抗虫基因载体的构建及白菜转基因研究
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摘要
蜘蛛杀虫蛋白(Spider insecticidal protein,SIP)基因可编码一种只有37个氨基酸的小肽，该多肽能杀死多种有害昆虫。质粒pBBBast-PinⅡ含有马铃薯蛋白酶抑制剂基因PinⅡ(Potato proteinase inhibitor Ⅱ)，PinⅡ基因编码的蛋白酶抑制剂具有广谱抗虫性。实验以蜘蛛杀虫肽基因为目的基因，将其转入质粒pBBBast-PinⅡ中，从而构建含有PinⅡ和SIP两个抗虫基因的双价植物表达载体——pBBBast-PinⅡ-SIP。同时，还将SIP基因转入pBBBast中，构建单价抗虫载体pBBBast-SIP。经过限制性内切酶酶切以及PCR目的片段扩增证明两个载体构建正确。
通过真空渗入和花序浸渍法，将上述双抗和单抗基因分别导入白菜不结球类型——49菜心中，获得了7棵抗性株。通过对标记性状——除草剂抗性的活体及离体检验(氯酚红法)、目的基因PCR扩增、PCR-Southern杂交等方法证实目的基因已整合到小白菜基因组中。其中2棵抗性株自交后代的遗传分析表明，真空渗入法所得的转化株为杂合体，且其分离比符合3：1的单基因分离规律。
对小白菜真空渗入转化法的转化条件进行了初步探索。结果表明植株生长阶段和真空渗入处理时间，所用菌株类型均对转化率有影响。其中以开花初期的植株，利用胭脂碱型农杆菌介导(如C58)，真空渗入5分钟的处理方法转化效率最高。
Spider insecticidal protein (SIP) gene can encode a small polypeptide containing 37 amino acids. The polypeptide has strong toxicity to many insects. Plasmid pBBBast-PinII has the Potato proteinase inhibitor II (Pin II ) gene which encodes potato proteinase inhibitor. The SIP gene was digested by restriction enzyme Hindlll and inserted into the vector pBBBast-Pin II . So a plant expression vector with two insect-resistant genes -pBBBast-Pin II -SIP was constructed. A plant expression vector - pBBBast-SIP with one insect-resistant gene was also constructed by inserting the SIP gene into the plasmid pBBBast. Restriction enzyme digestion analysis and PCR analysis showed that these two plant expression vectors constructed were correct.
49Caixin, one cultivar of the Brassica campestris L. ssp. chinensis , was transformed by vacuum infiltration method. 7 herbicide resistant plants were obtained. PCR, PCR-Southern blotting analysis showed that the target genes were integrated into the plant genome. The segregation ratio in T1 of two examed transformants was 3:1. It revealed that these two transformants were heterozygote, and the foreign gene inserted just to on locus in the plant genome. As the observed ratio of progeny of two transformants which on selfing was 3:1, it was proved to be heterozygote for two transformants.
Several factors influencing the vacuum infiltration transformation efficiency in Brassica campestris L. ssp. chinensis were also studied. The developmental status of seedlings, vacuum infiltration time, type of Agrobacterium tumefaciens used, all influence the transformation frequency. It was concluded that using the nopaline type of Agrobacterium (e.g. C58), 5min vacuum infiltration, and using the plants with just several blooming flowers would give the best results.
