of sperm function[J]. Indian J Exp Biol,1999,37(11):1085
[80]WALL RJ. Sperm-mediated gene transfer:advances in sperm cell research and applications[J]. Transgenic Res,1999,8(4):313
[81]郭冬生,孙亮先,曾志将等.中华蜜蜂精子介导egfp基因转移[J].昆虫学报,2007,50(9)：878-882.
[82]SU G M, LIU FY, QIN L, et al. The application of artificial insemination technology insistant hybridization between Antheraea pernyi and Dictyoploca japonica [J]. Acta Sericologica Sinica, 2011,37:544-548.
[83]郭秀洋.外源基因在家蚕汇总的插入与表达研究[D].中国农业科学院,2000.
[84]左正宏等.家蚕精子介导报告基因gfp和egfp转移技术的研究[J].厦门大学学报,2006,45(4)：580-584.
[85]GANDOLFIF. Sperm-mediated transgenesis[J]. Theriogenology,2000,53(1):127
[86]SPADAFORA C. Sperm cells and foreign DNA:a controversial relation [J]. Bioessays,1998,20: 955-964.
[87]LAVITRANO M, CAMAIONI A, FAZIO VM, et al. Sperm cells as vectors for introducing foreign DNA into eggs.genetic transformation of mice [J]. Cell,1989,57(5):717-723.
[88]BRINSTER R L, SANDGREN E P, BEHRINGER R R, et al. No simple solution for making transgenic mice [J].Cell,1989,59:239-241.
[89]SMITH K, SPADAFORA C. Sperm-mediated gene transfer:Applications and implications [J]. Bioessays,2005,27:551-562
[90]PHILLIPS D M. Insect sperm:their structure and morphogenesis[J]. The Journal of cell biology,1970, 44(2):243.
[91]FAWCETT D W, ANDERSON W A, PHILLIPS D M. Morphogenetic factors influencing the shape of the sperm head [J]. Developmental biology,1971,26(2):220-251.
[92]BACCETTIB. Insect sperm cells [J]. Advances in insect physiology,1972,9:315-397.
[93]LEVIATAN R, FRIEDLANDER M. The eupyrene-apyrene dichotomous spermatogenesis of Lepidoptera:I. The relationship with postembryonic development and the role of the decline in juvenile hormone titer toward pupation [J]. Developmental Biology,1979,68(2):515-524.
[94]FRIEDLANDER M, JANS P, BENZ G. Precocious reprogramming of eupyrene-apyrene spermatogenesis commitment induced by allatectomy of the penultimate larval instar of the moth Actias selene [J]. Journal of Insect Physiology,1981,27(4):267-269.
[95]FRIEDLANDER M, BENZ G. Control of spermatogenesis resumption in post-diapausing larvae of the codling moth [J]. Journal of Insect Physiology,1982,28(4):349-355.
[96]詹光杰,奚耕思.昆虫的精子[J].生物学教学,2007,32(5)：4-5.
[97]卢中建,王荫长,尤子平.小地老虎真核和无核精子的超微结构[J].南京农业大学学报,1993,16(3)：38-43.
[98]刘绪生.棉铃虫精子行为研究[D].南京农业大学,2001.
[99]孔海龙,梁醒财,罗佑珍.柑橘凤蝶生殖系统解剖学研究[J].云南农业大学学报,2006,21(4)：459-462.
[100]卢筝.小地老虎生殖系统的研究[J].昆虫学报,1982,25(3)：268-274.
[101]叶恭银,吴生泉.天蚕雄性内生殖器官的结构及其发育观察[J].浙江,农业学报,1996,8(6)：368-371.
[102]OMURA S. Studies on the reproductive system of the male of Bombys mori. Post-testicular organs and postiesticular behavious of the spermatozoa [J]. Fac Agr Hokkaido Imp Univ,1938,40: 129-170.
[103]OSANAI M, KASUGA H, AIGAKI T. The spermatophore and its structural changes with time in the bursa copulatrix of the silkworm, Bombyx mori[J]. Journal of morphology,1987,193(1):1-11.
[104]卢中建,王荫长,尤子平.小地老虎精子发生和形成的定量分析[J].南京农业大学学报,1992,16(3)：39-44.
[105]KATSUNO S. The Relation of the Behavioral and Morphological Changes of the Spermatozoa in the Male Reproductive Organ to Fertilization of Antheraea yamamai:(Lepidoptera Saturniidae) [J]. Journal of the Faculty of Agriculture, Hokkaido University,1983,61(2):272-278.
[106]小林腾,米川昌志,横山纪子,中恒雅,田中一行[J].应动昆,1991,36：231-237.
[107]叶恭银,龚和.保幼激素和蜕皮激素对天蚕卵巢发育的影响[J].浙江农业大学学报,1999,25(3)：276-280.
[108]MINORU OSANAI,梁振中.从家蚕蛾精液中分离出有核精束和无核精子[J].中国蚕业,1992,1.
