转Bt抗虫基因植物的酶联免疫检测技术研究
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摘要
培育和利用抗虫品种是防治作物害虫最经济有效的措施。通过转抗虫基因培育抗虫品种是重要方法之一。此项工作中需要对转基因植物进行鉴定和筛选，因此发展快速、准确、简便的检测技术具有重要的作用和意义。本研究以检测目前抗虫转基因育种中研究最多、获得成效最大的转Bt抗虫基因的表达产物(杀虫蛋白)为检测目标。为此研究了影响抗原和抗体制备的主要因素，制备出灵敏度高的特异抗体，在此基础上开展检测方法研究，掌握影响检测效果的有关因素，从而初步建立起灵敏度高的ELISA检测技术。本研究取得如下结果：
抗原是影响抗体特异性和效价的重要因素，为了得到产量高、质量好的杀虫晶体蛋白作为抗原，我们研究了Bt菌培养时间、伴孢晶体蛋白裂解液的种类和处理温度与时间对杀虫蛋白提取的影响。结果表明Bt HD-1菌株在趴生长繁殖培养基(Singer等，1968)上，30℃下培养41-55h均可获得大量孢晶混合物。采用50mMNa_2CO_3(含50mM EDTA，3％巯基乙醇，pH10)裂解液和0.04N Na0H的裂解液处理孢晶混合物，均可获得大量的130KD杀虫蛋白。但用0.04N NaOH提取的杀虫蛋白中含有较多杂蛋白。所研究的处理温度和时间对杀虫蛋白提取量和组成无明显影响。
抗原的免疫剂量是影响抗体产生的一个重要因素，免疫剂量过大会引起麻痹反应。为了提高免疫效果，获得较好的抗体。我们采用两种抗原剂量(750ug／只.次和375 ug／只.次)免疫家兔，结果表明所供试的两种剂量对抗血清效价和特异性无明显影响。因此采用任一种剂量免疫家兔均可。本研究制备的抗体用于检测标准抗原，灵敏度可达7.8-16.5ng。
在建立检测方法研究上，采用ELISA间接检测法，测定了两种酶标羊抗兔抗体(辣根过氧化物酶与碱性磷酸酶)和两种载体(聚苯乙烯酶标板和硝酸纤维膜)对检测灵敏度和样本检测效果的影响。结果表明，在采用纯的杀虫蛋白进行灵敏度检测时，二种酶标抗体和两种检测载体的测定结果非常相近，无明显差别。但在对植物样本检测时，不同酶标抗体和载体则存在差异。表现为碱性磷酸酶标记的抗体优于辣根过氧化物酶标记的抗体。这主要是由于植物样本中存在有内源过氧化物酶的干扰。在两种检测载体上，由于植物样本中存在叶绿素，采用硝酸纤维膜进行Dot-ELISA检测时，
叶绿素色斑干扰较大。如何排除叶绿素在D。t－ELISA检测中的影响，尚值得研究。
通过本研究，基本掌握了提取杀虫蛋白作为抗原和兔疫家兔制备抗血清的有关
因素和条件，制备出了特异性强的抗体，检测灵敏度可达 7．8刁．6ng。通过检测方法
研究，明确了影响检测的一些因素并初步建立起了检测棉叶样本中Bt杀虫蛋白的相
关技术。
Breeding and growing resistant culture was one of elements in the Integrated Pest Management (IPM). Genetic engineering can be used to breed resistant crop. The Bt gene producing insecticidal protein was transferred into cotton and other crops to improve crop resistance to pest.
