甘蓝转crylIa8和crylBa3双价Bt基因的研究
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摘要
结球甘蓝(Brassica oleracea L. var. capitata L.)是一种重要的十字花科蔬菜,在蔬菜周年供应中占有重要地位。近年来,随着栽培面积的逐年扩大,甘蓝的虫害日趋严重,其中以鳞翅目的小菜蛾(Plutella xylostella)和菜青虫(Pieris rapae)等为害最为严重。目前生产上害虫的防治以施用化学药剂为主,但化学农药污染环境、对人畜有害,且一些害虫已对多种化学杀虫剂产生了抗药性。培育抗虫品种是防治害虫最有效的方法,但在甘蓝中抗虫资源缺乏。利用植物基因工程技术将外源抗虫基因转入甘蓝,可为甘蓝育种提供有效的抗虫种质资源。
cry1Ia8和cry1Ba3基因是由中国农业科学院植物保护研究所自主分离克隆的。Cry1Ia8毒蛋白对小菜蛾和亚洲玉米螟具有很强的杀虫活性,Cry1Ba3毒蛋白对小菜蛾、小猿叶甲和家蝇具有很好的控制作用,并且Cry1Ia8和Cry1Ba3与生产上常用的Cry1A类毒蛋白之间没有交互抗性。创制同时含有cry1Ia8和cry1Ba3双价基因的Bt甘蓝,可以有效地抑制小菜蛾抗性的产生,为小菜蛾的抗性治理提供强有力的支持。本研究优化了甘蓝的再生体系和遗传转化体系,利用根癌农杆菌介导法将cry1Ia8和cry1Ba3双价基因导入甘蓝高代自交系,主要研究结果如下:
1.甘蓝再生体系的优化以4个甘蓝自交系为材料进行了再生体系的优化:苗龄控制在5 d,选用下胚轴为外植体,激素配比为6-BA 2 mg/L + NAA 0.1 mg/L的MS培养基,并添加有AgNO_3 3 mg/L时,可显著提高不定芽的分化率,最高再生频率达到96.87%。
2.甘蓝高效遗传转化体系的建立以重组质粒表达载体pCSIaN(含cry1Ia8基因)转化甘蓝,优化了农杆菌介导的甘蓝遗传转化体系:当选择培养基中激素配比为6-BA 2 mg/L + NAA 0.1 mg/L,并添加有AgNO_3 3 mg/L、Timentin 150 mg/L时能显著促进不定芽的再生,转化频率最高可达到10.42%。
3.甘蓝转cry1Ia8和cry1Ba3双价Bt抗虫基因植株的获得利用根癌农杆菌介导法将cry1Ia8和cry1Ba3双价基因导入甘蓝中,共获得41株卡那霉素抗性植株;分子检测证明cry1Ia8和cry1Ba3双价基因已被成功整合到甘蓝基因组中并得到表达;其中PCR检测阳性植株33株,Southern blot检测阳性植株27株,RT-PCR和Western blot检测均为阳性的植株有24株;抗虫性鉴定结果表明部分转基因植株不仅对敏感小菜蛾具有很强的杀虫活性,而且对Cry1Ac抗性小菜蛾也有很好的控制作用。
Cabbage(Brassica oleracea L. var. capitata L.)is one of the most important vegetables belonging to the Cruciferae. It plays an important role in supply of the vegetable all year-around. Along with the expansion of cabbage cultivation in recent years, cabbage is damaged by pests more and more seriously. Among them diamondback moth(Plutella xylostella)and cabbage worm(Pieris rapae)are the primary pests. Chemical prevention is one of the most important methods of pest contol at the present stage. But chemical insecticides cause severely environmental pollution and bring about adverse effects on people and animals. Moreover, pests have evolved resistance to many insecticides. The best way to control pests is to breed insect-resistant cultivars. However, insect-resistant germplasm resource available for cabbage breeding is limited. Bt cabbage can be obtained through genetic engineering. The transgenic plants may provide germplasm resources for cabbage breeding.
Both cry1Ia8 and cry1Ba3 gene were cloned by the Institute of Plant Protection, Chinese Academy of Agriculture Sciences. Cry1Ia8 toxin is toxic to diamondback, corn borer, and soybean moth. Cry1Ba3 toxin is toxic to diamondback moth, potato beetle, and housefly. Bioactivity results of Cry1Ia8 and Cry1Ba3 toxins showed that there was no cross resistance with Cry1Ac which is widely used for pest control in crop production. Pyramiding two Bt genes in cabbage is promising way to inhibit the evolution of insect resistance.
In this study, we optimized the systems of regeneration and genetic transformation of cabbage. cry1Ia8 and cry1Ba3 were transferred into cabbage line by Agrobacterium tumefaciens-mediated transformation method and transgenic plants with pyramided cry1Ia8 and cry1Ba3 were obtained. The main results were as follows:
1. Optimization of the regeneration system in cabbage
The system of regeneration was optimized with four cabbage lines. Hypocotyl from seedlings after 5 days from sowing was optimal. MS medium supplemented with 3 mg/L AgNO_3,150 mg/L Timentin, and 2 mg/L 6-BA in combination with 0.1 mg/L NAA could lead to generation of more adventitious buds . The highest regeneration frequency is 96.87%.
2. Optimization of the genetic transformation system in cabbage
The binary plasmid vector pCSIaN harboring cry1Ia8 gene was transferred into cabbage to optimize the genetic transformation system. The regeneration of kanamycin-resistant shoots has been obviously increased by using the selective medium supplemented with 2 mg/L 6-BA in combination with 0.1 mg/L NAA, 150 mg/L Timentin, and 3 mg/L AgNO_3 and the transformation frequency reached to 10.42%.
3. Transformation of cabbage with pyramided Bt genes cry1Ia8 and cry1Ba3
A total of 41 kanamycin-resistant plants with pyramided cry1Ia8 and cry1Ba3 transformation were obtained by Agrobacterium tumefaciens-mediated method. Molecular detections confirmed that cry1Ia8 and cry1Ba3 were successfully inserted into the genome of cabbage and also expressed in RNA and protein level. There are 33 PCR positive and 27 Southern blot positive plants. 24 RT-PCR and Western blot positive plants were obtained. Insect bioassay showed that the transgenic cabbage may control diamondback moth strains both susceptible to Bt toxin and resistant to Cry1Ac toxin.
cry1Ia8和cry1Ba3基因是由中国农业科学院植物保护研究所自主分离克隆的。Cry1Ia8毒蛋白对小菜蛾和亚洲玉米螟具有很强的杀虫活性,Cry1Ba3毒蛋白对小菜蛾、小猿叶甲和家蝇具有很好的控制作用,并且Cry1Ia8和Cry1Ba3与生产上常用的Cry1A类毒蛋白之间没有交互抗性。创制同时含有cry1Ia8和cry1Ba3双价基因的Bt甘蓝,可以有效地抑制小菜蛾抗性的产生,为小菜蛾的抗性治理提供强有力的支持。本研究优化了甘蓝的再生体系和遗传转化体系,利用根癌农杆菌介导法将cry1Ia8和cry1Ba3双价基因导入甘蓝高代自交系,主要研究结果如下:
1.甘蓝再生体系的优化以4个甘蓝自交系为材料进行了再生体系的优化:苗龄控制在5 d,选用下胚轴为外植体,激素配比为6-BA 2 mg/L + NAA 0.1 mg/L的MS培养基,并添加有AgNO_3 3 mg/L时,可显著提高不定芽的分化率,最高再生频率达到96.87%。
2.甘蓝高效遗传转化体系的建立以重组质粒表达载体pCSIaN(含cry1Ia8基因)转化甘蓝,优化了农杆菌介导的甘蓝遗传转化体系:当选择培养基中激素配比为6-BA 2 mg/L + NAA 0.1 mg/L,并添加有AgNO_3 3 mg/L、Timentin 150 mg/L时能显著促进不定芽的再生,转化频率最高可达到10.42%。
3.甘蓝转cry1Ia8和cry1Ba3双价Bt抗虫基因植株的获得利用根癌农杆菌介导法将cry1Ia8和cry1Ba3双价基因导入甘蓝中,共获得41株卡那霉素抗性植株;分子检测证明cry1Ia8和cry1Ba3双价基因已被成功整合到甘蓝基因组中并得到表达;其中PCR检测阳性植株33株,Southern blot检测阳性植株27株,RT-PCR和Western blot检测均为阳性的植株有24株;抗虫性鉴定结果表明部分转基因植株不仅对敏感小菜蛾具有很强的杀虫活性,而且对Cry1Ac抗性小菜蛾也有很好的控制作用。
Cabbage(Brassica oleracea L. var. capitata L.)is one of the most important vegetables belonging to the Cruciferae. It plays an important role in supply of the vegetable all year-around. Along with the expansion of cabbage cultivation in recent years, cabbage is damaged by pests more and more seriously. Among them diamondback moth(Plutella xylostella)and cabbage worm(Pieris rapae)are the primary pests. Chemical prevention is one of the most important methods of pest contol at the present stage. But chemical insecticides cause severely environmental pollution and bring about adverse effects on people and animals. Moreover, pests have evolved resistance to many insecticides. The best way to control pests is to breed insect-resistant cultivars. However, insect-resistant germplasm resource available for cabbage breeding is limited. Bt cabbage can be obtained through genetic engineering. The transgenic plants may provide germplasm resources for cabbage breeding.
