抗虫基因cry1C~*对早粳稻的转化
|
摘要
为了改良黑龙江水稻抗二化螟的遗传特性,采用农杆菌介导外源基因遗传转化的方法,以bar基因为筛选标记,将在ubi启动子调控下、经过密码子优化及DNA序列改造的、人工合成的cry1C~*基因,转入寒区优良水稻品种空育131核基因组中,获得了一批遗传转化苗。在实验过程中,对农杆菌菌株EHA105B(来自中国科学院遗传与发育生物学研究所植物发育研究室的农杆菌菌株EHA105)和EHA105N(来自东北林业大学教育部林木遗传育种与生物技术重点实验室的农杆菌菌株EHA105)的活力进行了比较,还对遗传转化水稻再生苗炼苗方法进行了优化。主要研究结果如下:
1.对水稻遗传转化载体pBar-cry1C~*进行酶切和PCR检测验证表明,载体和目的基因均准确无误,将携带目的基因cry1C~*基因的质粒pBar-cry1C~*电转化导入农杆菌,获得了农杆菌转化子EHA105(pBar-cry1C~*)。
2.根瘤农杆菌菌株EHA105B的活力比EHA105N强,EHA105B不能用于寒区水稻遗传转化工作,活力较弱的农杆菌菌株EHA105N可以介导外源基因转化北方粳稻。
3.黑龙江地区严冬季节产生的遗传转化再生水稻苗采用优化法炼苗,可以确保转基因再生水稻苗的成活率。优化法炼苗在组织培养室炼苗后,将转化苗置于水稻专用营养液中,在光照培养箱中25℃高湿条件下进行无土栽培3-4d,然后再移栽到土壤。
4.利用农杆菌介导的遗传转化方法,转化水稻空育131胚性愈伤1800块,共获来自20块独立抗性愈伤的分化再生苗123株,最终成活115株,成活率达到了93.5%。
5.对水稻空育131(cry1C~*)T_0代进行PCR分析,123株转化植株中阳性株120株,阳性率97.6%。cry1C~*基因已经整合到黑龙江水稻核基因组中。
Stemborers and leaffolders were two groups of Lepidopteran pests that caused severe damage to rice in many areas of the world. Kongyu131, being the most popular rice variety of Heilongjiang province, was highly susceptible to Lepidopteran insects, which greatly reduced the expected yield of Kongyu131. In this study, cry1C~* gene encoding Bacillus thuringiensis(Bt) d-endotoxin was synthesized based on codon optimization as one step toward gene stacking in the resistance management strategy of transgenic rice. The gene was transformed into Kongyu131 (Oryza sativa L.) via Agrobacterium-mediated method. The integration of the cry1C~* gene in rice genome was detected with PCR analyses. Meanwhile, the activity of EHA105B was compared to EHA105N and the optimized hardening method of regenerated plantlets in winter was improved in Heilongjiang province. The main results were as follows:
1. The vector pBar-cry1C~* was confirmed in E.coli DH5αwith restriction enzyme digestion and PCR detection.
2. The activity of the stain EHA105B of Agrobacterium tumefaciens was more powerful than the strain EHA105N and the EHA105N was suitable for the Agrobacterium-mediated transformation.
3. The optimized hardening method of regenerated plantlets elevated the living rate of transgenic plants in winter in Heilongjiang province.
4. 123 plants of the rice Kongyu131 transformed with cry1C~* gene from 20 independent resistant calli were regenerated and 115 living transgenic plants were obtained after optimized hardening.
5. PCR detection indicated that the cry1C~* gene was integrated into the genome of 120 transgenic plants of T_0 generation of the rice variety Kongyu131.