The goal of this research was to study on the preparation of Antigen of Bt insecticidal protein, production of antibody, and the test method technique for assay of the Bt toxin protein in transgenic cotton and other crop plants by using ELISA. The result was as follows: 1.Preparation of Bt insecticidal
The Bt isolate HD-1 was used in this study for producing toxin protein. The Bt bacterium was cultivated on solid medium at 30癈for 41-55hr. When the crystal protein formed and separated from bacterium cells, the colony on the plate was washed with water and centrifuged 5000g and 4 to collect the preparation of spore and crystal protein. The mixture of spore and crystal protein was washed three times with PBS pH7.4 and d-HaO and dissolved the crystal protein in alkaline solution(0.04N NaOH at 4癈 2-3hr or 50mM NaCO3,50mM EDTA,3% & -rnercaptoethanol,pH10 at 37癈 Ihr or 4癈 4hr) . The solution was centrifuged at SOOOg 20min. The supemate was collected and adjusted the pH to 4.4-5 with NaAc-HAc buffer pH4.4 or Acetic acid to precipitate the toxin protein overnight at 4癈. The precipitate of protein was gained by centrifuge at 5000g 20min 4癈 and washed three time. The protein was used for Antigen. 2.Antiserum of Bt production
Antibodies specific to Bt toxin protein were produced in six New Zealand white male rabbits by immunization procedure. The animals immunized with approximately 750ng or 375ng of antigen per/rabbit in Freund's complete adjuvant. After 30 days of first immunization, subsequent immunizations were conducted using same dosage of Antigen in Freund's incomplete adjuvant at 10 days intervals (three immunizations). Blood was drawn from the heart 10 days after last injection.
Blood Samples were allowed to coagulate, then, after overnight storage in a refrigerator,
-35-
the serum was decanted and clarified by low speed centrifugation. The title of Antibody was tested by ouchterlony gel diffusion. In my experiment, the title was 1/80-1/120 depending on the individual of the rabbit. The IgG fraction from rabbit Antiserum was precipitate by salting out with ammonium sulphate. 3.Establishment of ELISA assay for detection of Bt toxin protein
Two ELISA assay (indirect ELISA and Dot ELISA) were used in this research. The ELISA procedure consisted five general steps:
1.Preparing the test samples and diluting standards antigen in 0.1% BSA, 0.05M PBST pH7.4, 1%PVP.
2.Add test samples or antigen to plate well nitrocellulose membrane, incubate overnight at 4癈 or dry at room temperature for at least lh..
3 .Block the plate well or membrane with PBS pH7.4 containing 10 mg/ml BSA.
4.Add specific antibody to the plate well or membrane and incubate for 2-3h at room temperature.
5.Add enzyme-labeled anti-rabbit Antibody and incubate for 2h at room temperature. Plate wells or membrane were washed between each step from 2-5. The washing was accomplished by 4-5 washes with PBST pH7.4, at room temperature.
6.Add enzyme substrate to develop the color at room temperature and dark condition for about 30 min. Stop the reaction and evaluate the OD value.
The detectable level of purified Bt toxin protein by indirect ELISA and Dot ELISA was 7.8-15.6ng. Five samples of Bt transgenic cotton and two contrastive cottons (non-transgenic cotton) were tested using indirect ELISA and Dot ELISA. The results showed that the two assays could be used for detecting Bt toxin protein in transgenic cotton, but some problems (such as interference of sample green color) need to be solved.
抗原是影响抗体特异性和效价的重要因素，为了得到产量高、质量好的杀虫晶体蛋白作为抗原，我们研究了Bt菌培养时间、伴孢晶体蛋白裂解液的种类和处理温度与时间对杀虫蛋白提取的影响。结果表明Bt HD-1菌株在趴生长繁殖培养基(Singer等，1968)上，30℃下培养41-55h均可获得大量孢晶混合物。采用50mMNa_2CO_3(含50mM EDTA，3％巯基乙醇，pH10)裂解液和0.04N Na0H的裂解液处理孢晶混合物，均可获得大量的130KD杀虫蛋白。但用0.04N NaOH提取的杀虫蛋白中含有较多杂蛋白。所研究的处理温度和时间对杀虫蛋白提取量和组成无明显影响。
抗原的免疫剂量是影响抗体产生的一个重要因素，免疫剂量过大会引起麻痹反应。为了提高免疫效果，获得较好的抗体。我们采用两种抗原剂量(750ug／只.次和375 ug／只.次)免疫家兔，结果表明所供试的两种剂量对抗血清效价和特异性无明显影响。因此采用任一种剂量免疫家兔均可。本研究制备的抗体用于检测标准抗原，灵敏度可达7.8-16.5ng。
在建立检测方法研究上，采用ELISA间接检测法，测定了两种酶标羊抗兔抗体(辣根过氧化物酶与碱性磷酸酶)和两种载体(聚苯乙烯酶标板和硝酸纤维膜)对检测灵敏度和样本检测效果的影响。结果表明，在采用纯的杀虫蛋白进行灵敏度检测时，二种酶标抗体和两种检测载体的测定结果非常相近，无明显差别。但在对植物样本检测时，不同酶标抗体和载体则存在差异。表现为碱性磷酸酶标记的抗体优于辣根过氧化物酶标记的抗体。这主要是由于植物样本中存在有内源过氧化物酶的干扰。在两种检测载体上，由于植物样本中存在叶绿素，采用硝酸纤维膜进行Dot-ELISA检测时，
叶绿素色斑干扰较大。如何排除叶绿素在D。t－ELISA检测中的影响，尚值得研究。
通过本研究，基本掌握了提取杀虫蛋白作为抗原和兔疫家兔制备抗血清的有关
因素和条件，制备出了特异性强的抗体，检测灵敏度可达 7．8刁．6ng。通过检测方法
研究，明确了影响检测的一些因素并初步建立起了检测棉叶样本中Bt杀虫蛋白的相
关技术。
Breeding and growing resistant culture was one of elements in the Integrated Pest Management (IPM). Genetic engineering can be used to breed resistant crop. The Bt gene producing insecticidal protein was transferred into cotton and other crops to improve crop resistance to pest.