Both cry1Ia8 and cry1Ba3 gene were cloned by the Institute of Plant Protection, Chinese Academy of Agriculture Sciences. Cry1Ia8 toxin is toxic to diamondback, corn borer, and soybean moth. Cry1Ba3 toxin is toxic to diamondback moth, potato beetle, and housefly. Bioactivity results of Cry1Ia8 and Cry1Ba3 toxins showed that there was no cross resistance with Cry1Ac which is widely used for pest control in crop production. Pyramiding two Bt genes in cabbage is promising way to inhibit the evolution of insect resistance.
In this study, we optimized the systems of regeneration and genetic transformation of cabbage. cry1Ia8 and cry1Ba3 were transferred into cabbage line by Agrobacterium tumefaciens-mediated transformation method and transgenic plants with pyramided cry1Ia8 and cry1Ba3 were obtained. The main results were as follows:
1. Optimization of the regeneration system in cabbage
The system of regeneration was optimized with four cabbage lines. Hypocotyl from seedlings after 5 days from sowing was optimal. MS medium supplemented with 3 mg/L AgNO_3,150 mg/L Timentin, and 2 mg/L 6-BA in combination with 0.1 mg/L NAA could lead to generation of more adventitious buds . The highest regeneration frequency is 96.87%.
2. Optimization of the genetic transformation system in cabbage
The binary plasmid vector pCSIaN harboring cry1Ia8 gene was transferred into cabbage to optimize the genetic transformation system. The regeneration of kanamycin-resistant shoots has been obviously increased by using the selective medium supplemented with 2 mg/L 6-BA in combination with 0.1 mg/L NAA, 150 mg/L Timentin, and 3 mg/L AgNO_3 and the transformation frequency reached to 10.42%.
3. Transformation of cabbage with pyramided Bt genes cry1Ia8 and cry1Ba3
A total of 41 kanamycin-resistant plants with pyramided cry1Ia8 and cry1Ba3 transformation were obtained by Agrobacterium tumefaciens-mediated method. Molecular detections confirmed that cry1Ia8 and cry1Ba3 were successfully inserted into the genome of cabbage and also expressed in RNA and protein level. There are 33 PCR positive and 27 Southern blot positive plants. 24 RT-PCR and Western blot positive plants were obtained. Insect bioassay showed that the transgenic cabbage may control diamondback moth strains both susceptible to Bt toxin and resistant to Cry1Ac toxin.
引文
1.陈国胜.植物转基因沉默的机制及克服方法.河北农业科学,2008,12(3):75～76.
2.陈红梅,李昆志,陈丽梅.植物来源抗虫基因的研究进展.中国生物工程杂志,2008,28(11):116～121.
3.陈之浩,刘传秀,李凤良等.小菜蛾对杀虫双的抗药性监测交互抗性测定及药剂防治研究.西南农业学报, 1994,7(3):68～74.
4.陈中义,李丽华,张杰,管宇,吴限,韩岚岚,黄大蔥.双价苏云金芽孢杆菌cry基因载体构建及其在大肠杆菌中表达杀虫活性.农业生物技术学报,2002,10(3):272～277.
5.程家胜,鄂超苏,田颖川,孟秀美,莽克强.转Bt抗虫基因苹果植株的再生.中国果树,1994(4);14～15.
6.崔洪志,郭三堆. Bt毒蛋白抗虫植物基因工程研究(综述).农业生物技术学报,1998,6(2):166～172.
7.崔磊,杨丽梅,方智远,刘玉梅,庄木,张扬勇,孙培田.农杆菌介导的Bt基因cry1Ah转化结球甘蓝的研究.见:中国十字花科蔬菜研究进展.中国园艺学会十字花科蔬菜分会第六届学术研讨会论文集.武汉:中国园艺学会十字花科蔬菜分会, 2008,328～334.
8.崔磊. Bt cry1Ia8抗虫基因对结球甘蓝的转化及其表达. [中国农业科学院学位论文].北京:中国农业科学院,2009.
9.崔磊,杨丽梅,刘楠,郎志宏,刘玉梅,庄木,张扬勇,张友军,黄大,方智远. Bt cry1Ia8抗虫基因对结球甘蓝的转化及其表达.园艺学报,2009,36(8):1161-1168.
10.丁强,杨培龙,黄火清,王亚茹,姚斌.植酸酶发展性状和研究趋势.中国农业科技导报,2010,12(3):27～33.
11.丁之铨,张杰,宋福平等.双价杀虫晶体蛋白基因在荧光假单胞菌中的表达及增效.微生物学报,2000,40(6):573～578.
12.窦黎明,韩岚岚,张杰,何康来,赵奎军,黄大,宋福平.苏云金芽胞杆菌cry1Ia基因的克隆、表达与活性研究.农业生物技术学报,2007,15(6):1053～1057.
13.高丽,崔崇士,屈淑平.白菜类蔬菜转基因研究进展.中国蔬菜,2010,(12):7～13.
14.郭三堆,倪万潮.双价抗虫转基因棉花研究.中国农业科学,1999,32:1～7.
15.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.
16.华学军,陈晓邦,范云六. Bacillus thuringiensis基因杀虫在花椰菜愈伤组织的整合与表达.中国农业科学,1992,25(4):82～87.
17.黄东林,夏锦瑜,柳慧,王伟,季正文,王倩.转CpTI+Bt基因棉和转Bt基因棉对甜菜夜蛾种群增长和行为的影响.植物保护学报,2007,34(5):34～38.
18.黄剑,吴文君.小菜蛾抗药性研究进展.贵州大学学报,2003,20(1):97～104.
19.贾士荣,曹冬孙.转基因植物.植物学通报, 1992,9(2):3-15.
20.贾士荣,郭三堆,安道昌.转基因棉花.北京:科学出版社,2001,6～79.
21.靳贵晓,王中康,高贵芳,王宇,殷幼平.小菜蛾幼虫cDNA文库的构建及序列初步分析.农业生物技术学报,2010,18(6):1197～1202.
22.孔维文,邓仲香,王倩,唐克轩,张健.含双边界序列植物双价表达载体的构建.分子植物育种,3:801～806.
23.李汉霞,尹若贺,陆芽春,张俊红. Cry1A(c)转基因结球甘蓝的抗虫性研究.农业生物技术学报,2006,14(4):546～550.
24.李汉霞,张晓辉,付雪林,张余洋,张俊红,王涛涛,叶志彪.双Bt基因提高青花菜对菜粉蝶和小菜蛾抗性.农业生物技术学报,2010,18(4):654～662.
25.李文凤,季静,王罡,王海勇,牛宝龙.提高转基因植物标记基因安全性策略的研究进展.中国农业科学,2010,43(9):1～10.
26.李晓兵,陈彩艳,翟文学.培育具有安全选择标记或无选择标记的转基因植物.遗传,2003, 25(3):345～349
27.练云,贾志伟,何康来,刘允军,宋福平,王保民,王国英.人工改造的Cry1Ac和Cry1Ie基因在烟草中共表达对棉铃虫有更好的杀虫活性.科学通报,2008,53(6):658～663.
28.刘楠,王少丽,宋福平,束长龙,高继国,张杰. Cry1Ba3、Cry1Ia8蛋白对Cry1Ac抗性小菜蛾的杀虫活性研究.植物保护,2010,36(2):66～70.
29.刘志,郭旺珍,朱协飞,朱祯,张天真.转Bt+GNA双价基因抗虫棉花中抗虫基因及其抗虫性的遗传稳定性.作物学报,2004,30(1):6～10.
30.卢永恩.抗虫基因转化白菜、甘蓝和番茄的研究. [华中农业大学学位论文].武汉:华中农业大学,2000.
31.路子显,常团结,朱祯.植物外源凝集素及其在植物基因工程中的应用.生物工程进展,2002,22(2):3～9.