1.对水稻遗传转化载体pBar-cry1C~*进行酶切和PCR检测验证表明,载体和目的基因均准确无误,将携带目的基因cry1C~*基因的质粒pBar-cry1C~*电转化导入农杆菌,获得了农杆菌转化子EHA105(pBar-cry1C~*)。
2.根瘤农杆菌菌株EHA105B的活力比EHA105N强,EHA105B不能用于寒区水稻遗传转化工作,活力较弱的农杆菌菌株EHA105N可以介导外源基因转化北方粳稻。
3.黑龙江地区严冬季节产生的遗传转化再生水稻苗采用优化法炼苗,可以确保转基因再生水稻苗的成活率。优化法炼苗在组织培养室炼苗后,将转化苗置于水稻专用营养液中,在光照培养箱中25℃高湿条件下进行无土栽培3-4d,然后再移栽到土壤。
4.利用农杆菌介导的遗传转化方法,转化水稻空育131胚性愈伤1800块,共获来自20块独立抗性愈伤的分化再生苗123株,最终成活115株,成活率达到了93.5%。
5.对水稻空育131(cry1C~*)T_0代进行PCR分析,123株转化植株中阳性株120株,阳性率97.6%。cry1C~*基因已经整合到黑龙江水稻核基因组中。
Stemborers and leaffolders were two groups of Lepidopteran pests that caused severe damage to rice in many areas of the world. Kongyu131, being the most popular rice variety of Heilongjiang province, was highly susceptible to Lepidopteran insects, which greatly reduced the expected yield of Kongyu131. In this study, cry1C~* gene encoding Bacillus thuringiensis(Bt) d-endotoxin was synthesized based on codon optimization as one step toward gene stacking in the resistance management strategy of transgenic rice. The gene was transformed into Kongyu131 (Oryza sativa L.) via Agrobacterium-mediated method. The integration of the cry1C~* gene in rice genome was detected with PCR analyses. Meanwhile, the activity of EHA105B was compared to EHA105N and the optimized hardening method of regenerated plantlets in winter was improved in Heilongjiang province. The main results were as follows:
1. The vector pBar-cry1C~* was confirmed in E.coli DH5αwith restriction enzyme digestion and PCR detection.
2. The activity of the stain EHA105B of Agrobacterium tumefaciens was more powerful than the strain EHA105N and the EHA105N was suitable for the Agrobacterium-mediated transformation.
3. The optimized hardening method of regenerated plantlets elevated the living rate of transgenic plants in winter in Heilongjiang province.
4. 123 plants of the rice Kongyu131 transformed with cry1C~* gene from 20 independent resistant calli were regenerated and 115 living transgenic plants were obtained after optimized hardening.
5. PCR detection indicated that the cry1C~* gene was integrated into the genome of 120 transgenic plants of T_0 generation of the rice variety Kongyu131.
引文
[1]陈云鹏,陈火英,庄天明. Bt毒蛋白基因在转基因抗虫植物中的应用[J].生物学通报, 2000, 35(5): 15-16.
[2] Hannay, C. L. Fitz-James, P. Can J Microbiol., 1955, (1): 674-710.
[3]李海燕,朱延明,马凤鸣.植物抗虫基因工程的研究进展[J].东北农业大学学报, 2000, 31(4): 399-405.
[4] VanRie, J. Mc Gaughey, W. H. and Johnson, D. E. Mechanism of Insect Resistance to the Micro Bial Insect Icid Bacillust-huringieneis[J]. Science, 1990, (247): 72-74.
[5] Dlamudi, R. K. and Weber, E. JiI. J. Biol. Chem. [J]. 1995, 270: 5490-5494.
[6]刘凯于,姚汉超,杨红,洪华珠.昆虫中肠Bt杀虫晶体蛋白毒素受体氨肽酶N的研究进展[J].昆虫知识, 2004, 41(3).
[7]谭声江,陈晓峰等.昆虫对Bt毒素的抗性机理研究进展[J].昆虫知识, 2001, 38(1).
[8] Tang W. Production of insect-resistant rice through Bt genes transformation[D]. Huazhong Agricul-tural University, 2006.
[9]朱玉贤.植物基因工程的现状与展望[J].北京大学学报(自然科学版), 1996, 32(2).
[10]贾士荣,植物基因工程综述, http:/www.chinainfo.gov.cn/data/200202/1-20020204-27909. html.
