人工改造的cry1Ac、cry1Ie基因在大肠杆菌、转基因烟草和玉米中的表达
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摘要
本研究根据植物所偏爱密码子原则，改造苏云金芽孢杆菌crylAc，crylIe野生型基因的编码区序列，人工合成crylAc，crylIe基因。将这两个基因构建到原核表达载体pET28b中，构建成原核表达载体pETAc和pETIe。将两个原核表达载体转入大肠杆菌BL21(DE3)中，并进行了诱导表达。诱导表达出两个目的蛋白并进行了纯化。纯化的目的蛋白免疫兔子，制备了这两种蛋白的多克隆抗体。纯化这两种蛋白的包涵体，用玉米螟进行虫试。虫试结果显示原核表达的两种包涵体蛋白对玉米螟有较好的杀虫活性。
进一步将crylAc，crylIe基因构建到真核表达载体p3301中，构建植物表达载体p3301ubiAc和p3301ubiIe。在这两个表达载体中，两个基因分别受ubiquitin启动子的调控。两个载体转化烟草并得到了转基因烟草，PCR、Southern杂交结果显示，crylAc，crylIe基因整合到转基因烟草的基因组中。用转基因烟草叶片进行虫试，结果显示转基因烟草能有效杀死玉米螟幼虫。
运用PCR技术克隆了马铃薯蛋白酶抑制剂基因Ⅱ的信号肽序列，并将其分别连到crylAc、gfp基因的5’端，构建植物转化载体p3301ubisigAc和p3301ubisigGFP。分别用这两个载体转化烟草，并得到转基因植株。荧光显微观察到GFP在植物细胞间隙高效表达，Western blot结果显示CrylAc蛋白也在植物细胞间隙表达。这些结果表明，马铃薯蛋白酶抑制剂基因Ⅱ的信号肽序列能将外源蛋白定位到植物细胞间隙。ELISA结果显示，马铃薯蛋白酶抑制剂基因Ⅱ的信号肽序列使crylAc基因在转基因烟草中的表达量显著提高。
通过PCR方法，克隆了玉米叶绿素a／b结合蛋白基因的启动子(cab启动子)和玉米伸展蛋白基因的启动子(silk启动子)。将此两个启动子、玉米ubiquitin启动子与crylIe，crylAc基因分别构建成表达载体p3301cabIeubiAc，p3301silkIe和p3301ubiAc。转化玉米得到转基因玉米植株。转基因玉米田间和温室玉米螟接虫鉴定结果表明，有些转基因玉米株系具有较好的抗虫性。
The coding regions of wild Bacillus thuringiensis cry1Ie and cry1Ac gene were modified according to plant preferring codons. The modified crylle and crylAc gene were artificially synthesized. Modified crylle and crylAc gene were cloned into prokaryotic expression vector pET28b, to construct plasmid pETIe and pETAc. These two proteins' expression in E. coli BL21(DE3) were confirmed by SDS-PAGE analysis. Proteins purified were injected into rabbit to prepare polyclonal antibodies. Bioassays using crude expression products in E. coli revealed that Cry1Ie and Cry1Ac proteins had a similar toxicity to corn borer as wild type proteins.
Modified cry1Ac and cry1Ie gene had also been cloned into plant expression vector p3301, to construct plasmid p3301ubiAc and p3301ubiIe in which two genes were under the control of maize ubiquitin-1 promoter respectively. Tobacco plant leaves were transformed with these two plasmids and transgenic tobacco plants were obtained. The results of PCR and Sourthern blot showed that crylAc and crylle gene have integrated into tobacco plants genome. The results of insect assay with Asian corn borer showed that transgenic tobacco plants carrying crylAc and crylle showed insecticidal activity against corn borer.