通过真空渗入和花序浸渍法，将上述双抗和单抗基因分别导入白菜不结球类型——49菜心中，获得了7棵抗性株。通过对标记性状——除草剂抗性的活体及离体检验(氯酚红法)、目的基因PCR扩增、PCR-Southern杂交等方法证实目的基因已整合到小白菜基因组中。其中2棵抗性株自交后代的遗传分析表明，真空渗入法所得的转化株为杂合体，且其分离比符合3：1的单基因分离规律。
对小白菜真空渗入转化法的转化条件进行了初步探索。结果表明植株生长阶段和真空渗入处理时间，所用菌株类型均对转化率有影响。其中以开花初期的植株，利用胭脂碱型农杆菌介导(如C58)，真空渗入5分钟的处理方法转化效率最高。
Spider insecticidal protein (SIP) gene can encode a small polypeptide containing 37 amino acids. The polypeptide has strong toxicity to many insects. Plasmid pBBBast-PinII has the Potato proteinase inhibitor II (Pin II ) gene which encodes potato proteinase inhibitor. The SIP gene was digested by restriction enzyme Hindlll and inserted into the vector pBBBast-Pin II . So a plant expression vector with two insect-resistant genes -pBBBast-Pin II -SIP was constructed. A plant expression vector - pBBBast-SIP with one insect-resistant gene was also constructed by inserting the SIP gene into the plasmid pBBBast. Restriction enzyme digestion analysis and PCR analysis showed that these two plant expression vectors constructed were correct.
49Caixin, one cultivar of the Brassica campestris L. ssp. chinensis , was transformed by vacuum infiltration method. 7 herbicide resistant plants were obtained. PCR, PCR-Southern blotting analysis showed that the target genes were integrated into the plant genome. The segregation ratio in T1 of two examed transformants was 3:1. It revealed that these two transformants were heterozygote, and the foreign gene inserted just to on locus in the plant genome. As the observed ratio of progeny of two transformants which on selfing was 3:1, it was proved to be heterozygote for two transformants.
Several factors influencing the vacuum infiltration transformation efficiency in Brassica campestris L. ssp. chinensis were also studied. The developmental status of seedlings, vacuum infiltration time, type of Agrobacterium tumefaciens used, all influence the transformation frequency. It was concluded that using the nopaline type of Agrobacterium (e.g. C58), 5min vacuum infiltration, and using the plants with just several blooming flowers would give the best results.
引文
1. Bechtold et al, 1993, In planta Agrobectrium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. CR Acad Sci Paris Life Sci, 316:1194-1199
2. Binns AN, 1990, Plant Physiology, 79:135-139
3. Birch RG, 1997, Plant transformaiton:problems and strategies for practical application. Annu Rev Plant Physiol Plant Mol Biol, 48:297-326
4. Bowman J, 1994, Arabidopsis:An atlas of morphology and development. Springer-Verlag, New York.
5. Bufter P, Stiegler G. 1995, Soluble lipoplysaccharide (LPS) receptor CD14 is released via two different mechanisms from human monocyte and CD 14 transfections. Eur J Immunol, 25(3):604-610
6. Cao MQ, Liu F, Yao L et al. 2000, Transformation of Pakchoi (Brassica rapa L. ssp. chinensis) by Agrobacterium infiltration. Molecular Breeding, 6:67-72
7. Chang SS et al., 1994, Stable genetic transformation of Arabidopsis thaliana by Agorbacterium inoculation in planta. Plant J 5:551-558
8. Chee et al., 1995, Transformaion of soybean (Glycine max)via Agrobecterium inoculation and analysisof transformed plants. In KMA Gartland, MR Davey, eds, Agrobectrium Protocols: Methods in Molecular Biology, Vol 44. Humana Press, Totowa, Nj, 101-119
9. Chowrira GM et al., 1995, Eelectroporation-mediated gene transfer into intact nodal meristems in planta:generating transgenic plants without in vitro tissue culture. Mol Biotechnol, 3:17-23
10. Christey MC, Sinclair BK, Braun RH, et al, 1997, Regeneration of transgenic vegetable brassicas(Brassica oleracea and B. campestris)via Ri-mediated transformation. Plant Cell Rep 16:587-593.