[11] TANG Wei, Vanessa Samuels. Genetic Transformation and its Practical Application in Plants[J]. Developmental&Reproductive, Biology, 2001, 10(2): 77-88.
[12]石晶盈,陈维信,刘爱媛.植物内生菌及其防治植物病害的研究进展[J].生态学报, 2006, 26(7).
[13]范长胜,陈永青,李爽,雷肇祖.超甜蛋白的基因工程及开发研究进展[J].工业微生物, 1999, 29(1).
[14]沈世华,荆玉祥.中国生物固氮研究现状和展望[J].科学通报, 2003, 48(6).
[15]梁美霞,刘守伟,李景富.农杆菌介导的番茄遗传转化研究进展[J].东北农业大学学报, 2004, 35(2): 244-248.
[16] Curtiss R, Cardineau GA. Oral immunization by transgenic plants[J]. World Intellectual Property Organization, 1990, PCT/US89/03799.
[17]贾士荣.转基因植物概述[A];农业生物技术进展与展望[M].合肥:中国科学技术大学出版社, 1993, 17-24.
[18]王关林,方宏筠.植物基因工程原理与技术[Ⅱ][M].北京:科学出版社, 2002, 295-406.
[19]胡忠,李庆云,曹军.药用植物基因工程的研究进展[J].热带亚热带植物学报, 2002, 10(4): 371-380.
[20]谢志兵,钟晓红,董静洲.农杆菌属介导的植物细胞遗传转化研究现状[J].生物技术通讯, 2006, 17(1): 101-104.
[21] Zupan JR, Zambryski P. Transfer of T-DNA from Agrobacterium to the plant cell[J]. Plant Physiol, 1995, 107: 1041-1047.
[22] Stachel SE, Messens E, Van Montagu M et al. Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens[J]. Nature, 1986, 318: 624-629.
[23] Ashby AM, Watson MD, Loake GJ et al. Ti-plasmid specified chemotaxis of Agrobacterium tumefaciens toward vir-induction phenolic compounds and soluble factors from onocotyledonous and dicotyledonous plants[J]. Bacteriol, 1988, 170: 4181-4187.
[24] Song YN, Shibuya M, Ebizuka Y et al. Synergistic action of phenolic signal compounds and carbohydrates in the induction of virukence gene expression in Agrobacterium tumefaciens[J]. Chem Pharm Bull, 1991, 39: 2347-2350.
[25] Shimoda N, Toyoda-Yamamoto A, Nagamine J et al. Control of expression of Agrobacterium vir genes by synergistic actions of phenolic signal molecules andmonsaccharides[J]. Pros Natl Acad Sci USA, 1990, 87: 6684-6688.
[26] Alt-Moerbe J, Neddermann P, Von Lintig J et al. Temperature-sensitive steps in Ti plasmid vir region induction and correction with cytokinin secretion by Agrobacterium[J]. Mol Gen Genet, 1988, 213: 1-8.
[27] Stachel SE, Zambryski PC. VirA and VirG control the plant-induced activation of the T-DNA transfer process of Agrobacterium tumefaciens[J]. Cell, 1986, 46: 325-333.
[28]梁革梅,王桂荣等.昆虫Bt毒素受体蛋白的研究进展[J].昆虫学报, 2003, 46 (3): 390–396.
[29] Hooykas P J, Schilperoort R A. Agrobacterium and plant genetic engineering[J]. Plant Mol Biol, 1992, 19: 15-18.
[30] Tzfira T, Citovsky V. From host recognition to T-DNA integration: the function of bacterial and plant genes in the Agrobacterium-Plant cell interaction[J]. Mol Plant Pathol, 2000, 1(4): 201-212.
[31] Matthysse A G. Initial interactions of Agrobacterium tumefaciens with plant host cells[J]. Crit Rev Microbiol, 1986, 13: 281-307.