Potato proteinase inhibitor II (pinll) signal peptide sequence was obtained by the method of PCR. To allow secretion of the CrylAc protein into the intercellular space, potato proteinase inhibitor II (pinll) signal peptide sequence was N-terminally fused to the crylAc coding region to construct plasmid p3301ubisigAc. Expression of CrylAc in transgenic tobacco plants was assayed with ELISA. The results showed that pinll signal peptide sequence enhanced the expression of CrylAc protein in transgenic tobacco. GFP gene was also fused behind the signal peptide sequence to construct plasmid p3301ubisigGFP and transformed to tobacco. The results of fluorescent detection showed GFP localization in the apoplast.
The promoters of maize chlorophyll a/b binding protein (cab promoter) and maize extension protein gene (silk promoter) were cloned by the methods of PCR. These two promoters, maize ubiquitin promoter and crylle, crylAc gene were used to construct plant expression plasmid p3301cabIeubiAc, p3301silkIe and p3301ubiAc. These plasmids were used to transform maize Primary embryogenic calli and transgenic maize plants were obtained. The results of evaluating of the resistance to Asian corn borers showed
some Bt transgenic maize lines showed good insect-resistance.
进一步将crylAc，crylIe基因构建到真核表达载体p3301中，构建植物表达载体p3301ubiAc和p3301ubiIe。在这两个表达载体中，两个基因分别受ubiquitin启动子的调控。两个载体转化烟草并得到了转基因烟草，PCR、Southern杂交结果显示，crylAc，crylIe基因整合到转基因烟草的基因组中。用转基因烟草叶片进行虫试，结果显示转基因烟草能有效杀死玉米螟幼虫。
运用PCR技术克隆了马铃薯蛋白酶抑制剂基因Ⅱ的信号肽序列，并将其分别连到crylAc、gfp基因的5’端，构建植物转化载体p3301ubisigAc和p3301ubisigGFP。分别用这两个载体转化烟草，并得到转基因植株。荧光显微观察到GFP在植物细胞间隙高效表达，Western blot结果显示CrylAc蛋白也在植物细胞间隙表达。这些结果表明，马铃薯蛋白酶抑制剂基因Ⅱ的信号肽序列能将外源蛋白定位到植物细胞间隙。ELISA结果显示，马铃薯蛋白酶抑制剂基因Ⅱ的信号肽序列使crylAc基因在转基因烟草中的表达量显著提高。
通过PCR方法，克隆了玉米叶绿素a／b结合蛋白基因的启动子(cab启动子)和玉米伸展蛋白基因的启动子(silk启动子)。将此两个启动子、玉米ubiquitin启动子与crylIe，crylAc基因分别构建成表达载体p3301cabIeubiAc，p3301silkIe和p3301ubiAc。转化玉米得到转基因玉米植株。转基因玉米田间和温室玉米螟接虫鉴定结果表明，有些转基因玉米株系具有较好的抗虫性。
The coding regions of wild Bacillus thuringiensis cry1Ie and cry1Ac gene were modified according to plant preferring codons. The modified crylle and crylAc gene were artificially synthesized. Modified crylle and crylAc gene were cloned into prokaryotic expression vector pET28b, to construct plasmid pETIe and pETAc. These two proteins' expression in E. coli BL21(DE3) were confirmed by SDS-PAGE analysis. Proteins purified were injected into rabbit to prepare polyclonal antibodies. Bioassays using crude expression products in E. coli revealed that Cry1Ie and Cry1Ac proteins had a similar toxicity to corn borer as wild type proteins.
Modified cry1Ac and cry1Ie gene had also been cloned into plant expression vector p3301, to construct plasmid p3301ubiAc and p3301ubiIe in which two genes were under the control of maize ubiquitin-1 promoter respectively. Tobacco plant leaves were transformed with these two plasmids and transgenic tobacco plants were obtained. The results of PCR and Sourthern blot showed that crylAc and crylle gene have integrated into tobacco plants genome. The results of insect assay with Asian corn borer showed that transgenic tobacco plants carrying crylAc and crylle showed insecticidal activity against corn borer.