The goal of this research was to study on the preparation of Antigen of Bt insecticidal protein, production of antibody, and the test method technique for assay of the Bt toxin protein in transgenic cotton and other crop plants by using ELISA. The result was as follows: 1.Preparation of Bt insecticidal
The Bt isolate HD-1 was used in this study for producing toxin protein. The Bt bacterium was cultivated on solid medium at 30癈for 41-55hr. When the crystal protein formed and separated from bacterium cells, the colony on the plate was washed with water and centrifuged 5000g and 4 to collect the preparation of spore and crystal protein. The mixture of spore and crystal protein was washed three times with PBS pH7.4 and d-HaO and dissolved the crystal protein in alkaline solution(0.04N NaOH at 4癈 2-3hr or 50mM NaCO3,50mM EDTA,3% & -rnercaptoethanol,pH10 at 37癈 Ihr or 4癈 4hr) . The solution was centrifuged at SOOOg 20min. The supemate was collected and adjusted the pH to 4.4-5 with NaAc-HAc buffer pH4.4 or Acetic acid to precipitate the toxin protein overnight at 4癈. The precipitate of protein was gained by centrifuge at 5000g 20min 4癈 and washed three time. The protein was used for Antigen. 2.Antiserum of Bt production
Antibodies specific to Bt toxin protein were produced in six New Zealand white male rabbits by immunization procedure. The animals immunized with approximately 750ng or 375ng of antigen per/rabbit in Freund's complete adjuvant. After 30 days of first immunization, subsequent immunizations were conducted using same dosage of Antigen in Freund's incomplete adjuvant at 10 days intervals (three immunizations). Blood was drawn from the heart 10 days after last injection.
Blood Samples were allowed to coagulate, then, after overnight storage in a refrigerator,
-35-
the serum was decanted and clarified by low speed centrifugation. The title of Antibody was tested by ouchterlony gel diffusion. In my experiment, the title was 1/80-1/120 depending on the individual of the rabbit. The IgG fraction from rabbit Antiserum was precipitate by salting out with ammonium sulphate. 3.Establishment of ELISA assay for detection of Bt toxin protein
Two ELISA assay (indirect ELISA and Dot ELISA) were used in this research. The ELISA procedure consisted five general steps:
1.Preparing the test samples and diluting standards antigen in 0.1% BSA, 0.05M PBST pH7.4, 1%PVP.
2.Add test samples or antigen to plate well nitrocellulose membrane, incubate overnight at 4癈 or dry at room temperature for at least lh..
3 .Block the plate well or membrane with PBS pH7.4 containing 10 mg/ml BSA.
4.Add specific antibody to the plate well or membrane and incubate for 2-3h at room temperature.
5.Add enzyme-labeled anti-rabbit Antibody and incubate for 2h at room temperature. Plate wells or membrane were washed between each step from 2-5. The washing was accomplished by 4-5 washes with PBST pH7.4, at room temperature.
6.Add enzyme substrate to develop the color at room temperature and dark condition for about 30 min. Stop the reaction and evaluate the OD value.
The detectable level of purified Bt toxin protein by indirect ELISA and Dot ELISA was 7.8-15.6ng. Five samples of Bt transgenic cotton and two contrastive cottons (non-transgenic cotton) were tested using indirect ELISA and Dot ELISA. The results showed that the two assays could be used for detecting Bt toxin protein in transgenic cotton, but some problems (such as interference of sample green color) need to be solved.