[32] Cangelosi G A, Ankenbaner R G, Nester E W. Sugars induce the Agrobacterium virulence genes through a periplasmic binding protein and a transmembrane signal protein[J]. Proc Natl Acad Sci USA, 1990, 87: 6708-6712.
[33] Chen L S, Li C M, Nester E W, Transferred DNA (T-DNA)-associated proteins of Agrobacterium tumefaciens are exported independently of VirB[J]. Proc Natl Acad Sci USA, 2000, 97: 7545-7550.
[34] Stachel S, Messens E, Montagu M, et al. Identification of the signal molecules-produced by wounded plat cells that activate T-DNA transfer in Agrobacterium tumefaciens[J]. Nature, 1985, 318: 624-629.
[35] Mayerhofer R, Koncz-Kalman Z, Nawrath c, et al. T-DNA integration: a mode of illegitimate recombination in plants[J]. The EMBO Journal, 1991, 10(3): 697-704.
[36]贾敬芬. Ti质粒及其在植物遗传操作中的应用遗传[J].遗传学报, 1985, 7(6): 41-45.
[37]罗小敏.棉花组织培养与雪花莲凝集素基因转化[D].河北大学, 2004.
[38]侍福梅.农杆菌介导的彩色棉花遗传转化影响因素研究[D].四川农业大学, 2004.
[39] Smith R, Hood E. Agrobacterium-mediated transformation of monocotyledons[J]. Crop Sci. 1995, 35: 301-309.
[40]李卫,郭光沁.根癌农杆菌介导遗传转化研究的若干新进展[J].科学通报. 2000, 45(8): 798-805.
[41] Vijayachandra K, Palanichelvam, Veluthambi K. Rice scutellum induces Agrobacterium tumefaciens vir genes and T-strand generation[J]. Plant Mol Biol. 1995, 29: 125-133.
[42] Hiei Y, Ohta S, Komari T et al. Efficient Transformation of rice (Oryza sativa L.)mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA[J]. Plant J. 1994, 6: 271-282.
[43]刘巧泉,张景六,王宗阳等.根癌农杆菌介导的水稻高效转化系统的建立[J].植物生理学报. 1998, 24(3): 259-27.
[44] Dekeyser R, Claes B, Marichal M et al. Evaluation of selectable markers for rice transformation[J]. Plant Physiol, 1989, 90: 217-233.
[45] Toriyama K, Arimoto Y, Uchimiya H et al. Transgenic rice plants after direct gene transfer into protoplasts[J]. Bio Technol. 1988, 6: 1072-1074.
[46] DeBlock MD, Botterman J, Vandewiele M et al. Engineering herbicide resistance in plants by expression of a detoxifying enzyme[J]. EMBOJ, 1987, 6: 2513-2518.
[47]易自力,曹守云,王力等.提高农杆菌转化水稻频率的研究[J].遗传学报. 2001, 28(4): 352-358.
[48] Cao J, Zhao J Z, Tang J D, Shelton A M, Earle E D. Broccoli plants with pyramided cry1Ac and cry1C Bt genes control diamondback moths resistant toCry1A and Cry1C proteins[J]. Theor Appl Genet, 2002, 105: 258-264.
[49] Chen H, Tang W, Xu C G, Li X H, Lin Y J, Zhang Q. Transgenic indica rice plants harboring a synthetic cry2A* gene of Bacillus thuringiensis exhibit enhanced resistance against rice lepidopteran pests[J]. Theor Appl Genet, 2005, 111: 1330-1337.
[50] Ferre J, Van Rie Z. Biochemistry and genetics of insect resistance to Bacillus thuringiensis[J]. Annu Rev Entdmol, 2002, 47: 501-533.
[51] Lin Y.J. The studies on Agrobacterium-mediated transformation of rice[D]. Huazhong Agricultural University, 2001.
[52] Lin Y.J., Chen H., Cao Y.L., Wu C.Y., Wen J., Li Y.F., and Hua H.X. Establishment of high-efficiency Agrobacterium-mediated genetic transformation system of Mudanjiang8[J]. Zuowu Xuebao(Acta Agronomica Sinica), 2002, 28: 294-300.(林拥军,陈浩,曹应龙,吴昌银,文静,李亚芳,华红霞.农杆菌介导的牡丹江8号高效转基因体系的建立,作物学报, 2002, 28: 294-300.)