Potato proteinase inhibitor II (pinll) signal peptide sequence was obtained by the method of PCR. To allow secretion of the CrylAc protein into the intercellular space, potato proteinase inhibitor II (pinll) signal peptide sequence was N-terminally fused to the crylAc coding region to construct plasmid p3301ubisigAc. Expression of CrylAc in transgenic tobacco plants was assayed with ELISA. The results showed that pinll signal peptide sequence enhanced the expression of CrylAc protein in transgenic tobacco. GFP gene was also fused behind the signal peptide sequence to construct plasmid p3301ubisigGFP and transformed to tobacco. The results of fluorescent detection showed GFP localization in the apoplast.
The promoters of maize chlorophyll a/b binding protein (cab promoter) and maize extension protein gene (silk promoter) were cloned by the methods of PCR. These two promoters, maize ubiquitin promoter and crylle, crylAc gene were used to construct plant expression plasmid p3301cabIeubiAc, p3301silkIe and p3301ubiAc. These plasmids were used to transform maize Primary embryogenic calli and transgenic maize plants were obtained. The results of evaluating of the resistance to Asian corn borers showed
some Bt transgenic maize lines showed good insect-resistance.
引文
1 丁群星等，1993，用子房注射法将Bt毒蛋白基因导入玉米的研究，中国科学(B辑)，23(7)：707-713
2 郭三堆等，1999，双价抗虫转基因棉花研究，中国农业科学，32(3)：1-7
3 王国英等，1995，用基因枪将Bt毒蛋白基因转入玉米及转基因植株再生，中国科学(B辑)，25(1)：71-76
4 谢迎秋、朱祯等，一种强启动子的分离与功能，植物学报，2000，42(1)：50-54
5 晏小兰、邱国华、李宝健等，启动子对外源基因在转基因植物中表达的影响，中山大学学报(自然科学版)，1995，4(2)：60-67
6 周逢勇等，1998，玉米自交系P9-10遗传转化体系的建立，科学通报，43(23)：2517-2521
7 吴志平，徐步进．转基因植物释放后在环境中成为杂草的风险性[J]．生物工程进展，1999，19(1)：9-13．
8 钱迎倩 转基因作物的利弊分析 生物技术通报，1999，(5)：7-11
9 Adang MJ, Brody M S, Cardineau G. The reconstruction and expression of a Bacillus thuringiensis cryIIIA gene in protoplasts and potato plants. Plant Mol Biol, 1993,21: 1131-1145
10 Ali S and Taylor WC. Quantitative regulation of the Flaveria Mel gene is controlled by the 3'-untranslated region and sequences near the amino terminus. Plant Molecular Biology, 2001,46:251-261
11 Ali S and Taylor WC. The 3' non-coding region of a C4 photosynthesis gene increase transgene expression when combined with heterologous promoters. Plant Molecular Biology, 2001,46:325-333
12 Armstrong CL, Parker GB, Pershing JC, Brown SM, Sanders PR, Duncan DR, Stone T et al. Field evaluation of European corn borer control in progeny of 173 transgenie corn events expressing an insecticidal protein from Bacillus thuringiensis. Crop Sci, 1995, 35:550-557
13 Ballester V, Escriche B, Mensua jl, Riethmacher GW, Ferre J. Lack of cross-resistance to other Bacillus thuringiensis crystal proteins in a population of Plutella xylostella highly resistant to CryIA(b). Biocontrol Sci Technol, 1994, 4:437-443
14 Barton K, Whitely H., et al. Bacillus thuringensis δ-endotoxin in transgenic Nicotiana tabacum provides resistance to lepidopteran insets. Plant Physiol, 1987,85:1103-1109
15 Bergvinson D, Willcox M, Hoisington D. Efficacy and deployment of transgenic plants for stemborer management. Insect Sci Applic, 1997,(17) 1:157-167
16 Bohorova N, Frutos R, Royer M, Estaol P, Pacheco M, Rascón Q, McLean S, Hoisington D. Novel synthetic Bacillus thuringiensis cry1Bgene and the cry1B-cry1Abtranslational fusion confer resistance to southwestern corn borer, sugarcane borer and fall armyworm in transgenic tropical maize. Theor Appl Genet, 2001, 103:817-826