引文
1.Krattiger. A.F. Insect resistance in crops: A case study of Bacillus thuringiensis(Bt) and its transfer to developing countries. ISAAA Briefs No. 2. ISAAA:I thaca, N Y. 1997, pp. 4-11
2．束春娥、柏立新、孙以文，等：转基因抗虫棉的抗性鉴定，[J]，江苏农业学报，1997，13(1)：22-26
3．高丽洁、薛亚杰、杨正书，等：转Bt基因抗虫棉的研究进展，沈阳农业大学学报，2000-12，31(6)：600-603
4.Fromm ME, Taylor Lp, Walbot V. Stable transformation of maize after gene transfer by electroporation [J]. Nature, 1986,319:791-793
5.Klein TM, Wolf ED, Wu R, et al. High-velocity microjectiles for delivering nuleic acids into living cells [J].Nature, 1987,327:70-73
6.Horsch RB, Fry JE, Hotfman NL, et al. A simple and general method for transferring genes into plants [J].Science, 1985,227:1229-1231
7.Zhou GY, et al. Introduction of exogenous DNA into cotton embryos [J].Meth. Enzymol., 1983, 101:433-448
8．丁群新、谢友菊、戴景瑞，等：用子房注射法将Bt毒蛋白基因导入玉米的研究[J]。中国科学(B)，1993，23(7)：707—713
9．周兆斓、朱祯：植物抗虫基因工程研究进展，生物工程进展，1994，Vol．14，N．4：18—24；
10．张松、魏毓棠：植物抗虫基因工程研究进展，沈阳农业大学学报，1997—10，28(4)：332—337；
11．文学、张宝红：转基因抗虫棉研究现状与展望，农业生物技术学报，2000，8(2)：194—199；
12.Schuler TH, Poppy GM, Kerry BR, et al. Insect-resistant transgenic plants. TIBTECH, 1998,16:168-175
13.Peferoen M, progress and prospects for field use of Bt genes in crops. TIBTECH, 1997,15:17377
14.Benedict JH, Sachs ES, Altman DW, et al. Field performance of cottons expressing transgenic CryIA insecticidal proteins for resistance to Heliothis virescens and Helicoverpa zea (Lepido ptera:Noctuidae).J Econ Entomolo, 1996,89:230-238
15.Parlak FJ, Deaton RW, Armstrong TA, et al. Insect resistant cotton plants, Bio/technology, 1990,8:93943
16.Jenkins JN, Mccarty JC, Buehler RE, et al. Resistance of cotton with δ-endotoxin genes from Bacillus thuringiensis var. kurstaki on selected Lepidopteran insects. Agronomy Journal. 1997,89:768-780
17.Jenkins JN, Parrott WC, Mccarty JC, et al. Field test of transgenic cotton containing a Bacillus thuringfensJs gene. Miss Agric For Exp Stn Tech Bull, 174
18.Per FJ, Fuchs DA, Dean SL, et al. Modification of the coding sequence enhances plant expression of insect control protein genes. Proc Natl Acad Sci USA. 1991,88:3324-3328
19.Sachs ES, Benedict JH, Stelly DM, et al. Expression and segregation of genes encoding CryIA insecticidal protein in cotton. Crop Science, 1998(1):11-20
20.Cousins YL, Lyon BR, Llewellgn DJ, Transformation of an Australian cotton cultivar:prospects for cotton improvement through genetic engineering. Australian Journal of Plant Physiology, 1991, 18:481-494
21.Constable GA, Fitt GP. Transgenic cotton in the field and marketplace. In: Larkin PJ(ed.) Agricultural Biotechnology. Canberra, UTC Publishing, 1998:402-405