32.罗雁婕,吴文伟,杨祚斌,浦恩堂,郭志祥,尹可锁,何成兴.小菜蛾抗药性及治理的研究进展.云南大学学报,2008,30(S1):178～182.
33.马员春,马维亮,宋晓华,林志珊.植物凝集素在作物转基因中的利用现状.宁夏农林科技,2010,3:60～62
34.毛慧珠,唐惕,曹湘岭,白永延.抗虫转基因甘蓝及其后代的研究.中国科学C辑,1996,26(4):339～347.
35.帅应恒,冯夏,陈焕玉.广东供港菜区小菜蛾抗药性研究初报.广东农业科学,1994,(3):31～34.
36.孙磊,张启翔,周琳,陆苗,蔡明.利用双T-DNA载体系统获得无选择标记转基因菊花.园艺学报,2008,35(5):727～734.
37.唐克轩,徐亚男,李旭峰,孙小芬.表达雪花莲外源凝集素基因的油菜转基因植株的获得.复旦大学学报(自然科学版),2001,40(6):683～689.
38.唐振华,周成理.抗性小菜蛾的乙酰胆碱酯酶敏感性.昆虫学报, 1992,35(4):385～392.
39.王广君,张杰,吴隽,宋福平,黄大昉.苏云金芽孢杆菌杀虫晶体蛋白Cry1Ba3活性区的研究.中国农业科学,2005,38(8):1585～1590.
40.王伟,朱祯,邓朝阳等.陆地棉栽培品种新陆早1号转基因抗虫植株的获得.高技术通讯,1998,8(5):1～5.
41.王志斌,郭三堆.表达CryIA/GNA双价抗虫基因烟草兼抗棉铃虫和蚜虫.科学通报,1999(S3),44:2068～2075.
42.王志斌,张秀梅,郭三堆.在转基因植物中利用植物凝集素防治害虫的研究.植物学通报,2000,17(2):108～113.
43.卫志明,黄健秋,徐淑萍.甘蓝下胚轴的高效再生和农杆菌介导Bt基因转化甘蓝.上海农业学报,1998,14(2):11～18.
44.吴昌银,叶志彪,李汉霞,唐克轩.雪花莲凝集素基因转化番茄.植物学报,2000,12(7):719～723.
45.吴乃虎.基因工程原理.北京:科学出版社,1998,171～290.
46.谢道昕,范云六,倪万潮等.苏云金芽孢杆菌(Bt)杀虫晶体蛋白基因导人棉花获得转基因植株.中国科学B辑,1991,(4):367～373.
47.谢友菊,王国英,林爱星.遗传工程概论.北京:中国农业大学出版社,2004,233～248.
48.闫艳春,乔传令,钱传范.小菜蛾抗药性研究进展.昆虫知识,1997,34(5):310～314.
49.杨广东,李艳娥,上官晓霞.大白菜遗传转化系统优化的研究.北京:中国农业出版社,2001,300～304.
50.杨广东,朱祯,李燕娥,朱祝军,肖桂芳,魏晓丽.利用根癌农杆菌介导法和基因枪法转化获得转抗虫融合蛋白基因(Bt-CpTI)甘蓝植株.实验生物学报,2002,35(2):117～122.
51.姚斌,范云六.表达昆虫特异性神经毒素AaIT基因的转基因烟草的抗虫性.生物工程学报,1996,12(2):113～118.
52.尹若贺. Bt转基因结球甘蓝的遗传分析和检测. [华中农业大学学位论文].武汉:华中农业大学,2004.
53.余小林,曹家树,许立奎,齐文雯,王晓静等.优化白菜类蔬菜遗传转化体系的研究.浙江大学学报,2005,31(5):529～534.
54.喻子牛,孙明,刘子铎等.苏云金芽孢杆菌杀虫晶体蛋白及其基因的研究与应用.北京:农业出版社,1993:170～179.
55.张磊,朱祯.转基因水稻对生物多样性的影响.遗传,2011,21(3):1～12.
56.张七仙,敖光明.根癌农杆菌介导的甘蓝高效稳定的遗传转化系统的建立及对CpTI基因转化的研究.农业生物技术学报,2001,9(1):72～76.
57.张文玲,李凌,刘凡.农杆菌介导羽衣甘蓝转化体系的建立.西南大学学报,2010,32(4):61～65.
58.张小霞,曹素梅,梁振普,乔冠华,许锋,陈晓慧.植物凝集素在抗刺吸式害虫转基因工程中的应用.植物保护,2010,36(4):23～28.
59.张雪燕,何捷.云南省主要菜区小菜蛾抗药性研究初报.云南农业科技,1998,(4):10～13.
60.张扬勇,李汉霞,叶志彪,卢永恩,陆芽春.农杆菌介导GNA基因转化菜薹.园艺学报,2003,30(4):473～475.
61.周静,弭晓菊,崔继哲.植物转基因的非孟德尔遗传.分子植物育种,2006,4(5):614～620.
62.朱玉,吴茜,高越峰等.雪花莲外源凝集素基因的克隆、序列分析和植物表达载体的构建.农业生物技术学报,1997,5(4):331～338.
63.赵建周,卢美光,范贤林.转双价基因抗虫烟草延缓棉铃虫抗性的作用评价.科学通报,1999,44(15):1635-1639.
64. Allen G C, Hall G, Michalioski J, et al. High level transgene expression in plant cells: Effects of a strong SAR from tobacco. Plant Cell, 1996, 8: 899-913.
65. Baekj H, Kim I, Lee D.W, et al. Identification and characterization of ace1-type acetylcholinesterase likely associated with organophosphate resistance in Plutella xylostella. Pestic Biochem Physiol, 2005, 81:164-175.
66. Bagla P. Hardy cotton-munching pests are latest blow to GM crops. Science, 2010, 19: 1439.
67. Bates S L, Zhao J Z, Roush R T, et al. Insect resistance management in GM crops: past, present and future. Nat Biotechnol, 2005, 23:57-62.
68. Benedict J H. Behavior growth survival and plant injury by Heliothis viresences on transgenic Bt cotton. J. Econ. Entomol, 1992, 85:589-593.
69. Bertolla F, Kay E, Simonet P. Potential dissemination of antibiotic resistance genes from transgenic plants to microorganisms. Infect Control Hosp Epidemiol, 2000, 21:390-393.
70. Bhattacharya R C, Viswakarma N, Bhat S R, et al. Development of insect-resistant transgenic cabbage plants expressing a synthetic cryIA(b) gene from Bacillus thuringiensis. Curr Sci India, 2002, 83:146-150.
71. Bravo A, Soberón M. How to cope with insect resistance to Bt toxins? Trends in Biotechnol, 2008, 26:573-579.
72. Cao, J Earle E D. Transgenne expression in broccoli (Brassica oleracea var. italica) clones propagated in vitro via leaf explants. Plant Cell Reports, 2003, 21:789-796.
73. Cao J, Shelton A M, Earle E D. Development of transgenic collards (Brassica oleracea L., var. acephala) expressing a cry1Ac or cry1C Bt gene for control of the diamondback moth. Crop Prot, 2005, 24:804-813.
74. Cao J, Shelton A M., Earle E D. Gene expression and insect resistance in transgenic broccoli containing a Bacillus thuringiensis cry1Ab gene wih the chemically inducible PR-1a promoter. Molecular Breeding, 2001, 8:207-216.
75. Cao J, Shelton A M, Earle E D. Sequential transformation to pyramid two Bt genes in vegetable Indian mustard (Brassica juncea L.) and its potential for control of diamondback moth larvae. Plant Cell Rep, 2008, 27: 479-487.
76. Cao J, Tang J D., Strizhov N, et al. Transgenic broccoli with high levels of Bacillus thuringiensis Cry1C protein control diamondback moth larvae resistant to Cry1A of Cry1C. Molecular Breeding, 1999, 5:131-141.
77. Cao J, Zhao J Z, Tang J D, Shelton A M. Broccoli plants with pyramided cry1Ac and cry1C Bt genes control diamondback moths resistant to Cry1A and Cry1C proteins. Theor Appl Genet, 2002, 105:258-264.
78. Cao M Q, Liu F, Yao L, et al. Transformation of Pakchoi(Brassica rapa L. ssp. Chinesis) by Agrobacterium infiltration. Molecular breeding, 2000, 6:67-72.
79. Cho H S, Cao J, Ren J P, Earle E D. Control of Lepidopteran insect in transgenic Chinese cabbage (Brassica rapa ssp. pekinensis) transformed with a synthetic Bacillus thuringiensis cry1C gene. Plant Cell Rep, 2001, 20:1-7.