17 Bohorova N., Zhang W., Julstrum P., et al, Production of transgenic tropical maize with cry1A(b) and cry1A(c) genes via microprojectile bombardment of immatureembryos. Theor. Appl. Genet.,1999, 99:437-444
18 Brnke F, Hajirezaei M, Heineke D, Melzer M, Herbers K, Sonnewald U. High-level production of the noncariogenic sucrose isomer palatinose in transgenic tobacco plants strongly impairs development. Planta, 2002, 214:356-364
19 Bosch D., Schipper B, et al. Recombinant Bacillus thuringiensis crystal proteins with new properties: possibilities for resistance management. Bio/Technology, 12:915-918
20 Bruce E, et al. Global variation in the genetic and biochemical basis of diamondback moth resistance to bacillus thuringiensis, 1997, 94:12780-12785
21 Callis J, Fromm M, Walbot V. Introns increase gene expression in cultured maize cells. Genes. Devel., 1987,1: 1183-1200
22 Cao J, Zhao JZ, Tang JD, Shelton AM, Earle ED. Broccoli plants with pyramided crylAc and crylC Bt genes control diamondback moths resistant to Cry1A and Cry1C proteins. Theor. Appl. Genet, 2002,105:258-264
23 Cheng X Y, Sardana R, et al. Agrobacterium-transformed rice plants expressing synthetic CryIA(b) and CryIA(c) genes are highly toxic to striped stem borer and yellow stem borer. Proc. Natl. Acad. Sci. USA, 1998,95:2767-2772
24 Choi SK, Shin BS, Kong EM, Rho HM and Park SH. Cloning of a new Bacillus thuringiensis cry1I-type crystal protein gene. Crrrent Microbiology, 2000,41:65-69
25 Christensen AH, Sharrock RA, Quail PH. Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing and promoter activity following transfer to protoplasts by electroporation. Plant Mol Biol, 1992,18:675-689
26 Crickmore N. 2004 http://www.biols.susx.ac.uk/home/Neil_Crickmore/Bt/toxins2.html)
27 Dean C et al, Nuleic. Acids Research, 1986,14:2221-2240
28 Dowd PF. Indirect reduction of ear molds and associated mycotoxins in Bacillus thuringiensis corn under controlled and open field conditions: utility and limitation. Journal of Economic Entomology, 2000,93(6): 1669-1679
29 Estruch J. J., Warren G. W., Muillins M. A., et al. Vip3A, a novel bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidoteran insects, 1996,93:5389-5394
30 Farmer EE, Ryan CA. Octadecanoid precursors of jasmonie acid activate the synthesis of wound-inducible proteinase inhibitors. Plant Cell, 1992,4:371-382
31 Farran I, Snchez-Serrano JJ, Medina JF, Prieto J and Mingo-Castel AM. Targeted expression of human serum albumin to potato tubers. Transgenic Research, 2002, 11:337-346
32 Fischhoof et al. Insect tolerant transgenic tomato plants. Bio/Technology, 1987,5:807-812
33 Frutos R, Rang C, Royer M. Managing insect resistance to plants producing Bacillus thuringiensis toxins. Crit Rev Biotechnol, 1999, 19:227-276
34 Gahan LJ, Gould F, Heckel DG. Identification of a gene associated with Bt resistance in Heliothis virescens. Science, 2001,293:857-860
35 Gleave AP, Mitra DS, Markwick NP, et al. Enhanced expression of the Bacillus thuringiensis cry9Aa2 gene in transgenic plants by nucleotide sequence modification confers resistance to potato rubber moth. Molecular Breeding, 1998,4:459-472