[53] Xia L.Q., Wang Y., and Guo S.D. The stability of the expression of foreign genes in transgenic plants[J]. ShengwuJishu Tongxun(Biotechnology Information), 2000, 3: 8-12.(夏兰芹,王远,郭三堆.外源基因在转基因植物中的表达与稳定性,生物技术通报, 2000, 3: 8-12.)
[54] Kain W C, Zhao J Z, Janmaat A F, Myers J, Shelton A M, Wang P. Inheritance of resistance to Bacillus thuringiensis CrylAc toxin in a greenhouse-derived strain of cabbage looper (Lepidoptera: Noctuidae)[J]. J Econ Entomol, 2004, 97: 2073-2078.
[55] Lin Y J, Zhang Q. Optimizing the tissue culture conditions for high efficiency transformation of indica rice[J]. Plant Cell Rep, 2005, 35: 540-547.
[56] Ramesh S, Nagadhara D, Pasalu I C, Kumari A P, Sarma N P, Reddy V D, Rao K V Development of stem borer resistant transgenic parental lines involved in the production of hybrid rice[J]. J Biotech, 2004, 111: 131-141.
[57] Vain P, Afolabi A S, Worland J, Snape W. Transgene behaviour in populations of rice plants transformed using a new dual binary vector system: pGreen/pSoup[J]. Theor Appl Genet, 2003, 107: 210-217.
[58] Hayashi H, Czaja I, Lubenow H, et al. Activation of a plant gene by T-DNA tagging: auxin-independent growth in vitro[J]. Science, 1992, 258: 1350-1353.
[59] Smith R., Hood E. Agrobacterium-mediated trans-formation of monocotyledons[J]. Crop Sci., 1996, 35: 301-309.
[60] Lin Y.J., Zhang Q.F. Optimizing the tissue culture conditions for high efficiency transformation of indica rice[J]. Plant Cell Rep., 2005, 23(8): 540-547.
[61]侯文胜,郭三堆,路明. cry1A基因小麦高效表达载体的构建[J].西北农林科技大学学报, 2002, 30(6): 121-124.
[62]郭殿京,傅荣昭,李文彬等.小麦中外源基因瞬间表达调控研究及兔防御素(NP-l)基因的转化[J].遗传学报, 1999, 26(2): 168-173.
[63]刘根齐,李秀丽,杨春玲等.不同调控序列控制下的GUS基因在水稻和毛白杨愈伤组织中的瞬时表达[J].植物生理与分子生物学学报, 2005, 31(3): 327-330.
[64] Tang W., Chen H., Xu C.G., Li X.H., Lin Y.J., and Zhang Q.F. Development of insect resistant transgenic indica rice with a synthetic cry1C* gene[J]. Mol. Breed., 2006, 18(1): 1-10.
[65]贾士荣,郭三堆,安道昌.转基因棉花[M].科学出版社, 2001.
[66] Zhao J Z, Cao J, Li Y, Collins H L, Roush R T, Earle E D, Shelton A M. Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution[J]. Nat Biotechnol, 2003, 21, 1493-1497.
[2] Hannay, C. L. Fitz-James, P. Can J Microbiol., 1955, (1): 674-710.
[3]李海燕,朱延明,马凤鸣.植物抗虫基因工程的研究进展[J].东北农业大学学报, 2000, 31(4): 399-405.
[4] VanRie, J. Mc Gaughey, W. H. and Johnson, D. E. Mechanism of Insect Resistance to the Micro Bial Insect Icid Bacillust-huringieneis[J]. Science, 1990, (247): 72-74.
[5] Dlamudi, R. K. and Weber, E. JiI. J. Biol. Chem. [J]. 1995, 270: 5490-5494.