80. Chrispeels M J, Raikhel N V. Lectins, lectin genes and their role in plant defense. The Plant Cell, 1991, 3:1-9.
81. Christou P, McCabe D E, Swain W F. Stable transformation of soybean callus by DNA-coated gold particles. Plant Physiol, 1988, 87:671-674.
82. Crickmore N, Zeigler D R, Feitelson J, et al. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins . Microbiol Mol Biol R, 1998, 62: 807-813.
83. Czapla T H, Lang B A. Effect of plant lectins on the larval development of European corn borer (Lepidoptera: Pyralidae) and Southern corn rootworm (coleopteran: Chrysomelidae). J Econ Etomol, 1990, 83,2481-2485.
84. David C and TempéJ. Genetic transformation of cauliflower (Brassica oleracea L.var. Botrytis) by Agrobacterium rhizogenes. Plant Cell Reports, 1988, 7:88-91.
85. Dou L M, Han L L, Zhang J, et al. Cloning, expressing and activity of cry1Ia gene from Bacillus thuringiensis isolate (Chinese). J Agr Biotechnol, 2007, 15:1053-1057.
86. Doyle J J, Doyle J L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull, 1987, 19:11-15.
87. Etzler M E. Plant lectins: Molecular and biological aspects. Ann Rev Plant Physiol, 1985, 36:209-234.
88. Finnegan J and McElroy D. Transgene inactivation: plant fight back! Nature Biotechnology, 1994, 12:883-888.
89. Fischhoff D A, Bowdish K S,Perlak, F J, et al. Insect tolerant transgenic tomato plants. Nature Biotechnology, 1987, 5:807-813.
90. Gatehouse A M R, Davison C M, Newell C A. Transgenic potato plants with enhanced resistance to the tomato moth, Lacanobia oleracea: growth room trials. Molecular Breeding, 1997, 3:49-63.
91. Gould F, Walter P, Jeanne R L. Polistes wasps (Hymenoptera: Vespidae) as control agents for lepidopterous cabbage pests. Environ Entomol, 1984, 13:150-156.
92. Gould F. Sustainability of transgenic insecticidal cultivars: integrating pest genetics and ecology. Annu Rev Entomol, 1998, 43:701-726.
93. Graham J M. Biomembrane protocols. In: Dunn MJ (ed) Analysis of membrane proteins by Western blotting and enhanced chemiluminescence, 19th edn. Springer-Verlag, New York, 1993, pp 211-218.
94. Halpin C. Gene stacking in transgenic plants-the challenge for 21st century plant biotechnology. Plant Biotechnol J. 2005, 3:141-155.
95. Hernández C S, FerréJ. Common receptor for Bacillus thuringiensis toxins Cry1Ac, Cry1Fa, and Cry1Ja in Helicoverpa armigera,Helicoverpa zea, and Spodoptera exigua. Appl Environ Microbiol, 2005, 71(9):5627-5629.
96. Hider V A, Barker R F, Samour R A, et al. Protein and cDNA sequences of Bowman-Birk protease inhibitors from the cowpea(vigna unguicuata W aIp). Plant Mol Bio, 1989, 13:701-710.
97. Hider V A, Powell K S, Gatehouse A M R, Gatehouse J A, Gatehouse LN, Shi Y, Hamilton WDO, Merryweather A, Newell CA, Timans JC, Peumans WJ, Damme EV, Boulter D, 1995.
98. Holbrook L A, Miki B L. Brassica grown gall tumourigenesis and in vitro of transformed tissue. Plant Cell Rep, 1985, 4:329-332.
99. Iglesias V A, Moscone E A, Papp I. Molecular and cytogenetics, analyses of stably and unstably expressed transgenic loci in tobacco. Plant Cell, 1997, 9:1251-1264.
100. Jackson R E, Bradley J R, van Duyn J W, et al. Comparative production of Helicoverpa zea (Lepidoptera: Noctuidae) from transgenic cotton expressing either one or two Bacillus thuringiensis proteins with and without insecticide oversprays. J Econ Entomol, 2004, 97: 1719-1725.
101. James C. Global status of commercialized biotech/GM crops: 2009. ISAAA Brief No. 30, ISAAA, Ithaca, New York.
102. James C. Global status of commercialized biotech/GM crops: 2010. ISAAA Brief No. 31, ISAAA, Ithaca, New York.
103. Jin R G, Liu Y B, Tabashinik B E, et al. Development of transgenic cabbage (Brassica Oleracea Var. Capitata) for insect resistance by Agrobacterium Tumefaciens-mediated transformation. In-Vitro Cell Dev-Pl, 2000, 36: 231-237.
104. Kleter G A, Bhula R, Bodnaruk K, et al. Altered pesticide use on transgenic crops and the associated general impact from an environmental perspective . Pest Manag Sci, 2007, 63:1107-1115.
105. Kuvshinov V, Koivu K, Kanerva A, Pehu E. Transgenic crop plants expressing synthetic cry9Aa gene are protected against insect damage. Plant Sci, 2001, 160(2):341-353.
106. Larson E, Howlett B, Jagendorf A. Artificial reductant enhancement of the lowry method for protein determination. Anal Biochem, 1986, 155:243-248.
107. Lee M K, Curfiss A, Alcantara E, et al. Synergistic effect of the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac on the gypsy moth, Lymantria dispar . Appl Environ Microbiol, 1996, 62:583-586.
108. Li G P, Wu K M, Gould F. Increasing tolerance to Cry1Ac cotton from cotton bollworm, Helicoverpa armigera, was confirmed in Bt cotton farming area of China. Ecological Entomology, 2007, 32:366-375.
109. Lu H, Zhou X, Gong Z, et al. Generation of selectable marker-free transgenic rice using double right-border (DBR) binary vectors. J Plant Physiol, 2001, 28:241-248.
110. Lu Y H , Wu K M, Jiang Y Y, Xia B, Li P, Feng H Q, Wyckhuys K A G, Guo Y Y. Mirid bug outbreaks in multiple crops correlated with wide-scale adoption of Bt cotton in China. Science, 2010, 328(5892):1151-1154.
111. Maqbool S B, Christou P. Multiple traits of agronomic importance in transgenic indica rice plants:analysis of transgene integration patterns, expression levels and stability. Molecular Breeding, 1999, 5:471-480.
112. Matten S.R, Head G.P, and Quemada H.D, 2008, How governmental regulation can help or hinder the integration of Bt crops within IPM programs.(eds)Romeis J, Shelton AM, Kennedy GG (Springer, New York), pp 27–39.
113. Matzke M A, Mette M F, Matzke A J M. Transgene silencing by the host genome defense: implications for the evolution of epigenetic control mechanisms in plants and vertebrates. Plant Mol Biol, 2000, 43:401-415.
114. McGaughey W H. Insect resistance to the biological insecticide Bacillus thuringiensis. Science, 1985, 229:193-195.
115. Mersereau M, Pazour GJ, Das A. Efficient transformation of Agrobacterium tumefaciens by electroporation. Gene, 1990, 90:149-151.
116. Metz T D, Dixit R, Earle E D. Agrobacterium tumefaciens-mediated transformation of broccoli (Brassica oleracea var. italica) and cabbage (Brassica oleracea var. capitata). Plant Cell Rep, 1995, 15:287-292.
117. Meyer P. Stabilities and instabilities in transgene expression. In: Lindsey K (ed) Trangenic plant research. Harwood Academic, Zurich, 1998, pp 263-275.
118. Murry E E, Rochleau T R, Eberle M, Stock C, Sckar V, Adang M J. Analysis of unstable RNA transcripts of insecticidal crystal protein genes of Bacillus thuringiensis in transgenic plants and electroporated protoplast. Plant Mol Biol, 1991, 16:1035-1060.
119. Paul S, Sikdar S R. Expression of nptⅡmarker and gus reporter genes and their inheritance in subsequent of transgenic Brassica developed through Agrobacterium-mediated gene transfer. Curr Sci India, 1999, 76:1569-1573.
120. Perez C J, Shelton A M. Field applications, leaf-dip assay, diet-incorporated diagnostic assays used against Bacillus thuringiensis-susceptible and resistant diamondback moth. J Econ Entomol, 1996, 89:1364-1371.
121. Perlak F J, Stone T B, Muskopf Y M, Petersen LJ, Parker G B, Mcpherson S A, Wyman J, Love S, Reed G, Biever. Geneticlly improved potatoes: protection from damage by Colorado potato beetles. Plant Mol Biol, 1993, 22:313-321.