36 Greenlate JT, Penn SR, Shappley Z, Oppenhuizen M, Mann J, Reich B, Osborn J. Bollgard Ⅱ efficacy: quantification of total lepidopteran activity in a two-gene product. Proc Beltwide Cotton Conf, 2000,vol2:1041-1043
37 Griffitts JS, Whitacre JL, Stevens DE, Aroian RV. Bt toxin resistance from loss of a prtative carbohydrate-modifying enzyme. Science, 2001,293:860-863
38 Grochuske P., Masson L, Borisova S., et al. Bacillus thuringensis cry1A(a) insecticidal toxin: cyustal structure and channel formation. Journal of Molecular Biology, 1995,254(3):447-464
39 Herbers K, Wilke I, Sonnewaid U. A thermostable xylanase from clostridium thermocellum expressed at high levels in the apoplast of transgenic tobacco has no detrimental effects and is easily purified. Bio/Technology,1995,13:63-66
40 Hilder V A et al. A novel mechanism of insect resistance engineered into tobacco. Nature, 1987,330:160-163
41 Honee G, Vriezen W, Visser B., et al. A translation fusion product of two different insecticidal crystal protein genes of Bacillus thuringiensis exhibits an enlarger insecticidal spectrum. Appl. And Envi. Mico, 1991,56(3):923-925
42 Hood EE, Murphy JM, Pendleton RC. Molecular characterization of maize extensin expression. Plant Molecular Biology, 1993,23:685-695
43 Huang E, Buschman L L, Higgins R.A., et al. Inheritance of resistance to bacillus thuringiensis toxin in the European corn borer. Science, 1999,284:965-967
44 Hudspeth R. Structure and expression of the maize gene encoding the phosphoenolpyruvate carboxylaase isozyme involved in C4 photosynthesis. Plant Molecular Biology, 1989,12:579-589
45 Jansens S, van Vliet A, Dickburt C, Buysse L, Piens C, Saey B, Wulf AD et al. Transgenic corn expressing a Cry9C insecticidal protein from Bacillus thuringiensis protected from European corn bore damage. Crop Sci, 1997, 37:1616-1624
46 Joachim W., Andreas K., et al. Transgenic Indica rice breeding line IR58 expressing a synthetic CryIA(b) gene from Bacillus thuringiensis provides effective insect pest control. Bio/Technology, 1996,14:171-176
47 Juan F., et al. Resistance to the bacillus thuringiensis bioinsecticide in a field population of plutella xylostella is due to a change in a midgut membrane receptor. Proc. Natl. Acad. Sci. USA, 1991, 88:5119-5123
48 Kato T, Ttoh E, Whittien R F, et al. Increase of foreign gene expression in monocot and dicot cells by and intron in the 5' untranslated region of a soybean phosphoenolyruvate carboxylase gene. Bioscience Biotechnology Biochemistry, 1998,62(1): 152-1-153
49 Knight PJ, Crickmore N, Ellar DJ. The receptor for Bacillus thuringiensis Cry1A delta-endotoxin in the brush border membrane of the lepidopteran Manduca Sexta is aminopeptidase. N. Mol Microbiol, 1994,11:429-436
50 Knowles B and Dow J A T. The crystal δ-endotoxin of bacillus thuringiensis: models for their mechanism of action on the insect gut. Bioassays, 1993,15:469-476
51 Knowles BH. Mechanism of action of Bacillus thuringiensis insecticidal endotoxins. Adv Insect Physiol, 1994:24:275-308
52 Kostichka K, GW Warren, Mullins M, Mullins AD, Craig JA, Koziel MG and Estruch JJ. Cloning of a cryV-type insecticidal protein gene from Bacillus thuringiensis: the cryV-encoded protein is expressed early in stationary phase. J. Bacteriol. 1996,178:2141-2144