[6]刘凯于,姚汉超,杨红,洪华珠.昆虫中肠Bt杀虫晶体蛋白毒素受体氨肽酶N的研究进展[J].昆虫知识, 2004, 41(3).
[7]谭声江,陈晓峰等.昆虫对Bt毒素的抗性机理研究进展[J].昆虫知识, 2001, 38(1).
[8] Tang W. Production of insect-resistant rice through Bt genes transformation[D]. Huazhong Agricul-tural University, 2006.
[9]朱玉贤.植物基因工程的现状与展望[J].北京大学学报(自然科学版), 1996, 32(2).
[10]贾士荣,植物基因工程综述, http:/www.chinainfo.gov.cn/data/200202/1-20020204-27909. html.
[11] TANG Wei, Vanessa Samuels. Genetic Transformation and its Practical Application in Plants[J]. Developmental&Reproductive, Biology, 2001, 10(2): 77-88.
[12]石晶盈,陈维信,刘爱媛.植物内生菌及其防治植物病害的研究进展[J].生态学报, 2006, 26(7).
[13]范长胜,陈永青,李爽,雷肇祖.超甜蛋白的基因工程及开发研究进展[J].工业微生物, 1999, 29(1).
[14]沈世华,荆玉祥.中国生物固氮研究现状和展望[J].科学通报, 2003, 48(6).
[15]梁美霞,刘守伟,李景富.农杆菌介导的番茄遗传转化研究进展[J].东北农业大学学报, 2004, 35(2): 244-248.
[16] Curtiss R, Cardineau GA. Oral immunization by transgenic plants[J]. World Intellectual Property Organization, 1990, PCT/US89/03799.
[17]贾士荣.转基因植物概述[A];农业生物技术进展与展望[M].合肥:中国科学技术大学出版社, 1993, 17-24.
[18]王关林,方宏筠.植物基因工程原理与技术[Ⅱ][M].北京:科学出版社, 2002, 295-406.
[19]胡忠,李庆云,曹军.药用植物基因工程的研究进展[J].热带亚热带植物学报, 2002, 10(4): 371-380.
[20]谢志兵,钟晓红,董静洲.农杆菌属介导的植物细胞遗传转化研究现状[J].生物技术通讯, 2006, 17(1): 101-104.
[21] Zupan JR, Zambryski P. Transfer of T-DNA from Agrobacterium to the plant cell[J]. Plant Physiol, 1995, 107: 1041-1047.
[22] Stachel SE, Messens E, Van Montagu M et al. Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens[J]. Nature, 1986, 318: 624-629.
[23] Ashby AM, Watson MD, Loake GJ et al. Ti-plasmid specified chemotaxis of Agrobacterium tumefaciens toward vir-induction phenolic compounds and soluble factors from onocotyledonous and dicotyledonous plants[J]. Bacteriol, 1988, 170: 4181-4187.
[24] Song YN, Shibuya M, Ebizuka Y et al. Synergistic action of phenolic signal compounds and carbohydrates in the induction of virukence gene expression in Agrobacterium tumefaciens[J]. Chem Pharm Bull, 1991, 39: 2347-2350.
[25] Shimoda N, Toyoda-Yamamoto A, Nagamine J et al. Control of expression of Agrobacterium vir genes by synergistic actions of phenolic signal molecules andmonsaccharides[J]. Pros Natl Acad Sci USA, 1990, 87: 6684-6688.
[26] Alt-Moerbe J, Neddermann P, Von Lintig J et al. Temperature-sensitive steps in Ti plasmid vir region induction and correction with cytokinin secretion by Agrobacterium[J]. Mol Gen Genet, 1988, 213: 1-8.
[27] Stachel SE, Zambryski PC. VirA and VirG control the plant-induced activation of the T-DNA transfer process of Agrobacterium tumefaciens[J]. Cell, 1986, 46: 325-333.
[28]梁革梅,王桂荣等.昆虫Bt毒素受体蛋白的研究进展[J].昆虫学报, 2003, 46 (3): 390–396.