22．倪万潮、张震林、郭三堆：转墓因抗虫棉的培育，中国农业科学，1998，31(2)：83
23．谢道昕，范六云、倪万潮，等：苏云金芽孢杆菌杀虫蛋白基因导入棉花获得转基因植株，中国科学(B)，1991，(4)：367-373
24.Wang W, Zhu Z, Deng CY, et al. Obtaining of pest resistant cotton by transforming mediated Agrobacterium. New Frontiers in Cotton Research, Word Cotton Research conference-2. Athens, Greece, 1998:119
25．李燕娥、朱帧、陈志贤，等，豇豆胰蛋白酶抑制剂转基因棉花的获得，棉花学报，1998，10(5)：237—243
26.Thomas JC, Adams DG, Keppenne VD. Et al. Protease inhibitors of Manduce Sexta expressed in transgenic cotton. Plant Cell Reporters.1995.14:758-762
27.Cleveland TE, et al. Molecular characterization of a wound-inducible inhibitor I gene from potato and processing of its mRNA and protein, Plant Mol, Biol.,1987,8:199-207
28.Hussing JE, et al. Rice and stinging nettle lectins:insecticidal activity similar to wheat germ agglutinin. Phytochem, 1991,30(11):3565-3568
29．喻子牛：苏云金杆菌，科学出版社，1990；
30．郭三堆：植物Bt抗虫基因工程研究进展，中国农业科学，1995，28(5)：8-13；
31．林良斌、官春云：Bt毒蛋白基因与植物抗虫基因工程，生物工程进展，1997，Vol．17，N．2：51—55；
32．吴刚、崔海瑞、舒庆尧，等：Bt 杀虫晶体蛋白墓因及其转基因育种研究进展，生物工程进展，2000，Vol．20，NO．2：45-48；
33．严建民：作物转Bt抗虫基因育种研究进展，核农学报，2000，14(3)：184—188
34．罗志军：Bt毒蛋白基因工程进展，重庆师专学报，2000，19(2)：89—91
35．赵久然、郭景伦、腾海涛，等：玉米转基因研究进展，玉米科学，2000，8(3)：14-17
36．孙万仓：转基因油菜研究进展，西北农业学报，2000，9(3)：117—121
37．施爱民：我国转基因抗虫棉研究利用现状与展望，江西棉花，2000，20(5)：3-6
38．尚泓泉、刘建明、景丽：转Bt基因抗虫棉研究利用现状及其展望，河南农业科学，2000(7)：11-13
39．聂练兵：转基因作物及其检测技术与安全性评价问题，种子科技，2001(3)：152-154
40.Linn F, Heidmann L, Saedler H, et al. Epigenetic xhanges in the expression of the mainze A1 gene in Petinia hybrida: role of number intergrated genes copies and state of methylation[J]. Mol Gen Genet, 1990, (222):329-336
41.Assaad F F, Tucker K L, Singer E R. Epignetic repeat-induced gene silencing(RIGS) in ArabidopsJs[J].Plant Mol Bio1,1993, (22):1067-1085
42.Carolyn N, Lemieux C, Jorgensen R. Itroduction of a chimetic chalcone synthase gene into petunia results in reversible cosuppression of homologous genes in trans[J],Plant Cell,1990, (23):279-289
43.Jacobs John JMR, Litiere K, Van Dijk V, et al. Post-transcriptional β-1,3-glucanase gene silencing involves increased transcript turnover that is translation-independent [J]. Plant J, 1997,12(4):885-893
44．陈松、吴敬音、何小兰等：转基因抗虫棉组织中Bt毒蛋白表达量的ELISA测定，江苏农业学报，1997，13(3)：154-156；
45.CHEN Song, WU Jing-yin, CHENG De-rong et. al:On the Enzyme-linked Immunosorbent Assay of Bacillus thuringiensis Insecticidal Protein Expressed in Transgenic Cotton, 棉花学报，1999, 11 (5): 259-267
46．陈松、吴敬音、周宝良，等：转基因抗虫棉Bt毒蛋白表达量的时空变化，棉花学报，2000，12(4)：189—193；
47．张小四、李松岗、许崇任等：转Bt棉不同生长期及不同器官杀虫蛋白表达量的免疫学方法测定，北京大学学报(自然科学版)，Vol．36，No．4：477-484；