[109]须贝悦治.家蚕人工生殖及其利用——Ⅳ：精子和卵细胞的成熟[J].广东蚕业,1992,2.
[110]KAWAMURA N, YAMASHIKI N, SAITOH H, et al. Peristaltic squeezing of sperm bundles at the late stage of spermatogenesis in the silkworm, Bombyx mori[J]. Journal of Morphology,2000, 246(2):53-58.
[111]张波等.柞蚕精子形态的显微观察[J].北方蚕业,2008,29(2)：7-8
[112]张波,王林美,叶博等.柞蚕蛹精巢内精子形成过程的显微观察[J].北方蚕业,2010,31(2)：13-16.
[113]HIGHTOWER R C, MEAGHER R B. The molecular evolution of actin [J]. Genetics,1986,114: 315-322.
[114]MOUNIER N, GOUY M, MOUCHIROUD D, et al. Insect muscle actins differ distinctly from invertebrate and vertebrate cyto-plasmic actins [J]. J Mol Evol,1992,34:406-415.
[115]BUNGER M K, MORAN S M, GLOVER E, et al. Resistance to 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity and abnormal liver development in mice carrying a mutation in the nuclear localization sequence of the aryl hydrocarbon receptor[J]. J Biol Chem,2003,278:17767-17774.
[116]KITADE Y, YAMAZAKI S, SAGA N. A method for extraction of high molecular weight DNA from the macroalga porphyra yezoensis (Rhodophyta) [J]. Journal of Phycology,1996,32(3):496-498.
[117]MOUNIER N, PRUDHOMME J C. Isolation of actin genes in Bombyx mori:the coding sequence of a cytoplasmic actin gene expressed in the silk gland is interrupted by a single intron in an unusual position [J]. Biochimie,1986,68(9):1053-1061.
[118]MANGE A, PRUDHOMME J C. Comparison of Bombyx mori and Helicoverpa armigera cytoplasmic actin genes provides clues to the evolution of actin genes in insects [J]. Mol Biol Evol, 1999,16:165-172.
[119]MANGE A, JULIEN E, PRUDHOMME J C, et al. A Strong Inhibitory Element Down-regulates SRE-stimulated Transcription of the A3 Cytoplasmic Actin Gene of Bombyx mori [J]. J Mol Biol, 1997,265:266-274.
[120]ZHU SL, GUO HL, Hu ZY, et al. Characterization of the promoter elements of Bombyx mori bidensovirus nonstructural gene 1 [J]. Curr Microbiol,2012,65:643-648.
[121]张小辉,祁艳霞.真核生物启动子TATA-box, GC-box和CAAT-box的分析[J].安徽农业科学,2008,36(4)：1380-1381.
[122]LIN YS, GREEN MR. Similarities between prokaryotic and eukaryotic cyclic AMP-responsive promoter elements [J]. Nature,1989,340:656-659.
[123]TIMMERS H T, MEYERS R E, SHARP P A. Composition of transcription factor B-TFIID [J]. Proceedings of the National Academy of Sciences,1992,89(17):8140-8144.
[124]GARCIA J A, OU S H, WU F, et al. Cloning and chromosomal mapping of a human immunodeficiency virus 1 TATA element modulatory factor [J]. Proceedings of the National Academy of Sciences,1992,89(20):9372-9376.
[125]KAGEYAMA R, PASTAN I. Molecular cloning and characterization of a human DNA binding factor that represses transcription [J]. Cell,1989,59:815-825.
[126]LEMAIRE P, VESQUE C, SCHMITT J, et al. The serum-inducible mouse gene Krox-24 encodes a sequence-specific transcriptional activator [J]. Molecular and cellular biology, 1990,10(7):3456-3467.
[127]DING H, BENOTMANE A M, SUSKE G, et al. Functional interactions between Spl or Sp3 and the helicase-like transcription factor mediate basal expression from the human plasminogen activator inhibitor-1 gene [J]. Journal of Biological Chemistry,1999,274(28):19573-19580.
[128]MCVEY D, STRAUSS M, Gluzman Y. Properties of the DNA-binding domain of the simian virus 40 large T antigen[J]. Molecular and cellular biology,1989,9(12):5525-5536.
[129]WU F K, GARCIA J A, HARRICH D, et al. Purification of the human immunodeficiency virus typel enhancer and TAR binding proteins EBP-1 and UBP-1 [J]. The EMBO journal,1988, 7(7):2117-2129.
[130]MOSER M, IMHOF A, PSCHERER A., et al. Cloning and characterization of a second AP-2 transcription factor:AP-2 beta[J]. Development,1995,121(9):2779-2788.
[131]PEDONE P. V., GHIRLANDO R., CLORE G. M., et al. The single Cys2-His2 finger domain of the GAGA protein flanked by basic residues is sufficient for high-affinity specific DNA binding[J]. Proceedings of the National Academy of Sciences,1996,93(7):2822-2826.