53 Kota M, Varma H, et al. Overexpression of the bacillus thuringiensis cry2A protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects. Proc. Natl. Acad. Sci. USA, 1999,96:1840-1845
54 Kozak M. Point mutations define a sequence flanding the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell, 1986,44:283-292
55 Krattiger A F. Insect resistance in crops: a case study of bacillus thuringiensis and its transfer to developing countries. ISAAA Briefs, 1997, No.2. ISAAA. Ithaca, New York
56 Leroy T, Henry A M, Royer M, Altosaar I, Frutos T, Duris D, Philippe R. Genetically modified coffee plants expressing the Bacillus thuringiensis cry1Ac gene for resistance to leaf miner. Plant Cell Reports, 2000, 19:382-389
57 Li J. D, Koni P. A, Ellar D J. Structure of the mosquitocidal δ-endotoxin CytB from Bacillus thuringensis SP. Kyushuensis and implications for membrane poration. Journal of Molecular Biology, 1996,257(1): 129-152
58 Li J. D., Carroll J., Ellar D.J. Crystal structure of insecticidal δ-endotoxin from Bacillus thuringensis at 2.5 A resolution, Nature,1991,353(31):815-821
59 Liu Y. B., Tabashnik B.E., Dennehy T.J., et al. Development time and resistance to Bt crops. Nature, 1999,400:519
60 Liu YJ, Wang GY. The inheritance and expression of Cry1A gene in transgenie maize. Acta Botanica Sinica, 2003, 45:253-256
61 Luehrsen KR, Walbot V. The impact of AUG start codon context on maize gene expression in vivo. Mol. Gen. Genet., 1994,13:454-458
62 Luehrsen KR, Waibot V. Inntron enhancement of gene expression and the splicing efficiency of introns in maize cells. Mol. Gen. Genet., 1991,225:81-93
63 Maas C, Laufs J, Grant S, et al. The combination of a novel stimulatory element in the first exon of the maize shrunken-1 gene with the following intron 1 enhance reporter gene expression up to 1000-fold. Plant Molecular Biology, 1991,16:199-207
64 Maqbool SB, Riazuddin S, Loc NT, Gatehouse AMR, Gatehouse JA, Christou E Expression of multiple insecticidal genes confers broad resistance against a range of different rice pests. Mol Breeding, 2001,7:85-93
65 Marnix P., Progress and prospects for field use of Bt genes in crops, 1997,15(5):173-177
66 Mascerenhas D, Mettler IJ, Pierce DA, et al. Intron mediated enhancement of heterologous gene expression in maize. Plant Mol. Biol., 1990,15:913-920
67 McBride K. E., Suab I., Schaaf D. E, et al. Amplification of and chimeric bacillus gene in chloroplasts leads to an extraordinary level of an insecticide protein in tobacco. Biotechnology, 1995, 13:362-365
68 Mcelroy D, Zhang W, Wu R. Isolation of an efficient promoter for use in rice transformation. Plant Cell, 1990,2:163-171
69 McElroy D., Blowers A., Jenes R, et al. Construction of expression vectors based on the rice actinl 5' region for use in monocot transformation. Mol. Gen. Genet., 1991,231:150-160
70 McGaughey W H and Whalon M E. Managing insect resistance to Bacillus thuringiensis toxins. 1992,258:1451
71 McGaughey W H. Insect resistance to the biological insecticide bacillus thuringiensis. Science, 1985,229:193-195
72 McGaughey WH, Gould F, Gelernter W. Bt resistance management. Nature Biotechnology, 1998, 16: 144-146
73 Mett V., Lochhead, et al. Copper-controllable gene expression system for whole plants. Proc. Natl. Acad. Sci. USA, 1993,90:4567-4571
74 Michael G, et al. Field performance of elite transgenie maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. 1993,11: 194-200