11．Clark M S．植物分子生物学——实验手册 高等教育出版社，施普林格出版社 1998 3-6．
12. Clough SJ et al., 1998, Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735-743
13. Desfeux C et al., 2000, Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method. Plant Physiol 123:895-904
14. Dunwell J M. 1981, Invitro regeneration from excised leaf discs of three Brassica species. J EXP Bot, 32:789-799
15. Feldmann KA, Marks MD, 1987, Agrobectrium-mediated transformation of germinating seeds of Arabidopsis thaliana: a non-tissue culture approach. Mol Gen Genet 208:1-9
16. Feldmann, 1991, Prospects for plant genome modification by nonconventional methods. Annu Rev Genet. 11: 79-101
17. Golz C et al., 1988, Progress in Plant Protoplast Research, p.353.
18. Golz C et al., 1990, Molecular Biology. 15:475.
19. Guerche P et al., 1987, Genetic transformation of oilseed rape (Brassica napus) by the Ri-T-DAN of Agrobacterum rhizogenes and analysis of the transformed phenotype. Mol. Gen. Genet, 206:382.
20. Helena Mathews, N. Bharathan 1990, Transgenic plants of mustard Brassica juncea (L.) Czern and Coss. Plant Science 72:245-252
21. Helena Mathews, N. Bharathan. 1990, Transgenic plants of mustard Brassica juncea (L.) Czern and Coss Plant Science 72: 245-252
22. Hess D. 1969, Versuche zur transformation a hoheren Pflanzen:Wiederholung der Anthocyan-Induktion bei petunia und erste Charakterisierung des transformierenden prinzips. Z. Pflanzenphysiol 61:286-298
23. Hu C-Y et al, 1999, In planta transformation etchnologies developed in China: procedure, confirmation and field performance. In Vitro Cell Dev Biol-Plant 35:417-420
24. I. J. Puddephat, T. J. Riggs and T. M. Fenning, 1996, Transformation of Brassica oleracea L:a crotoca; review Molecular Breeding 2:185-210,
25．J. Sambrook E. P. Fritsch T. Maniatis分子克隆实验指南(第二版) 1992 科学出版社
26. Jacq B, Le sobre, Sangwan RS et al. 1993, Factors influencing T-DNA transfer in Agrobacterium-mediated Transformation of sugarbeet. Plant Cell Reports 12:621-624.
27. Joao K, Brown T, 1993, Enhanced transformation of tomato co-cultivated with Agrobacterium tumefaciens C58CIRif: pGSFR1161 in the presence of acetosyringone. Plant Cell Reports 12: 422-425.
28. Jun SI, Kwon SY, Paek KY et al. 1995, Agrobacterium-mediated transformation and regeneration of fertile transgenic plants of Chinese Cabbage (Brassica campestfis ssp.pekinensis cv. spring flavor). Plant Cell Reports 14: 620-625.
29. Katavic V et al., 1994, In planta transformation of Arabidopsis thaliana. Mol Gen Genet 245:363-370
30. Katavic V, Haugghn G. W., Reed D et al. 1994, In planta teansformation of Arabidopsis thaliana. Mol. Gen. Genet. 245, 363-370.
31. Kohler Fet al., 1989, Plan Molecular Biology, 12:189.
32. Kuvshinov V, Koivu K, Kmerva A et al. 1999, Agrobacterium tumefaciens-mediated Transformation of greenhouse-grown Brassica rapa ssp. oleifera. Plant Cell Reports 18: 773-777.
33. Lim HT, You YS, Park EJ et al. 1997, High Plant Regeneration, Genetic Stability of Regenerants, and Genetic Transformation of Herbicide resistance Gene (bar) in Chinese Cabbage (Brassica campestris ssp. pekinensis). Proceeding of the International Symposium on Brassicas p199-208.