[132]FATYOL K, ILLES K, SZALAY A. An alternative intronic promoter of the Bombyx A3 cytoplasmic actin gene exhibits a high level of transcriptional activity in mammalian cells [J]. Mol Gen Genet 1999,261:337-345.
[133]MOHUN T, TAYLOR M, GARRETT N, et al.The CArG promoter sequence is necessary for muscle-specific transcription of the cardiac actin gene in Xenopus embryos [J]. The EMBO Journal, 1989,8(4):1153-1161.
[134]CASES I, DE LORENZO V. Promoters in the environment:transcriptional regulation in its natural context [J]. Nat RevMicrobiol,2005,3(5):105-118.
[135]LIU YQ, LI Y, LI X, et al. The origin and dispersal of the domesticated Chinese oak silkworm, Antheraea pernyi, in China:A reconstruction based on ancient texts[J]. Journal of Insect Science, 2010,10:1-10.
[136]吴雪峰.家蚕转基因载体pBacA3E, pBacA4R在昆虫培养细胞中的表达研究[D].西南大学,2006.
[137]HOCHI S, NINOMIYA T, MIZUNO A, et al. Fate of exogenous DNA carried into mouse eggs by spermatozoa [J]. Anim Biotechnol,1990,1:21-31.
[138]LAVITRANO M, BUSNELLI M, CERRITO M G, et al. Sperm-mediated gene transfer [J]. Reprod Fert Develop,2005,18:19-23.
[139]GARCIA-VAZQUEZ FA, RUIZ S, GRULLON LA., et al. Factors affecting porcine sperm mediated gene transfer [J]. Res Vet Sci,2011,91:446-453.
[140]TAMURA T, KANDA Y, TAKIYA S, et al. Transient expression of chimeric CAT genes injected into early embryos of the domesticated silk worm Bombyx mori [J]. Jpn J Genet,1990,65:401-410.
[141]SHAMILA Y, MATHAVAN S. Sperm-mediated gene transfer in the silkworm Bombyx mori [J]. Arch Insect Biochem,1998,37:168-177.
[142]ZHAO Y, LI X, CAO G, et al. Expression of hIGF-I in the silk glands of transgenic silkworms and in transformed silkworm cells [J].Sci China Ser C,2009,52:1131-1139.
[143]CONG L, CAO G, REN YX, et al. Reducing blood glucose level in TIDM mice by orally administering the silk glands of transgenic hIGF-I silkworms [J]. Biochem Biophys Res Commun, 2011,410:721-725.
[144]XUE R, CHEN H, CUI L, et al. Expression of hGM-CSF in silk glands of transgenic silkworms using gene targeting vector [J]. Transgenic Res,2012,21:101-111.
[145]CAO GL, XUE RY, SHENG WD, et al. Research on hGM-CSF transgenetic silkworm with piggyback transposon [J]. Acta Sericologica Sinica,2006,32:324-327.
[146]LIU JF, SU Q, AN LJ, et al. Transfer of a minimal linear marker-free and vector-free smGFP cassette into soybean via ovary-drip transformation [J]. Biotechnol Lett,2009,31:295-303.
[147]CHANG KT, IKEDA A, HAYASHI K, et al. Possible mechanisms for the testis-mediated gene transfer as a new method for producing transgenic animals [J]. J Reprod Dev,1999,45:37-42.
[148]赵万源,陈德明.用大体放射自显影术研究家蚕精液在雌虫生殖系统中的分布和转移[J].昆虫学报,1966,1：006.
[149]SUZUKI N, OKUDA T, SHINBO H. Sperm precedence and sperm movement under different copulation intervals in the silkworm, Bombyx mori [J].Insect Physiol,1996,42:199-204.
[150]胡巧玲,李晓东,沈家骢.仿生结构材料的研究进展[J].材料研究学报,2003,17(4)：337-344.
[151]ARCIDIACONO S, MELLO C, KAPLAN D, et al. Purification and characterization of recombinant spider silk expressed in Escherichia coli [J]. Appl Microbiol Biotechnol,1998,49(1):31-38.
[152]FAHNESTOCK S R, AND BEDZYK L A. Production of synthetic spider dragline silk protein in Pichia pastoris.Appl [J]. Microbiol Biotechnol,1997,47(1):33-39.
[153]KANE J F. Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coil [J]. Current Opinion in Biotechnology,1995,6(5):494-500.
[154]马鹤雯,张立树等.外源丙氨酸提高蜘蛛牵引丝蛋白天然基因在原核系统中的表达[J].中国生物工程杂志,2007,27(1)：47-51.
[155]LAZARIS A, ARCIDIACONO S, HUANG Y, et al. Spider silk fibers spun from soluble recombinant silk produced in mammalian cells [J].Science,2002,295(5554):472-476.
[156]任洪林,柳增善,潘风光等.大腹圆蛛主壶腹腺cDNA文库构建和丝蛋白基因筛选[J].生物技术,2002,12(5)：1-3
[157]王宝玲.柞蚕种卵孵化率的影响因子研究[J].现代农业科技,2012,13：183.