48.WANG Bao-min, He Zhong-pei, TIAN Xiao-li:Preparation of Monoclonal Antibodies of CryIA Insecticidal Crystal Protein and Its Application in Determining Toxin Level in Bt Cotton, 棉花学报，2000, 12(1): 34-39;
49．方中达：植病研究方法(第三版)，中国农业出版社，1998：289-290
50．王保民、何钟佩、赵继勋：抗虫棉Bt杀虫晶体蛋白免疫检测方法的研究，棉花学报，1998，10(4)：220-221
51．陈松、吴敬音、沈新莲等：苏云金杆菌HD-73杀虫蛋白的纯化及其多克隆抗体的制备，江苏农业学报，1999，15(4)：203—205
52．李萍、刘丰茂：苏云金杆菌(B.t.)晶体毒素的分离与纯化，农药，1996，Vol(35)，No．12：13-16
53．左雅慧、丁之铨、张杰：苏云金芽孢杆菌培养条件及晶体蛋白提纯方法初探，植物保护，25(4)：32-34
54．沈法富、于元杰、王留明等：利用Dot-ELISA检测Bt棉杀虫蛋白的研究，中国农业科学，1993，32(1)：15—19；
55．鲁子贤：蛋白质和酶学研究方法(第一册)，科学出版社，1989：1-7
56．袁玉荪、朱婉华、陈均辉：生物化学实验，高等教育出版社，1988：96—99
57．朱家柽、奚国良、易庆成等编译：使用蛋白质化学技术，1982：81-84
58．王世中：免疫化学技术，科学出版社，1980：158-161
59.Stephen A. Hill, Methods in plant Virology, 1984, Blackwell Scientific publication,101-105
60．陈建新、陈梅英、赵会杰：免疫学技术在植物科学中的应用，中国农业大学出版社，1998：91—94
61.R.H. Burdon, P.H. van Knippenbery, Monoclonal antibody technology, 1984, Elsevier science publication company, INC, New York, USA
62．荣廷昭等，田间试验与统计分析，四川大学出版社，2001
63.Hofte, H. and Whiteley, H.R. Microbiol. Rev. 1989(53),242-255
64.Feitelson J S, Payne J, Kim L, Bacillus thuringiensis: Insects and Beyond. Bio/Technology, 1992,10:271-275
65．李萍、刘丰茂、钱传范：苏云金杆菌(B.t.)的分析测定技术，农药科学与管理，1998，3：6—8；
66．[美]J.萨姆布鲁克，E.F.弗里奇，T.曼尼阿蒂斯：分子克隆实验指南，第二版(中译本)：852-855
67．M.W.斯图尔德(著)、张世馥(译)：抗体的结构与功能，科学出版社，1987：7-9；
68．梁训生、张成良、张作芳：植物病毒血清学技术，农业出版社，1985：57—63
69．裘维蕃：植物病毒学(修订版)，农业出版社，1984：273—275
70．徐宜为：实验免疫学技术，科学出版社，1979：165—182；
2．束春娥、柏立新、孙以文，等：转基因抗虫棉的抗性鉴定，[J]，江苏农业学报，1997，13(1)：22-26
3．高丽洁、薛亚杰、杨正书，等：转Bt基因抗虫棉的研究进展，沈阳农业大学学报，2000-12，31(6)：600-603
4.Fromm ME, Taylor Lp, Walbot V. Stable transformation of maize after gene transfer by electroporation [J]. Nature, 1986,319:791-793
5.Klein TM, Wolf ED, Wu R, et al. High-velocity microjectiles for delivering nuleic acids into living cells [J].Nature, 1987,327:70-73
6.Horsch RB, Fry JE, Hotfman NL, et al. A simple and general method for transferring genes into plants [J].Science, 1985,227:1229-1231
7.Zhou GY, et al. Introduction of exogenous DNA into cotton embryos [J].Meth. Enzymol., 1983, 101:433-448
8．丁群新、谢友菊、戴景瑞，等：用子房注射法将Bt毒蛋白基因导入玉米的研究[J]。中国科学(B)，1993，23(7)：707—713
9．周兆斓、朱祯：植物抗虫基因工程研究进展，生物工程进展，1994，Vol．14，N．4：18—24；
10．张松、魏毓棠：植物抗虫基因工程研究进展，沈阳农业大学学报，1997—10，28(4)：332—337；
11．文学、张宝红：转基因抗虫棉研究现状与展望，农业生物技术学报，2000，8(2)：194—199；
12.Schuler TH, Poppy GM, Kerry BR, et al. Insect-resistant transgenic plants. TIBTECH, 1998,16:168-175
13.Peferoen M, progress and prospects for field use of Bt genes in crops. TIBTECH, 1997,15:17377
14.Benedict JH, Sachs ES, Altman DW, et al. Field performance of cottons expressing transgenic CryIA insecticidal proteins for resistance to Heliothis virescens and Helicoverpa zea (Lepido ptera:Noctuidae).J Econ Entomolo, 1996,89:230-238