34. Liu F et al., 1998, In planta transformation of pakchoi (Brassica campestris L. ssp. Chinensis) by infiltration of adult plants with Agrobacterium. Acta Hort 467:187-192
35. Marc De Block. 1990, Factors Influencing the Tissue Culture and the Agrobacterium tumefaciens-Mediated Transformation of Hybird Aspen and Poplar Clones Plant Physiol. 93:1110-1116
36. Marc De Block, Dirk De brouwer. 1989, Transformation of Brassica napus and
Brassica oleracea Using Agrobacterium tumefaciens and the Expression of the bar and neo Genes in the Transgenic Plants Plant Physiol. 91:694-701
37. Mengiste T et al., 1997, High-efficiency transformation of Arabidopsis thaliana with a selectable marker gene regulated by the T-DNA 1' promoter. Plant J. 12:945-948
38. Ming C et al, 1997, Plant Physiology, 115:971-980
39. Mukhopadhyay A, Arumugam N, Nandakumar PBA, et al, 1992, Agrobacterium-mediated genetic Transformation of oilseed Brassica campestris: Transformation frequency is strongly influenced by the mode of shoot regeneration. Plant Cell Reports 11: 506-513.
40. Murata M et al., 1987, Callus initiation and regeneration capacities in Brassica species. Plant Cell Tiss Org Cult 11:111-123
41. Neuhaus Get al., 1987, Transgenic rape seed plants obtained by the microinjection of DNA into microspore-derived embryoids. Theor Appl Genet.75:30.
42. Paszkawski J, Pisan B, Shilito R D Plant molecular Biology, 1986, 6:303
43. Paul J J et al, 1992, Plant Molecular Biology. 19:15-38.
44. Pierre J. Charest, V. N. Iyer. 1989, Virulence of Agrobacterium tumefaciens strains with Brassica napus and Brassicajuncea Plant Cell Reports 8:303-306
45. Rakousk S, Kocbek T, Vincenciov R, et al., 1997/98, Transient β-glucuronidase activity after infiltration of Arabidopsis thaliana by agrobacteium tumefaciens. Biologia Plantarum (40)1:33-41
46. Richardson K et al., 1998, T-DNA tagging of a flowering-time gene and improved gene transfer by in planta transformation of Arabidopsis. Aust. J. Plant Physiol. 25:125-130
47. Se Ⅱ Jun, Seok Yoon Kwon. 1995, Agrobacterium-mediated transformation and regeneration of fertile transgenic plants of Chinese cabbage (brassica campestris ssp. pekinensis cv. 'spring flavor') Plant Cell Reports 14:620-625
48. Steven. J. Cloigh and Anerew F, Bent. 1998, Floral dip: a simplified methods for Agrobacterium-mediated transformation of Arabidopsis thaliana The Plant Journal 16(6), 745-743
49. Stroun M, Anker P, Charles P, Ledoux L, 1966, Fate of bacterial deoxyribonucleic acid in Lycopersicon esculentum. Nature 121:397-398.
50. Takasaki T, et al., 1997, Factors influencing Agrobacterium-mediated transformation of Brassica rapa L. Breeding Science 47:127-134
51. Trieu AT et al., 2000, Transformation of Medicago tmncatula via infiltration of seedlings or flowering plants with Agrotacterium. Plant J 22:531-541
52. Vaughan A. Hilder, Angharad M. R. Gatehouse. 1987, A novel mechanism of insect resistance engineered into tobacco Nature Vol, 300 12 November
53. Vernade D, Herrera-Estrlla A, Wang K et al. 1988, Glycine betaine allows enhanced induction of the Agrobacterium Tumefaciens vir genes by As at low pH. J. Bacteriol 170: 5822-29.