122. Peumans W J, Van Damme E J M. Lectins as plant defense proteins. Plant Physiol, 1995, 109:347-352.
123. Pius P K, Achar P N. Agrobacterium tumefaciens-mediated transformation and plant regeneration of Brassica oleracea var. capitata. Plant Cell Rep, 2000, 19:888-892.
124. Qaim M, Zilberman D. Yield effects of genetically modified crops in developing countries. Science, 2003, 299:900-902.
125. Qian Y. Analysis of advantages and disadvantages on transgenic crops. Biotechnology Information, 1999, 5:7-11.
126. Rafat A, Aziz M A, Rashid A A, et al. Optimization of Agrobacterium tumefaciens-mediatedtransformation and shoot regeneration after co-cultivation of cabbage (Brassica oleracea subsp. capitata) cv KY Cross with AtHSP101 gene. Sci Hortic, 2010, 124:1-8.
127. Randhawa G J, Chhabra R,Singh M. Molecular characterization of Bt cauliflower with multiplex PCR and validation of endogenous reference gene in Brassicaceae family. Current Science, 95(12):1729-1731.
128. Rao K V, Rathore K S, Hodges T K, et al. Expression of snowdrop lectin (GNA) in transgenic rice plants confers resistance to rice brown planthopper. The Plant Journal, 1998, 15:469-477.
129. Roush R T. Bt-transgenic crops: just another pretty insecticide or a chance for a new start in resistance management? Pestic Sci, 1997b, 51:328-334.
130. Roush R T. Managing resistance to transgenic crops. In: Carozzi N, Koziel M (eds) Advances in insect control: the role of transgenic plants. Taylor and Francis, London, 1997a, pp 271-294.
131. Roush R T. Two-toxin strategies for management of insect resistant transgenic crops: can pyramiding succeed where pesticide mixtures have not? Phil Trans Proc R Soc London Ser B, 1998, 353: 1777-1786
132. Ryan C A. Proteinase inhibitors in plants: genes for improving defenses against insects and pathogens. Annual Review of Phytopathology, 1990, 28:425-449.
133. Sache E S, Benedict J H, Stelly D M, et al. Expression and segregation of gene encoding Cry LA insecticidal proteins in cotton. Crop Science, 1998, 38:1-11.
134. Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory, New York, 1989.
135. Schnepf H E, and Whiteley H R. Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coli. Proc Natl Acad Sci, 1981, 78: 2893-2897.
136. Shelton A M, Andalora J T, Barnards J. Effects of cabbage looper, imported cabbageworm, and diamondback moth on fresh market and processing cabbage. J Econ Entomol, 1982, 75:742-745.
137. Shelton A M, Roberson J L, Tang J D, et al. Resistance of diamondback moth (Lepidoptera: Plutellidae) to Bacillus thuringiensis subspecies in the field. J Econ Entomol, 1993, 86:697-705.
138. Shelton A M, Wyman J A, Cushing N L, et al. Insecticide resistance of diamondback moth in North America. J Econ Entomol, 1993, 86:11-19.
139. Srivastava V, Reddy A S, Guha-Mukherjee S. Transformation and regeneration of Brassica oleracea mediated by an oncogenic Agrobacterium tumefaciens. Plant Cell Reports, 1988, 7:504-507.
140. Steward S D, Adamczyk J J, Jr Knighten K S, et al. Inpack of Bt cottons expressing one or two insecticidal proteins of Bacillus thuringiensis Berliner on growth and survival of noctuid (Lepidoptera) larvae. J Econ Entomol, 2001, 94:752-760.
141. Tabashnik B E, Cushing N L, Finson N, et al. Field development of resistance to Bacillus thuringiensis in diamondback moth (Lepidoptera: Plutellidae). J Econ Entomol, 1990, 83(5): 1671-1676.
142. Tabashnik B E, Bruce E, Van R, et al. Field-evolved insect resistance to Bt crops: definition, theory, and data. J Econ Entomol, 2009, 102:2011-2025.
143. Tabashnik B E, Carrière Y, Dennehy T J, et al. Insect resistance to transgenic Bt crops: lessons from the laboratory and field. J Econ Entomol, 2003, 96:1031-1038.
144. Tabashnik B E, Carrière Y. Evolution of insect resistance to transgenic plants. University of California Press, 2008, 267 - 279.
145. Tabashnik B E, Gassmann A J, Crowder D W, et al. Insect resistance to Bt crops: evidence versus theory. Nat Biotechnol, 2008, 26:199-202.
146. Theunissen J, Booij C J H, Lotz L A P. Effects of intercropping white cabbage with clovers on pest infestation and yield. Entomol Exp Appl, 1995, 74:7-16.
147. Ulian E C, Magill J M, Smith R H. Expression and inheritance pattern of two foreign genes in petunia. Theor Appl Genet, 1994, 88:433-440.
148. Vaeck M, Reynaerts A, Hofte H S, et al. Transgenic plants protected from insect attack. Nature, 1987, 328:33-37.
149. van Rensburg J.B.J. First report of field resistance by the stem borer, Busseola fusca (Fuller) to Bt-transgenic maize, South African Journal of Plant Soil, 2007, 24(3): 147-151.
150. van Rie J, McGaughey W H, Johnson D E, Barnett B D, van Mellaert H. Mechanism of insect resistance to the microbial insecticide Bacillus thuringiensis . Science, 1990, 247(4938):72-74.
151. Zhang FL, Takahata Y, Xu J B. Medium and genotype factors influencing shoot regeneration from cotyledonary explants of Chinese cabbage (Brassica campestris L. ssp. pekinensis). Plant Cell Rep, 1998, 17:780-786.
152. Zhao J Z, Cao J, Collins H L, et al. Concurrent use of transgenic plants expressing a single and two Bacillus thuringiensis genes speeds insect adaption to pyramided plants. Proc Natl Acad Sci USA, 2005, 102:8426-8430.
153. Zhao J Z, Cao J, Li Y X, et al. Transgenic plant expressing two Bacillus thuringiensis toxins delay insect resistance evolution. Nature Biotechnology, 2003, 21:1493-1497.
2.陈红梅,李昆志,陈丽梅.植物来源抗虫基因的研究进展.中国生物工程杂志,2008,28(11):116～121.
3.陈之浩,刘传秀,李凤良等.小菜蛾对杀虫双的抗药性监测交互抗性测定及药剂防治研究.西南农业学报, 1994,7(3):68～74.
4.陈中义,李丽华,张杰,管宇,吴限,韩岚岚,黄大蔥.双价苏云金芽孢杆菌cry基因载体构建及其在大肠杆菌中表达杀虫活性.农业生物技术学报,2002,10(3):272～277.
5.程家胜,鄂超苏,田颖川,孟秀美,莽克强.转Bt抗虫基因苹果植株的再生.中国果树,1994(4);14～15.
6.崔洪志,郭三堆. Bt毒蛋白抗虫植物基因工程研究(综述).农业生物技术学报,1998,6(2):166～172.
7.崔磊,杨丽梅,方智远,刘玉梅,庄木,张扬勇,孙培田.农杆菌介导的Bt基因cry1Ah转化结球甘蓝的研究.见:中国十字花科蔬菜研究进展.中国园艺学会十字花科蔬菜分会第六届学术研讨会论文集.武汉:中国园艺学会十字花科蔬菜分会, 2008,328～334.
8.崔磊. Bt cry1Ia8抗虫基因对结球甘蓝的转化及其表达. [中国农业科学院学位论文].北京:中国农业科学院,2009.
9.崔磊,杨丽梅,刘楠,郎志宏,刘玉梅,庄木,张扬勇,张友军,黄大,方智远. Bt cry1Ia8抗虫基因对结球甘蓝的转化及其表达.园艺学报,2009,36(8):1161-1168.
10.丁强,杨培龙,黄火清,王亚茹,姚斌.植酸酶发展性状和研究趋势.中国农业科技导报,2010,12(3):27～33.
11.丁之铨,张杰,宋福平等.双价杀虫晶体蛋白基因在荧光假单胞菌中的表达及增效.微生物学报,2000,40(6):573～578.
12.窦黎明,韩岚岚,张杰,何康来,赵奎军,黄大,宋福平.苏云金芽胞杆菌cry1Ia基因的克隆、表达与活性研究.农业生物技术学报,2007,15(6):1053～1057.
13.高丽,崔崇士,屈淑平.白菜类蔬菜转基因研究进展.中国蔬菜,2010,(12):7～13.
14.郭三堆,倪万潮.双价抗虫转基因棉花研究.中国农业科学,1999,32:1～7.
15.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.
16.华学军,陈晓邦,范云六. Bacillus thuringiensis基因杀虫在花椰菜愈伤组织的整合与表达.中国农业科学,1992,25(4):82～87.