75 Murray C, Sutherland PW, Phung MM, Lester MT, Marshall RK and Christeller JT. Expression of biotin-binding proteins, avidin and streptavidin, in plant tissues using plant vacuolar targeting sequences. Transgenic Research, 2002, 11:199-214
76 Murray EE, Lotler J and Eberle M, Codon usage in plant genes, Nuleic Acids Research, 1989,17(2):477-491
77 Murray et al. Analysis of unstable RNA transcripts of insecticidal cyustal protein genes of bacillus thuringiensis in transgenic plants, and electroporated protoplasts. Plant Molecular Biology, 1991,16:1559-1564
78 Nayak P, Basu D, Das S, Basu A, Dipankar G, Ramakrishnan NA, Ghosh M, et al. Transgenic elite indica rice plants expressing CryIAc δ-endotoxin of Bacillus thuringiensis are resistant against yellow stem borer (Scirpophaga incertulas). Proc Natl Acad Sci USA, 1997, 94:2111-2116
79 Nicole CG, et al. Tissue-dependent enhancement of transgene expression by introns of replacement histone H3 henes of arabidopsis. Plant Molecular Biology, 2001,45:17-30
80 Ponstein AS, Bade JB, Verwoerd TC, Molendijk L, Storms J, Beudeker RF, Pen J. Stable expression of Phytase(phyA) in canola (Brassica napus) seeds: towards a commercial product. Mol. Breeding, 2002, 10:31-44
81 Rose AB and Beliakoff JA. Intron-mediated enhancement of gene expression independent of unique intron sequences and splicing. Plant Physiology, 2000,122:535-542
82 Rott R., Liveanu V, Drager RG, et al. The sequence and structure of the 3'-untranslated regions of chloroplast transcripts are important determinants of mRNA accumulation and stability. Plant Molecular Biology, 1998,36:307-314
83 Roush RT. Bt-transgenic crops: just another pretty insecticide or a chance for a new start in resistance management. Pesticide Science, 1997,51:328-334
84 Schaewen AV, Stitt M, Schmidt R, et al. Expression of a yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photosynthesis and strongly influences growth and phenotype of transgenic tobacco plants. The EMBO Journal, 1990,9(10):3033-3044
85 Song FP, Zhang J, Gu AX, Wu Y, Han LL. Identification of cry11-Type Genes from Bacillus thuringiensis Strains and Characterization of a Novel cry11-Type Gene. Applied Environmental Microbiology, 2003, 69:5207-5211
86 Spiker S and Thompson WF. Nuclear Matrix attachment regions and transgene expression in plants. Plant Physiology, 1996,110:15-21
87 Stewart SD, Adamczyk JJ, Knighten KS, Advis FM. Impact of Bt cottons expressing one or two insecticidal proteins of Bacillus thuringiensis Berliner on growth and survival of noctuid larvae. J Econ Entomol, 2001, 94:752-760
88 Stdzhov N, Keller M, Mathur J, et al, A synthetic crylc gene, encoding a Bacillus thuringiensis δ-endotoxin, confers spodoptera resistance in alfalfa and tobacco, Proc. Natl. Acad. Sci. USA, 1996,93:15012-15017
89 Sullivan TD, Christensen AH, Quail PH. Isolation and characterization of a maize chlorophyll a/b binding protein gene that produces high levels of mRNA in the dark. Mol Gen Genet,
1989,215:431-440
90 Tabashnik BE, Finson N, Johnson MW, Moar WJ. Resistance to toxins from Bacillus thuringiensis subsp. Kurstaki causes minimal cross-resistance to B. thuringiensis subsp. Aizawai in the diamondback moth (Lepidoptera: Plutellida). Appl Environ Microbiol, 1993, 59:1332-1335