54. Ye GN et al., 1999, Arabidopsis ovule is the target for Agrobacterium in planta vacuum infiltration transformation. Plant J 19:249-257
55. Zhang F. L. Y. Takahata J. B. Xu Medium and genotype factors influencing shoot regeneration from cotyledonary explants of Chinese cabbage(Brassica campestris
L. ssp. pekinensis) Plant Cell Reports(1998)17: 780～786
56. Zhang FL et al., 2000, Agrobacterium-mediated transformation of cotylydonary explants of Chinese cabbage(Brassica campestris L. ssp．pekinensis). Plant Cell Rep. 19: 569-575
57. Zhou GY, Weng J, Zeng Y, et al., 1983, Introduction of exogenous DNA into cotton embryos. Methods Enzymol. 101: 433-434
58．傅荣昭，孙勇如，贾士荣主编，1994，《植物遗传转化技术手册》北京：中国科学技术出版社
59．巩振辉 Milner J J 何玉科，1996，拟南芥基因转移新方法—真空渗入法的研究 西北植物学报 16(3)：277-283
60．候丙凯 陈正华，2000，植物抗虫基因工程研究进展 植物学通报 17 (5)：385-393
61．蒋红 朱玉贤 陈章良，1996，导入蜘蛛杀虫肽基因的烟草具有抗虫性 植物学报 38 (2)：95-99
62．刘凡，姚磊，李岩，曹鸣庆 1998，利用结球白菜小孢子胚状体获得抗除草剂转基因植株。华北农学报，13 (4)：93-98
63．卢圣栋 现代分子生物学实验技术(第二版) 1999中国协和医科大学出版社
64．毛立群 郭三堆，昆虫神经毒素基因工程研究进展 生物技术通报 1998，6
65．王从丽等，2002，农杆菌——植物间基因转移的分子基础。生命科学，14 (1)：1-5
66．王关林等，1996，遗传，18 (6)：37-41
67．王景雪 孙毅，农杆菌介导的植物基因转化研究进展 生物技术通报 1999年第一期
68．王凌健，倪迪安，王光运等 1999，青菜(Brassica campestris ssp．Chinensis L．)组织培养和转化体系的初步建立。实验生物学报，32 (1)：99。
69．许耀，贾敬芬，郑国倡等1988，酚类化合物促进根癌农杆菌对植物离体外植体的高效转化。科学通报，22：1745-48
70．印莉萍 刘祥林 分子细胞生物学实验技术 2001 首都师范大学出版社
71．余淑文，1992，《植物生理与分子生物学》北京：科学出版社，第一版，63-83
72．张宝红 1993，转基因植物研究进展。河南职技师院学报。21 (4)：26-31
73．张广辉，巩振辉，薛万新等 1998，大白菜和油菜真空渗入遗传转化法初报。西北农业大学学报，26 (4)
74．张桂华、巩振辉、张广辉 2000，农杆菌的芸苔属作物遗传转化研究进展 西北农业大学学报 28 (2)：80-84
75．王锡华 张峰 赵遵田 1999，我国转基因植物研究进展 山东科学 12 (2)：32-37
76．杨广东 朱桢 李燕娥 2002，大白菜转修饰豇豆胰蛋白酶抑制剂基因获得抗虫植株 园艺学报 29 (3)：224-228
77．佘建明 蔡小宁 朱卫民 2000，普通不结球白菜抗虫转基因植株的获得 江苏农业学报 16 (2)：79-82
2. Binns AN, 1990, Plant Physiology, 79:135-139
3. Birch RG, 1997, Plant transformaiton:problems and strategies for practical application. Annu Rev Plant Physiol Plant Mol Biol, 48:297-326