17.黄东林,夏锦瑜,柳慧,王伟,季正文,王倩.转CpTI+Bt基因棉和转Bt基因棉对甜菜夜蛾种群增长和行为的影响.植物保护学报,2007,34(5):34～38.
18.黄剑,吴文君.小菜蛾抗药性研究进展.贵州大学学报,2003,20(1):97～104.
19.贾士荣,曹冬孙.转基因植物.植物学通报, 1992,9(2):3-15.
20.贾士荣,郭三堆,安道昌.转基因棉花.北京:科学出版社,2001,6～79.
21.靳贵晓,王中康,高贵芳,王宇,殷幼平.小菜蛾幼虫cDNA文库的构建及序列初步分析.农业生物技术学报,2010,18(6):1197～1202.
22.孔维文,邓仲香,王倩,唐克轩,张健.含双边界序列植物双价表达载体的构建.分子植物育种,3:801～806.
23.李汉霞,尹若贺,陆芽春,张俊红. Cry1A(c)转基因结球甘蓝的抗虫性研究.农业生物技术学报,2006,14(4):546～550.
24.李汉霞,张晓辉,付雪林,张余洋,张俊红,王涛涛,叶志彪.双Bt基因提高青花菜对菜粉蝶和小菜蛾抗性.农业生物技术学报,2010,18(4):654～662.
25.李文凤,季静,王罡,王海勇,牛宝龙.提高转基因植物标记基因安全性策略的研究进展.中国农业科学,2010,43(9):1～10.
26.李晓兵,陈彩艳,翟文学.培育具有安全选择标记或无选择标记的转基因植物.遗传,2003, 25(3):345～349
27.练云,贾志伟,何康来,刘允军,宋福平,王保民,王国英.人工改造的Cry1Ac和Cry1Ie基因在烟草中共表达对棉铃虫有更好的杀虫活性.科学通报,2008,53(6):658～663.
28.刘楠,王少丽,宋福平,束长龙,高继国,张杰. Cry1Ba3、Cry1Ia8蛋白对Cry1Ac抗性小菜蛾的杀虫活性研究.植物保护,2010,36(2):66～70.
29.刘志,郭旺珍,朱协飞,朱祯,张天真.转Bt+GNA双价基因抗虫棉花中抗虫基因及其抗虫性的遗传稳定性.作物学报,2004,30(1):6～10.
30.卢永恩.抗虫基因转化白菜、甘蓝和番茄的研究. [华中农业大学学位论文].武汉:华中农业大学,2000.
31.路子显,常团结,朱祯.植物外源凝集素及其在植物基因工程中的应用.生物工程进展,2002,22(2):3～9.
32.罗雁婕,吴文伟,杨祚斌,浦恩堂,郭志祥,尹可锁,何成兴.小菜蛾抗药性及治理的研究进展.云南大学学报,2008,30(S1):178～182.
33.马员春,马维亮,宋晓华,林志珊.植物凝集素在作物转基因中的利用现状.宁夏农林科技,2010,3:60～62
34.毛慧珠,唐惕,曹湘岭,白永延.抗虫转基因甘蓝及其后代的研究.中国科学C辑,1996,26(4):339～347.
35.帅应恒,冯夏,陈焕玉.广东供港菜区小菜蛾抗药性研究初报.广东农业科学,1994,(3):31～34.
36.孙磊,张启翔,周琳,陆苗,蔡明.利用双T-DNA载体系统获得无选择标记转基因菊花.园艺学报,2008,35(5):727～734.
37.唐克轩,徐亚男,李旭峰,孙小芬.表达雪花莲外源凝集素基因的油菜转基因植株的获得.复旦大学学报(自然科学版),2001,40(6):683～689.
38.唐振华,周成理.抗性小菜蛾的乙酰胆碱酯酶敏感性.昆虫学报, 1992,35(4):385～392.
39.王广君,张杰,吴隽,宋福平,黄大昉.苏云金芽孢杆菌杀虫晶体蛋白Cry1Ba3活性区的研究.中国农业科学,2005,38(8):1585～1590.
40.王伟,朱祯,邓朝阳等.陆地棉栽培品种新陆早1号转基因抗虫植株的获得.高技术通讯,1998,8(5):1～5.
41.王志斌,郭三堆.表达CryIA/GNA双价抗虫基因烟草兼抗棉铃虫和蚜虫.科学通报,1999(S3),44:2068～2075.
42.王志斌,张秀梅,郭三堆.在转基因植物中利用植物凝集素防治害虫的研究.植物学通报,2000,17(2):108～113.
43.卫志明,黄健秋,徐淑萍.甘蓝下胚轴的高效再生和农杆菌介导Bt基因转化甘蓝.上海农业学报,1998,14(2):11～18.
44.吴昌银,叶志彪,李汉霞,唐克轩.雪花莲凝集素基因转化番茄.植物学报,2000,12(7):719～723.
45.吴乃虎.基因工程原理.北京:科学出版社,1998,171～290.
46.谢道昕,范云六,倪万潮等.苏云金芽孢杆菌(Bt)杀虫晶体蛋白基因导人棉花获得转基因植株.中国科学B辑,1991,(4):367～373.
47.谢友菊,王国英,林爱星.遗传工程概论.北京:中国农业大学出版社,2004,233～248.
48.闫艳春,乔传令,钱传范.小菜蛾抗药性研究进展.昆虫知识,1997,34(5):310～314.
49.杨广东,李艳娥,上官晓霞.大白菜遗传转化系统优化的研究.北京:中国农业出版社,2001,300～304.
50.杨广东,朱祯,李燕娥,朱祝军,肖桂芳,魏晓丽.利用根癌农杆菌介导法和基因枪法转化获得转抗虫融合蛋白基因(Bt-CpTI)甘蓝植株.实验生物学报,2002,35(2):117～122.
51.姚斌,范云六.表达昆虫特异性神经毒素AaIT基因的转基因烟草的抗虫性.生物工程学报,1996,12(2):113～118.
52.尹若贺. Bt转基因结球甘蓝的遗传分析和检测. [华中农业大学学位论文].武汉:华中农业大学,2004.
53.余小林,曹家树,许立奎,齐文雯,王晓静等.优化白菜类蔬菜遗传转化体系的研究.浙江大学学报,2005,31(5):529～534.
54.喻子牛,孙明,刘子铎等.苏云金芽孢杆菌杀虫晶体蛋白及其基因的研究与应用.北京:农业出版社,1993:170～179.
55.张磊,朱祯.转基因水稻对生物多样性的影响.遗传,2011,21(3):1～12.
56.张七仙,敖光明.根癌农杆菌介导的甘蓝高效稳定的遗传转化系统的建立及对CpTI基因转化的研究.农业生物技术学报,2001,9(1):72～76.
57.张文玲,李凌,刘凡.农杆菌介导羽衣甘蓝转化体系的建立.西南大学学报,2010,32(4):61～65.
58.张小霞,曹素梅,梁振普,乔冠华,许锋,陈晓慧.植物凝集素在抗刺吸式害虫转基因工程中的应用.植物保护,2010,36(4):23～28.
59.张雪燕,何捷.云南省主要菜区小菜蛾抗药性研究初报.云南农业科技,1998,(4):10～13.
60.张扬勇,李汉霞,叶志彪,卢永恩,陆芽春.农杆菌介导GNA基因转化菜薹.园艺学报,2003,30(4):473～475.
61.周静,弭晓菊,崔继哲.植物转基因的非孟德尔遗传.分子植物育种,2006,4(5):614～620.
62.朱玉,吴茜,高越峰等.雪花莲外源凝集素基因的克隆、序列分析和植物表达载体的构建.农业生物技术学报,1997,5(4):331～338.
63.赵建周,卢美光,范贤林.转双价基因抗虫烟草延缓棉铃虫抗性的作用评价.科学通报,1999,44(15):1635-1639.
64. Allen G C, Hall G, Michalioski J, et al. High level transgene expression in plant cells: Effects of a strong SAR from tobacco. Plant Cell, 1996, 8: 899-913.
65. Baekj H, Kim I, Lee D.W, et al. Identification and characterization of ace1-type acetylcholinesterase likely associated with organophosphate resistance in Plutella xylostella. Pestic Biochem Physiol, 2005, 81:164-175.
66. Bagla P. Hardy cotton-munching pests are latest blow to GM crops. Science, 2010, 19: 1439.
67. Bates S L, Zhao J Z, Roush R T, et al. Insect resistance management in GM crops: past, present and future. Nat Biotechnol, 2005, 23:57-62.