[29] Hooykas P J, Schilperoort R A. Agrobacterium and plant genetic engineering[J]. Plant Mol Biol, 1992, 19: 15-18.
[30] Tzfira T, Citovsky V. From host recognition to T-DNA integration: the function of bacterial and plant genes in the Agrobacterium-Plant cell interaction[J]. Mol Plant Pathol, 2000, 1(4): 201-212.
[31] Matthysse A G. Initial interactions of Agrobacterium tumefaciens with plant host cells[J]. Crit Rev Microbiol, 1986, 13: 281-307.
[32] Cangelosi G A, Ankenbaner R G, Nester E W. Sugars induce the Agrobacterium virulence genes through a periplasmic binding protein and a transmembrane signal protein[J]. Proc Natl Acad Sci USA, 1990, 87: 6708-6712.
[33] Chen L S, Li C M, Nester E W, Transferred DNA (T-DNA)-associated proteins of Agrobacterium tumefaciens are exported independently of VirB[J]. Proc Natl Acad Sci USA, 2000, 97: 7545-7550.
[34] Stachel S, Messens E, Montagu M, et al. Identification of the signal molecules-produced by wounded plat cells that activate T-DNA transfer in Agrobacterium tumefaciens[J]. Nature, 1985, 318: 624-629.
[35] Mayerhofer R, Koncz-Kalman Z, Nawrath c, et al. T-DNA integration: a mode of illegitimate recombination in plants[J]. The EMBO Journal, 1991, 10(3): 697-704.
[36]贾敬芬. Ti质粒及其在植物遗传操作中的应用遗传[J].遗传学报, 1985, 7(6): 41-45.
[37]罗小敏.棉花组织培养与雪花莲凝集素基因转化[D].河北大学, 2004.
[38]侍福梅.农杆菌介导的彩色棉花遗传转化影响因素研究[D].四川农业大学, 2004.
[39] Smith R, Hood E. Agrobacterium-mediated transformation of monocotyledons[J]. Crop Sci. 1995, 35: 301-309.
[40]李卫,郭光沁.根癌农杆菌介导遗传转化研究的若干新进展[J].科学通报. 2000, 45(8): 798-805.
[41] Vijayachandra K, Palanichelvam, Veluthambi K. Rice scutellum induces Agrobacterium tumefaciens vir genes and T-strand generation[J]. Plant Mol Biol. 1995, 29: 125-133.
[42] Hiei Y, Ohta S, Komari T et al. Efficient Transformation of rice (Oryza sativa L.)mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA[J]. Plant J. 1994, 6: 271-282.
[43]刘巧泉,张景六,王宗阳等.根癌农杆菌介导的水稻高效转化系统的建立[J].植物生理学报. 1998, 24(3): 259-27.
[44] Dekeyser R, Claes B, Marichal M et al. Evaluation of selectable markers for rice transformation[J]. Plant Physiol, 1989, 90: 217-233.
[45] Toriyama K, Arimoto Y, Uchimiya H et al. Transgenic rice plants after direct gene transfer into protoplasts[J]. Bio Technol. 1988, 6: 1072-1074.
[46] DeBlock MD, Botterman J, Vandewiele M et al. Engineering herbicide resistance in plants by expression of a detoxifying enzyme[J]. EMBOJ, 1987, 6: 2513-2518.
[47]易自力,曹守云,王力等.提高农杆菌转化水稻频率的研究[J].遗传学报. 2001, 28(4): 352-358.
[48] Cao J, Zhao J Z, Tang J D, Shelton A M, Earle E D. Broccoli plants with pyramided cry1Ac and cry1C Bt genes control diamondback moths resistant toCry1A and Cry1C proteins[J]. Theor Appl Genet, 2002, 105: 258-264.
[49] Chen H, Tang W, Xu C G, Li X H, Lin Y J, Zhang Q. Transgenic indica rice plants harboring a synthetic cry2A* gene of Bacillus thuringiensis exhibit enhanced resistance against rice lepidopteran pests[J]. Theor Appl Genet, 2005, 111: 1330-1337.