4. Bowman J, 1994, Arabidopsis:An atlas of morphology and development. Springer-Verlag, New York.
5. Bufter P, Stiegler G. 1995, Soluble lipoplysaccharide (LPS) receptor CD14 is released via two different mechanisms from human monocyte and CD 14 transfections. Eur J Immunol, 25(3):604-610
6. Cao MQ, Liu F, Yao L et al. 2000, Transformation of Pakchoi (Brassica rapa L. ssp. chinensis) by Agrobacterium infiltration. Molecular Breeding, 6:67-72
7. Chang SS et al., 1994, Stable genetic transformation of Arabidopsis thaliana by Agorbacterium inoculation in planta. Plant J 5:551-558
8. Chee et al., 1995, Transformaion of soybean (Glycine max)via Agrobecterium inoculation and analysisof transformed plants. In KMA Gartland, MR Davey, eds, Agrobectrium Protocols: Methods in Molecular Biology, Vol 44. Humana Press, Totowa, Nj, 101-119
9. Chowrira GM et al., 1995, Eelectroporation-mediated gene transfer into intact nodal meristems in planta:generating transgenic plants without in vitro tissue culture. Mol Biotechnol, 3:17-23
10. Christey MC, Sinclair BK, Braun RH, et al, 1997, Regeneration of transgenic vegetable brassicas(Brassica oleracea and B. campestris)via Ri-mediated transformation. Plant Cell Rep 16:587-593.
11．Clark M S．植物分子生物学——实验手册 高等教育出版社，施普林格出版社 1998 3-6．
12. Clough SJ et al., 1998, Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735-743
13. Desfeux C et al., 2000, Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method. Plant Physiol 123:895-904
14. Dunwell J M. 1981, Invitro regeneration from excised leaf discs of three Brassica species. J EXP Bot, 32:789-799
15. Feldmann KA, Marks MD, 1987, Agrobectrium-mediated transformation of germinating seeds of Arabidopsis thaliana: a non-tissue culture approach. Mol Gen Genet 208:1-9
16. Feldmann, 1991, Prospects for plant genome modification by nonconventional methods. Annu Rev Genet. 11: 79-101
17. Golz C et al., 1988, Progress in Plant Protoplast Research, p.353.
18. Golz C et al., 1990, Molecular Biology. 15:475.
19. Guerche P et al., 1987, Genetic transformation of oilseed rape (Brassica napus) by the Ri-T-DAN of Agrobacterum rhizogenes and analysis of the transformed phenotype. Mol. Gen. Genet, 206:382.
20. Helena Mathews, N. Bharathan 1990, Transgenic plants of mustard Brassica juncea (L.) Czern and Coss. Plant Science 72:245-252
21. Helena Mathews, N. Bharathan. 1990, Transgenic plants of mustard Brassica juncea (L.) Czern and Coss Plant Science 72: 245-252
22. Hess D. 1969, Versuche zur transformation a hoheren Pflanzen:Wiederholung der Anthocyan-Induktion bei petunia und erste Charakterisierung des transformierenden prinzips. Z. Pflanzenphysiol 61:286-298
23. Hu C-Y et al, 1999, In planta transformation etchnologies developed in China: procedure, confirmation and field performance. In Vitro Cell Dev Biol-Plant 35:417-420
24. I. J. Puddephat, T. J. Riggs and T. M. Fenning, 1996, Transformation of Brassica oleracea L:a crotoca; review Molecular Breeding 2:185-210,
25．J. Sambrook E. P. Fritsch T. Maniatis分子克隆实验指南(第二版) 1992 科学出版社
26. Jacq B, Le sobre, Sangwan RS et al. 1993, Factors influencing T-DNA transfer in Agrobacterium-mediated Transformation of sugarbeet. Plant Cell Reports 12:621-624.
27. Joao K, Brown T, 1993, Enhanced transformation of tomato co-cultivated with Agrobacterium tumefaciens C58CIRif: pGSFR1161 in the presence of acetosyringone. Plant Cell Reports 12: 422-425.
28. Jun SI, Kwon SY, Paek KY et al. 1995, Agrobacterium-mediated transformation and regeneration of fertile transgenic plants of Chinese Cabbage (Brassica campestfis ssp.pekinensis cv. spring flavor). Plant Cell Reports 14: 620-625.
29. Katavic V et al., 1994, In planta transformation of Arabidopsis thaliana. Mol Gen Genet 245:363-370
30. Katavic V, Haugghn G. W., Reed D et al. 1994, In planta teansformation of Arabidopsis thaliana. Mol. Gen. Genet. 245, 363-370.