68. Benedict J H. Behavior growth survival and plant injury by Heliothis viresences on transgenic Bt cotton. J. Econ. Entomol, 1992, 85:589-593.
69. Bertolla F, Kay E, Simonet P. Potential dissemination of antibiotic resistance genes from transgenic plants to microorganisms. Infect Control Hosp Epidemiol, 2000, 21:390-393.
70. Bhattacharya R C, Viswakarma N, Bhat S R, et al. Development of insect-resistant transgenic cabbage plants expressing a synthetic cryIA(b) gene from Bacillus thuringiensis. Curr Sci India, 2002, 83:146-150.
71. Bravo A, Soberón M. How to cope with insect resistance to Bt toxins? Trends in Biotechnol, 2008, 26:573-579.
72. Cao, J Earle E D. Transgenne expression in broccoli (Brassica oleracea var. italica) clones propagated in vitro via leaf explants. Plant Cell Reports, 2003, 21:789-796.
73. Cao J, Shelton A M, Earle E D. Development of transgenic collards (Brassica oleracea L., var. acephala) expressing a cry1Ac or cry1C Bt gene for control of the diamondback moth. Crop Prot, 2005, 24:804-813.
74. Cao J, Shelton A M., Earle E D. Gene expression and insect resistance in transgenic broccoli containing a Bacillus thuringiensis cry1Ab gene wih the chemically inducible PR-1a promoter. Molecular Breeding, 2001, 8:207-216.
75. Cao J, Shelton A M, Earle E D. Sequential transformation to pyramid two Bt genes in vegetable Indian mustard (Brassica juncea L.) and its potential for control of diamondback moth larvae. Plant Cell Rep, 2008, 27: 479-487.
76. Cao J, Tang J D., Strizhov N, et al. Transgenic broccoli with high levels of Bacillus thuringiensis Cry1C protein control diamondback moth larvae resistant to Cry1A of Cry1C. Molecular Breeding, 1999, 5:131-141.
77. Cao J, Zhao J Z, Tang J D, Shelton A M. Broccoli plants with pyramided cry1Ac and cry1C Bt genes control diamondback moths resistant to Cry1A and Cry1C proteins. Theor Appl Genet, 2002, 105:258-264.
78. Cao M Q, Liu F, Yao L, et al. Transformation of Pakchoi(Brassica rapa L. ssp. Chinesis) by Agrobacterium infiltration. Molecular breeding, 2000, 6:67-72.
79. Cho H S, Cao J, Ren J P, Earle E D. Control of Lepidopteran insect in transgenic Chinese cabbage (Brassica rapa ssp. pekinensis) transformed with a synthetic Bacillus thuringiensis cry1C gene. Plant Cell Rep, 2001, 20:1-7.
80. Chrispeels M J, Raikhel N V. Lectins, lectin genes and their role in plant defense. The Plant Cell, 1991, 3:1-9.
81. Christou P, McCabe D E, Swain W F. Stable transformation of soybean callus by DNA-coated gold particles. Plant Physiol, 1988, 87:671-674.
82. Crickmore N, Zeigler D R, Feitelson J, et al. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins . Microbiol Mol Biol R, 1998, 62: 807-813.
83. Czapla T H, Lang B A. Effect of plant lectins on the larval development of European corn borer (Lepidoptera: Pyralidae) and Southern corn rootworm (coleopteran: Chrysomelidae). J Econ Etomol, 1990, 83,2481-2485.
84. David C and TempéJ. Genetic transformation of cauliflower (Brassica oleracea L.var. Botrytis) by Agrobacterium rhizogenes. Plant Cell Reports, 1988, 7:88-91.
85. Dou L M, Han L L, Zhang J, et al. Cloning, expressing and activity of cry1Ia gene from Bacillus thuringiensis isolate (Chinese). J Agr Biotechnol, 2007, 15:1053-1057.
86. Doyle J J, Doyle J L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull, 1987, 19:11-15.
87. Etzler M E. Plant lectins: Molecular and biological aspects. Ann Rev Plant Physiol, 1985, 36:209-234.
88. Finnegan J and McElroy D. Transgene inactivation: plant fight back! Nature Biotechnology, 1994, 12:883-888.
89. Fischhoff D A, Bowdish K S,Perlak, F J, et al. Insect tolerant transgenic tomato plants. Nature Biotechnology, 1987, 5:807-813.
90. Gatehouse A M R, Davison C M, Newell C A. Transgenic potato plants with enhanced resistance to the tomato moth, Lacanobia oleracea: growth room trials. Molecular Breeding, 1997, 3:49-63.
91. Gould F, Walter P, Jeanne R L. Polistes wasps (Hymenoptera: Vespidae) as control agents for lepidopterous cabbage pests. Environ Entomol, 1984, 13:150-156.
92. Gould F. Sustainability of transgenic insecticidal cultivars: integrating pest genetics and ecology. Annu Rev Entomol, 1998, 43:701-726.
93. Graham J M. Biomembrane protocols. In: Dunn MJ (ed) Analysis of membrane proteins by Western blotting and enhanced chemiluminescence, 19th edn. Springer-Verlag, New York, 1993, pp 211-218.
94. Halpin C. Gene stacking in transgenic plants-the challenge for 21st century plant biotechnology. Plant Biotechnol J. 2005, 3:141-155.
95. Hernández C S, FerréJ. Common receptor for Bacillus thuringiensis toxins Cry1Ac, Cry1Fa, and Cry1Ja in Helicoverpa armigera,Helicoverpa zea, and Spodoptera exigua. Appl Environ Microbiol, 2005, 71(9):5627-5629.
96. Hider V A, Barker R F, Samour R A, et al. Protein and cDNA sequences of Bowman-Birk protease inhibitors from the cowpea(vigna unguicuata W aIp). Plant Mol Bio, 1989, 13:701-710.
97. Hider V A, Powell K S, Gatehouse A M R, Gatehouse J A, Gatehouse LN, Shi Y, Hamilton WDO, Merryweather A, Newell CA, Timans JC, Peumans WJ, Damme EV, Boulter D, 1995.
98. Holbrook L A, Miki B L. Brassica grown gall tumourigenesis and in vitro of transformed tissue. Plant Cell Rep, 1985, 4:329-332.
99. Iglesias V A, Moscone E A, Papp I. Molecular and cytogenetics, analyses of stably and unstably expressed transgenic loci in tobacco. Plant Cell, 1997, 9:1251-1264.
100. Jackson R E, Bradley J R, van Duyn J W, et al. Comparative production of Helicoverpa zea (Lepidoptera: Noctuidae) from transgenic cotton expressing either one or two Bacillus thuringiensis proteins with and without insecticide oversprays. J Econ Entomol, 2004, 97: 1719-1725.
101. James C. Global status of commercialized biotech/GM crops: 2009. ISAAA Brief No. 30, ISAAA, Ithaca, New York.
102. James C. Global status of commercialized biotech/GM crops: 2010. ISAAA Brief No. 31, ISAAA, Ithaca, New York.
103. Jin R G, Liu Y B, Tabashinik B E, et al. Development of transgenic cabbage (Brassica Oleracea Var. Capitata) for insect resistance by Agrobacterium Tumefaciens-mediated transformation. In-Vitro Cell Dev-Pl, 2000, 36: 231-237.
104. Kleter G A, Bhula R, Bodnaruk K, et al. Altered pesticide use on transgenic crops and the associated general impact from an environmental perspective . Pest Manag Sci, 2007, 63:1107-1115.
105. Kuvshinov V, Koivu K, Kanerva A, Pehu E. Transgenic crop plants expressing synthetic cry9Aa gene are protected against insect damage. Plant Sci, 2001, 160(2):341-353.
106. Larson E, Howlett B, Jagendorf A. Artificial reductant enhancement of the lowry method for protein determination. Anal Biochem, 1986, 155:243-248.
107. Lee M K, Curfiss A, Alcantara E, et al. Synergistic effect of the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac on the gypsy moth, Lymantria dispar . Appl Environ Microbiol, 1996, 62:583-586.
108. Li G P, Wu K M, Gould F. Increasing tolerance to Cry1Ac cotton from cotton bollworm, Helicoverpa armigera, was confirmed in Bt cotton farming area of China. Ecological Entomology, 2007, 32:366-375.
109. Lu H, Zhou X, Gong Z, et al. Generation of selectable marker-free transgenic rice using double right-border (DBR) binary vectors. J Plant Physiol, 2001, 28:241-248.
110. Lu Y H , Wu K M, Jiang Y Y, Xia B, Li P, Feng H Q, Wyckhuys K A G, Guo Y Y. Mirid bug outbreaks in multiple crops correlated with wide-scale adoption of Bt cotton in China. Science, 2010, 328(5892):1151-1154.