[50] Ferre J, Van Rie Z. Biochemistry and genetics of insect resistance to Bacillus thuringiensis[J]. Annu Rev Entdmol, 2002, 47: 501-533.
[51] Lin Y.J. The studies on Agrobacterium-mediated transformation of rice[D]. Huazhong Agricultural University, 2001.
[52] Lin Y.J., Chen H., Cao Y.L., Wu C.Y., Wen J., Li Y.F., and Hua H.X. Establishment of high-efficiency Agrobacterium-mediated genetic transformation system of Mudanjiang8[J]. Zuowu Xuebao(Acta Agronomica Sinica), 2002, 28: 294-300.(林拥军,陈浩,曹应龙,吴昌银,文静,李亚芳,华红霞.农杆菌介导的牡丹江8号高效转基因体系的建立,作物学报, 2002, 28: 294-300.)
[53] Xia L.Q., Wang Y., and Guo S.D. The stability of the expression of foreign genes in transgenic plants[J]. ShengwuJishu Tongxun(Biotechnology Information), 2000, 3: 8-12.(夏兰芹,王远,郭三堆.外源基因在转基因植物中的表达与稳定性,生物技术通报, 2000, 3: 8-12.)
[54] Kain W C, Zhao J Z, Janmaat A F, Myers J, Shelton A M, Wang P. Inheritance of resistance to Bacillus thuringiensis CrylAc toxin in a greenhouse-derived strain of cabbage looper (Lepidoptera: Noctuidae)[J]. J Econ Entomol, 2004, 97: 2073-2078.
[55] Lin Y J, Zhang Q. Optimizing the tissue culture conditions for high efficiency transformation of indica rice[J]. Plant Cell Rep, 2005, 35: 540-547.
[56] Ramesh S, Nagadhara D, Pasalu I C, Kumari A P, Sarma N P, Reddy V D, Rao K V Development of stem borer resistant transgenic parental lines involved in the production of hybrid rice[J]. J Biotech, 2004, 111: 131-141.
[57] Vain P, Afolabi A S, Worland J, Snape W. Transgene behaviour in populations of rice plants transformed using a new dual binary vector system: pGreen/pSoup[J]. Theor Appl Genet, 2003, 107: 210-217.
[58] Hayashi H, Czaja I, Lubenow H, et al. Activation of a plant gene by T-DNA tagging: auxin-independent growth in vitro[J]. Science, 1992, 258: 1350-1353.
[59] Smith R., Hood E. Agrobacterium-mediated trans-formation of monocotyledons[J]. Crop Sci., 1996, 35: 301-309.
[60] Lin Y.J., Zhang Q.F. Optimizing the tissue culture conditions for high efficiency transformation of indica rice[J]. Plant Cell Rep., 2005, 23(8): 540-547.
[61]侯文胜,郭三堆,路明. cry1A基因小麦高效表达载体的构建[J].西北农林科技大学学报, 2002, 30(6): 121-124.
[62]郭殿京,傅荣昭,李文彬等.小麦中外源基因瞬间表达调控研究及兔防御素(NP-l)基因的转化[J].遗传学报, 1999, 26(2): 168-173.
[63]刘根齐,李秀丽,杨春玲等.不同调控序列控制下的GUS基因在水稻和毛白杨愈伤组织中的瞬时表达[J].植物生理与分子生物学学报, 2005, 31(3): 327-330.
[64] Tang W., Chen H., Xu C.G., Li X.H., Lin Y.J., and Zhang Q.F. Development of insect resistant transgenic indica rice with a synthetic cry1C* gene[J]. Mol. Breed., 2006, 18(1): 1-10.
[65]贾士荣,郭三堆,安道昌.转基因棉花[M].科学出版社, 2001.
[66] Zhao J Z, Cao J, Li Y, Collins H L, Roush R T, Earle E D, Shelton A M. Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution[J]. Nat Biotechnol, 2003, 21, 1493-1497.