31. Kohler Fet al., 1989, Plan Molecular Biology, 12:189.
32. Kuvshinov V, Koivu K, Kmerva A et al. 1999, Agrobacterium tumefaciens-mediated Transformation of greenhouse-grown Brassica rapa ssp. oleifera. Plant Cell Reports 18: 773-777.
33. Lim HT, You YS, Park EJ et al. 1997, High Plant Regeneration, Genetic Stability of Regenerants, and Genetic Transformation of Herbicide resistance Gene (bar) in Chinese Cabbage (Brassica campestris ssp. pekinensis). Proceeding of the International Symposium on Brassicas p199-208.
34. Liu F et al., 1998, In planta transformation of pakchoi (Brassica campestris L. ssp. Chinensis) by infiltration of adult plants with Agrobacterium. Acta Hort 467:187-192
35. Marc De Block. 1990, Factors Influencing the Tissue Culture and the Agrobacterium tumefaciens-Mediated Transformation of Hybird Aspen and Poplar Clones Plant Physiol. 93:1110-1116
36. Marc De Block, Dirk De brouwer. 1989, Transformation of Brassica napus and
Brassica oleracea Using Agrobacterium tumefaciens and the Expression of the bar and neo Genes in the Transgenic Plants Plant Physiol. 91:694-701
37. Mengiste T et al., 1997, High-efficiency transformation of Arabidopsis thaliana with a selectable marker gene regulated by the T-DNA 1' promoter. Plant J. 12:945-948
38. Ming C et al, 1997, Plant Physiology, 115:971-980
39. Mukhopadhyay A, Arumugam N, Nandakumar PBA, et al, 1992, Agrobacterium-mediated genetic Transformation of oilseed Brassica campestris: Transformation frequency is strongly influenced by the mode of shoot regeneration. Plant Cell Reports 11: 506-513.
40. Murata M et al., 1987, Callus initiation and regeneration capacities in Brassica species. Plant Cell Tiss Org Cult 11:111-123
41. Neuhaus Get al., 1987, Transgenic rape seed plants obtained by the microinjection of DNA into microspore-derived embryoids. Theor Appl Genet.75:30.
42. Paszkawski J, Pisan B, Shilito R D Plant molecular Biology, 1986, 6:303
43. Paul J J et al, 1992, Plant Molecular Biology. 19:15-38.
44. Pierre J. Charest, V. N. Iyer. 1989, Virulence of Agrobacterium tumefaciens strains with Brassica napus and Brassicajuncea Plant Cell Reports 8:303-306
45. Rakousk S, Kocbek T, Vincenciov R, et al., 1997/98, Transient β-glucuronidase activity after infiltration of Arabidopsis thaliana by agrobacteium tumefaciens. Biologia Plantarum (40)1:33-41
46. Richardson K et al., 1998, T-DNA tagging of a flowering-time gene and improved gene transfer by in planta transformation of Arabidopsis. Aust. J. Plant Physiol. 25:125-130
47. Se Ⅱ Jun, Seok Yoon Kwon. 1995, Agrobacterium-mediated transformation and regeneration of fertile transgenic plants of Chinese cabbage (brassica campestris ssp. pekinensis cv. 'spring flavor') Plant Cell Reports 14:620-625
48. Steven. J. Cloigh and Anerew F, Bent. 1998, Floral dip: a simplified methods for Agrobacterium-mediated transformation of Arabidopsis thaliana The Plant Journal 16(6), 745-743
49. Stroun M, Anker P, Charles P, Ledoux L, 1966, Fate of bacterial deoxyribonucleic acid in Lycopersicon esculentum. Nature 121:397-398.