111. Maqbool S B, Christou P. Multiple traits of agronomic importance in transgenic indica rice plants:analysis of transgene integration patterns, expression levels and stability. Molecular Breeding, 1999, 5:471-480.
112. Matten S.R, Head G.P, and Quemada H.D, 2008, How governmental regulation can help or hinder the integration of Bt crops within IPM programs.(eds)Romeis J, Shelton AM, Kennedy GG (Springer, New York), pp 27–39.
113. Matzke M A, Mette M F, Matzke A J M. Transgene silencing by the host genome defense: implications for the evolution of epigenetic control mechanisms in plants and vertebrates. Plant Mol Biol, 2000, 43:401-415.
114. McGaughey W H. Insect resistance to the biological insecticide Bacillus thuringiensis. Science, 1985, 229:193-195.
115. Mersereau M, Pazour GJ, Das A. Efficient transformation of Agrobacterium tumefaciens by electroporation. Gene, 1990, 90:149-151.
116. Metz T D, Dixit R, Earle E D. Agrobacterium tumefaciens-mediated transformation of broccoli (Brassica oleracea var. italica) and cabbage (Brassica oleracea var. capitata). Plant Cell Rep, 1995, 15:287-292.
117. Meyer P. Stabilities and instabilities in transgene expression. In: Lindsey K (ed) Trangenic plant research. Harwood Academic, Zurich, 1998, pp 263-275.
118. Murry E E, Rochleau T R, Eberle M, Stock C, Sckar V, Adang M J. Analysis of unstable RNA transcripts of insecticidal crystal protein genes of Bacillus thuringiensis in transgenic plants and electroporated protoplast. Plant Mol Biol, 1991, 16:1035-1060.
119. Paul S, Sikdar S R. Expression of nptⅡmarker and gus reporter genes and their inheritance in subsequent of transgenic Brassica developed through Agrobacterium-mediated gene transfer. Curr Sci India, 1999, 76:1569-1573.
120. Perez C J, Shelton A M. Field applications, leaf-dip assay, diet-incorporated diagnostic assays used against Bacillus thuringiensis-susceptible and resistant diamondback moth. J Econ Entomol, 1996, 89:1364-1371.
121. Perlak F J, Stone T B, Muskopf Y M, Petersen LJ, Parker G B, Mcpherson S A, Wyman J, Love S, Reed G, Biever. Geneticlly improved potatoes: protection from damage by Colorado potato beetles. Plant Mol Biol, 1993, 22:313-321.
122. Peumans W J, Van Damme E J M. Lectins as plant defense proteins. Plant Physiol, 1995, 109:347-352.
123. Pius P K, Achar P N. Agrobacterium tumefaciens-mediated transformation and plant regeneration of Brassica oleracea var. capitata. Plant Cell Rep, 2000, 19:888-892.
124. Qaim M, Zilberman D. Yield effects of genetically modified crops in developing countries. Science, 2003, 299:900-902.
125. Qian Y. Analysis of advantages and disadvantages on transgenic crops. Biotechnology Information, 1999, 5:7-11.
126. Rafat A, Aziz M A, Rashid A A, et al. Optimization of Agrobacterium tumefaciens-mediatedtransformation and shoot regeneration after co-cultivation of cabbage (Brassica oleracea subsp. capitata) cv KY Cross with AtHSP101 gene. Sci Hortic, 2010, 124:1-8.
127. Randhawa G J, Chhabra R,Singh M. Molecular characterization of Bt cauliflower with multiplex PCR and validation of endogenous reference gene in Brassicaceae family. Current Science, 95(12):1729-1731.
128. Rao K V, Rathore K S, Hodges T K, et al. Expression of snowdrop lectin (GNA) in transgenic rice plants confers resistance to rice brown planthopper. The Plant Journal, 1998, 15:469-477.
129. Roush R T. Bt-transgenic crops: just another pretty insecticide or a chance for a new start in resistance management? Pestic Sci, 1997b, 51:328-334.
130. Roush R T. Managing resistance to transgenic crops. In: Carozzi N, Koziel M (eds) Advances in insect control: the role of transgenic plants. Taylor and Francis, London, 1997a, pp 271-294.
131. Roush R T. Two-toxin strategies for management of insect resistant transgenic crops: can pyramiding succeed where pesticide mixtures have not? Phil Trans Proc R Soc London Ser B, 1998, 353: 1777-1786
132. Ryan C A. Proteinase inhibitors in plants: genes for improving defenses against insects and pathogens. Annual Review of Phytopathology, 1990, 28:425-449.
133. Sache E S, Benedict J H, Stelly D M, et al. Expression and segregation of gene encoding Cry LA insecticidal proteins in cotton. Crop Science, 1998, 38:1-11.
134. Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory, New York, 1989.
135. Schnepf H E, and Whiteley H R. Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coli. Proc Natl Acad Sci, 1981, 78: 2893-2897.
136. Shelton A M, Andalora J T, Barnards J. Effects of cabbage looper, imported cabbageworm, and diamondback moth on fresh market and processing cabbage. J Econ Entomol, 1982, 75:742-745.
137. Shelton A M, Roberson J L, Tang J D, et al. Resistance of diamondback moth (Lepidoptera: Plutellidae) to Bacillus thuringiensis subspecies in the field. J Econ Entomol, 1993, 86:697-705.
138. Shelton A M, Wyman J A, Cushing N L, et al. Insecticide resistance of diamondback moth in North America. J Econ Entomol, 1993, 86:11-19.
139. Srivastava V, Reddy A S, Guha-Mukherjee S. Transformation and regeneration of Brassica oleracea mediated by an oncogenic Agrobacterium tumefaciens. Plant Cell Reports, 1988, 7:504-507.
140. Steward S D, Adamczyk J J, Jr Knighten K S, et al. Inpack of Bt cottons expressing one or two insecticidal proteins of Bacillus thuringiensis Berliner on growth and survival of noctuid (Lepidoptera) larvae. J Econ Entomol, 2001, 94:752-760.
141. Tabashnik B E, Cushing N L, Finson N, et al. Field development of resistance to Bacillus thuringiensis in diamondback moth (Lepidoptera: Plutellidae). J Econ Entomol, 1990, 83(5): 1671-1676.
142. Tabashnik B E, Bruce E, Van R, et al. Field-evolved insect resistance to Bt crops: definition, theory, and data. J Econ Entomol, 2009, 102:2011-2025.
143. Tabashnik B E, Carrière Y, Dennehy T J, et al. Insect resistance to transgenic Bt crops: lessons from the laboratory and field. J Econ Entomol, 2003, 96:1031-1038.
144. Tabashnik B E, Carrière Y. Evolution of insect resistance to transgenic plants. University of California Press, 2008, 267 - 279.
145. Tabashnik B E, Gassmann A J, Crowder D W, et al. Insect resistance to Bt crops: evidence versus theory. Nat Biotechnol, 2008, 26:199-202.
146. Theunissen J, Booij C J H, Lotz L A P. Effects of intercropping white cabbage with clovers on pest infestation and yield. Entomol Exp Appl, 1995, 74:7-16.
147. Ulian E C, Magill J M, Smith R H. Expression and inheritance pattern of two foreign genes in petunia. Theor Appl Genet, 1994, 88:433-440.
148. Vaeck M, Reynaerts A, Hofte H S, et al. Transgenic plants protected from insect attack. Nature, 1987, 328:33-37.
149. van Rensburg J.B.J. First report of field resistance by the stem borer, Busseola fusca (Fuller) to Bt-transgenic maize, South African Journal of Plant Soil, 2007, 24(3): 147-151.
150. van Rie J, McGaughey W H, Johnson D E, Barnett B D, van Mellaert H. Mechanism of insect resistance to the microbial insecticide Bacillus thuringiensis . Science, 1990, 247(4938):72-74.
151. Zhang FL, Takahata Y, Xu J B. Medium and genotype factors influencing shoot regeneration from cotyledonary explants of Chinese cabbage (Brassica campestris L. ssp. pekinensis). Plant Cell Rep, 1998, 17:780-786.
152. Zhao J Z, Cao J, Collins H L, et al. Concurrent use of transgenic plants expressing a single and two Bacillus thuringiensis genes speeds insect adaption to pyramided plants. Proc Natl Acad Sci USA, 2005, 102:8426-8430.
153. Zhao J Z, Cao J, Li Y X, et al. Transgenic plant expressing two Bacillus thuringiensis toxins delay insect resistance evolution. Nature Biotechnology, 2003, 21:1493-1497.