15.Parlak FJ, Deaton RW, Armstrong TA, et al. Insect resistant cotton plants, Bio/technology, 1990,8:93943
16.Jenkins JN, Mccarty JC, Buehler RE, et al. Resistance of cotton with δ-endotoxin genes from Bacillus thuringiensis var. kurstaki on selected Lepidopteran insects. Agronomy Journal. 1997,89:768-780
17.Jenkins JN, Parrott WC, Mccarty JC, et al. Field test of transgenic cotton containing a Bacillus thuringfensJs gene. Miss Agric For Exp Stn Tech Bull, 174
18.Per FJ, Fuchs DA, Dean SL, et al. Modification of the coding sequence enhances plant expression of insect control protein genes. Proc Natl Acad Sci USA. 1991,88:3324-3328
19.Sachs ES, Benedict JH, Stelly DM, et al. Expression and segregation of genes encoding CryIA insecticidal protein in cotton. Crop Science, 1998(1):11-20
20.Cousins YL, Lyon BR, Llewellgn DJ, Transformation of an Australian cotton cultivar:prospects for cotton improvement through genetic engineering. Australian Journal of Plant Physiology, 1991, 18:481-494
21.Constable GA, Fitt GP. Transgenic cotton in the field and marketplace. In: Larkin PJ(ed.) Agricultural Biotechnology. Canberra, UTC Publishing, 1998:402-405
22．倪万潮、张震林、郭三堆：转墓因抗虫棉的培育，中国农业科学，1998，31(2)：83
23．谢道昕，范六云、倪万潮，等：苏云金芽孢杆菌杀虫蛋白基因导入棉花获得转基因植株，中国科学(B)，1991，(4)：367-373
24.Wang W, Zhu Z, Deng CY, et al. Obtaining of pest resistant cotton by transforming mediated Agrobacterium. New Frontiers in Cotton Research, Word Cotton Research conference-2. Athens, Greece, 1998:119
25．李燕娥、朱帧、陈志贤，等，豇豆胰蛋白酶抑制剂转基因棉花的获得，棉花学报，1998，10(5)：237—243
26.Thomas JC, Adams DG, Keppenne VD. Et al. Protease inhibitors of Manduce Sexta expressed in transgenic cotton. Plant Cell Reporters.1995.14:758-762
27.Cleveland TE, et al. Molecular characterization of a wound-inducible inhibitor I gene from potato and processing of its mRNA and protein, Plant Mol, Biol.,1987,8:199-207
28.Hussing JE, et al. Rice and stinging nettle lectins:insecticidal activity similar to wheat germ agglutinin. Phytochem, 1991,30(11):3565-3568
29．喻子牛：苏云金杆菌，科学出版社，1990；
30．郭三堆：植物Bt抗虫基因工程研究进展，中国农业科学，1995，28(5)：8-13；
31．林良斌、官春云：Bt毒蛋白基因与植物抗虫基因工程，生物工程进展，1997，Vol．17，N．2：51—55；
32．吴刚、崔海瑞、舒庆尧，等：Bt 杀虫晶体蛋白墓因及其转基因育种研究进展，生物工程进展，2000，Vol．20，NO．2：45-48；
33．严建民：作物转Bt抗虫基因育种研究进展，核农学报，2000，14(3)：184—188
34．罗志军：Bt毒蛋白基因工程进展，重庆师专学报，2000，19(2)：89—91
35．赵久然、郭景伦、腾海涛，等：玉米转基因研究进展，玉米科学，2000，8(3)：14-17
36．孙万仓：转基因油菜研究进展，西北农业学报，2000，9(3)：117—121
37．施爱民：我国转基因抗虫棉研究利用现状与展望，江西棉花，2000，20(5)：3-6
38．尚泓泉、刘建明、景丽：转Bt基因抗虫棉研究利用现状及其展望，河南农业科学，2000(7)：11-13
39．聂练兵：转基因作物及其检测技术与安全性评价问题，种子科技，2001(3)：152-154