50. Takasaki T, et al., 1997, Factors influencing Agrobacterium-mediated transformation of Brassica rapa L. Breeding Science 47:127-134
51. Trieu AT et al., 2000, Transformation of Medicago tmncatula via infiltration of seedlings or flowering plants with Agrotacterium. Plant J 22:531-541
52. Vaughan A. Hilder, Angharad M. R. Gatehouse. 1987, A novel mechanism of insect resistance engineered into tobacco Nature Vol, 300 12 November
53. Vernade D, Herrera-Estrlla A, Wang K et al. 1988, Glycine betaine allows enhanced induction of the Agrobacterium Tumefaciens vir genes by As at low pH. J. Bacteriol 170: 5822-29.
54. Ye GN et al., 1999, Arabidopsis ovule is the target for Agrobacterium in planta vacuum infiltration transformation. Plant J 19:249-257
55. Zhang F. L. Y. Takahata J. B. Xu Medium and genotype factors influencing shoot regeneration from cotyledonary explants of Chinese cabbage(Brassica campestris
L. ssp. pekinensis) Plant Cell Reports(1998)17: 780～786
56. Zhang FL et al., 2000, Agrobacterium-mediated transformation of cotylydonary explants of Chinese cabbage(Brassica campestris L. ssp．pekinensis). Plant Cell Rep. 19: 569-575
57. Zhou GY, Weng J, Zeng Y, et al., 1983, Introduction of exogenous DNA into cotton embryos. Methods Enzymol. 101: 433-434
58．傅荣昭，孙勇如，贾士荣主编，1994，《植物遗传转化技术手册》北京：中国科学技术出版社
59．巩振辉 Milner J J 何玉科，1996，拟南芥基因转移新方法—真空渗入法的研究 西北植物学报 16(3)：277-283
60．候丙凯 陈正华，2000，植物抗虫基因工程研究进展 植物学通报 17 (5)：385-393
61．蒋红 朱玉贤 陈章良，1996，导入蜘蛛杀虫肽基因的烟草具有抗虫性 植物学报 38 (2)：95-99
62．刘凡，姚磊，李岩，曹鸣庆 1998，利用结球白菜小孢子胚状体获得抗除草剂转基因植株。华北农学报，13 (4)：93-98
63．卢圣栋 现代分子生物学实验技术(第二版) 1999中国协和医科大学出版社
64．毛立群 郭三堆，昆虫神经毒素基因工程研究进展 生物技术通报 1998，6
65．王从丽等，2002，农杆菌——植物间基因转移的分子基础。生命科学，14 (1)：1-5
66．王关林等，1996，遗传，18 (6)：37-41
67．王景雪 孙毅，农杆菌介导的植物基因转化研究进展 生物技术通报 1999年第一期
68．王凌健，倪迪安，王光运等 1999，青菜(Brassica campestris ssp．Chinensis L．)组织培养和转化体系的初步建立。实验生物学报，32 (1)：99。
69．许耀，贾敬芬，郑国倡等1988，酚类化合物促进根癌农杆菌对植物离体外植体的高效转化。科学通报，22：1745-48
70．印莉萍 刘祥林 分子细胞生物学实验技术 2001 首都师范大学出版社
71．余淑文，1992，《植物生理与分子生物学》北京：科学出版社，第一版，63-83
72．张宝红 1993，转基因植物研究进展。河南职技师院学报。21 (4)：26-31
73．张广辉，巩振辉，薛万新等 1998，大白菜和油菜真空渗入遗传转化法初报。西北农业大学学报，26 (4)
74．张桂华、巩振辉、张广辉 2000，农杆菌的芸苔属作物遗传转化研究进展 西北农业大学学报 28 (2)：80-84
75．王锡华 张峰 赵遵田 1999，我国转基因植物研究进展 山东科学 12 (2)：32-37
76．杨广东 朱桢 李燕娥 2002，大白菜转修饰豇豆胰蛋白酶抑制剂基因获得抗虫植株 园艺学报 29 (3)：224-228
77．佘建明 蔡小宁 朱卫民 2000，普通不结球白菜抗虫转基因植株的获得 江苏农业学报 16 (2)：79-82