40.Linn F, Heidmann L, Saedler H, et al. Epigenetic xhanges in the expression of the mainze A1 gene in Petinia hybrida: role of number intergrated genes copies and state of methylation[J]. Mol Gen Genet, 1990, (222):329-336
41.Assaad F F, Tucker K L, Singer E R. Epignetic repeat-induced gene silencing(RIGS) in ArabidopsJs[J].Plant Mol Bio1,1993, (22):1067-1085
42.Carolyn N, Lemieux C, Jorgensen R. Itroduction of a chimetic chalcone synthase gene into petunia results in reversible cosuppression of homologous genes in trans[J],Plant Cell,1990, (23):279-289
43.Jacobs John JMR, Litiere K, Van Dijk V, et al. Post-transcriptional β-1,3-glucanase gene silencing involves increased transcript turnover that is translation-independent [J]. Plant J, 1997,12(4):885-893
44．陈松、吴敬音、何小兰等：转基因抗虫棉组织中Bt毒蛋白表达量的ELISA测定，江苏农业学报，1997，13(3)：154-156；
45.CHEN Song, WU Jing-yin, CHENG De-rong et. al:On the Enzyme-linked Immunosorbent Assay of Bacillus thuringiensis Insecticidal Protein Expressed in Transgenic Cotton, 棉花学报，1999, 11 (5): 259-267
46．陈松、吴敬音、周宝良，等：转基因抗虫棉Bt毒蛋白表达量的时空变化，棉花学报，2000，12(4)：189—193；
47．张小四、李松岗、许崇任等：转Bt棉不同生长期及不同器官杀虫蛋白表达量的免疫学方法测定，北京大学学报(自然科学版)，Vol．36，No．4：477-484；
48.WANG Bao-min, He Zhong-pei, TIAN Xiao-li:Preparation of Monoclonal Antibodies of CryIA Insecticidal Crystal Protein and Its Application in Determining Toxin Level in Bt Cotton, 棉花学报，2000, 12(1): 34-39;
49．方中达：植病研究方法(第三版)，中国农业出版社，1998：289-290
50．王保民、何钟佩、赵继勋：抗虫棉Bt杀虫晶体蛋白免疫检测方法的研究，棉花学报，1998，10(4)：220-221
51．陈松、吴敬音、沈新莲等：苏云金杆菌HD-73杀虫蛋白的纯化及其多克隆抗体的制备，江苏农业学报，1999，15(4)：203—205
52．李萍、刘丰茂：苏云金杆菌(B.t.)晶体毒素的分离与纯化，农药，1996，Vol(35)，No．12：13-16
53．左雅慧、丁之铨、张杰：苏云金芽孢杆菌培养条件及晶体蛋白提纯方法初探，植物保护，25(4)：32-34
54．沈法富、于元杰、王留明等：利用Dot-ELISA检测Bt棉杀虫蛋白的研究，中国农业科学，1993，32(1)：15—19；
55．鲁子贤：蛋白质和酶学研究方法(第一册)，科学出版社，1989：1-7
56．袁玉荪、朱婉华、陈均辉：生物化学实验，高等教育出版社，1988：96—99
57．朱家柽、奚国良、易庆成等编译：使用蛋白质化学技术，1982：81-84
58．王世中：免疫化学技术，科学出版社，1980：158-161
59.Stephen A. Hill, Methods in plant Virology, 1984, Blackwell Scientific publication,101-105
60．陈建新、陈梅英、赵会杰：免疫学技术在植物科学中的应用，中国农业大学出版社，1998：91—94
61.R.H. Burdon, P.H. van Knippenbery, Monoclonal antibody technology, 1984, Elsevier science publication company, INC, New York, USA
62．荣廷昭等，田间试验与统计分析，四川大学出版社，2001
63.Hofte, H. and Whiteley, H.R. Microbiol. Rev. 1989(53),242-255
64.Feitelson J S, Payne J, Kim L, Bacillus thuringiensis: Insects and Beyond. Bio/Technology, 1992,10:271-275
65．李萍、刘丰茂、钱传范：苏云金杆菌(B.t.)的分析测定技术，农药科学与管理，1998，3：6—8；
66．[美]J.萨姆布鲁克，E.F.弗里奇，T.曼尼阿蒂斯：分子克隆实验指南，第二版(中译本)：852-855
67．M.W.斯图尔德(著)、张世馥(译)：抗体的结构与功能，科学出版社，1987：7-9；
68．梁训生、张成良、张作芳：植物病毒血清学技术，农业出版社，1985：57—63
69．裘维蕃：植物病毒学(修订版)，农业出版社，1984：273—275
70．徐宜为：实验免疫学技术，科学出版社，1979：165—182；