91 Tabashnik BE, Liu YB, Finson N, Masson L, Heckel DG. One gene in diamondback moth confers resistance to four Bacillus thuringiensis toxins. Proc Natl Acad Sci USA, 1997, 94:1640-1644
92 Tabe L. M., Higgins C.M., McNabb W. C., et al. Genetic engineering of grain and pasture legumes for improved nutritive valve. Genetical, 1993,90:181-200
93 Tailor R, Tippett J, Gibb G, Pells S, Pike D, Jordon L and S. Ely. Identification and characterization of a novel Bacillus thuringiensis delta-endotoxin entomocidai to coleopteran ans lepidopteran larvae. Mol. Microbiol. 1992,6; 1211-1217
94 Takagi MO, Taylor CB, Newman TC, et al, The effect of sequences with high AU content on mRNA stability in tobacco, Proc. Natl. Acad. Sci., 1993,90:11811-11815
95 Tang JD, Smadar G, Roush RT, Shelton AM. Inheritance, stability, and lack-of-fitness costs of field-selected resistance to bacillus thuringiensis in diamondback moth (lepidoptera: Plutellidae) from Florida. J Econ Entomol, 1997, 90:732-741
96 Taylor JL, Jones JDG, Sandier S et al. Optimizing the expression of chimeric genes in plant cells. Mol. Gen. Genet., 1987,210:572-577
97 Vadlamudi RK, Weber E, et al. Cloning and expression of a rcceptor for an insecticidal toxin of Bacillus thuringiensis. J Biol Chem, 1995,270:5490-5494
98 Vaeck M, Reynaerts A, Hofte H, et al. Transgenic plants protected from insect attack. Nature, 1987,328:33-37
99 Van Rie J, McGaughey W H, Johnson D E, et al. Mechanism of insect resistance to the microbial insecticide bacillus thuringiensis. Science, 1990,247:72-74
100 Vankan P, EMBO J, 1988,7:791-799
101 Ward ER et al. The chemical regulation of plant transgene. Plant Molecular Biology, 1993,22:361-366
102 Wklliams S, Friedrich L, Dincher S, et al. Chemical Regulation of Bacillus thuringiensis δ-endotoxin expression in transgenic plants. Bio/Technology, 1992,10:540-543
103 Wong EY, Hironaka CM, Fischhoff DA. Arabidopsis thaliana small subunit leader and transit peptide enhance the expression of Bacillus thruingiensis proteins in transgenic plants. Plant Molecular Biology, 1992,20:81-93
104 Zhao JZ, Collins H, Tang JD, Cao J, Earle ED, et al. Development and characterization of diamondback moth resistance to transgenie broccoli expressing high levels of Cry1C. Appl Environ Microbiol, 2000, 66:3784-3789
105 Zhao JZ, Fan YL, Fan XL, Shi XP, Lu MG. Evaluation of transgenic tobacco expressing two insecticidal genes to delay resistance development of Helicoverpa armigera. Chinese Sci Bull, 1999,44:1871-1874
106 Zhao JZ, Li YX, Collins H, Cao J, Earle ED, et al. Different cross-resistance patterns in the diamondback moth (lepidoptera: Plutellidae) resistant to bacillus thuringiensis toxin Cry1C. J Econ Entomol, 2001, 94:1547-155
107 Zhou FY, Wang GY, Xie YJ, Cui HZ, Guo SD, Dai JR. Establishment of a genetic transformation system for maize inbred P9-10. Chinese Sci Bull, 1998, 44:624-627
2 郭三堆等，1999，双价抗虫转基因棉花研究，中国农业科学，32(3)：1-7
3 王国英等，1995，用基因枪将Bt毒蛋白基因转入玉米及转基因植株再生，中国科学(B辑)，25(1)：71-76
4 谢迎秋、朱祯等，一种强启动子的分离与功能，植物学报，2000，42(1)：50-54
5 晏小兰、邱国华、李宝健等，启动子对外源基因在转基因植物中表达的影响，中山大学学报(自然科学版)，1995，4(2)：60-67
6 周逢勇等，1998，玉米自交系P9-10遗传转化体系的建立，科学通报，43(23)：2517-2521
7 吴志平，徐步进．转基因植物释放后在环境中成为杂草的风险性[J]．生物工程进展，1999，19(1)：9-13．
8 钱迎倩 转基因作物的利弊分析 生物技术通报，1999，(5)：7-11
9 Adang MJ, Brody M S, Cardineau G. The reconstruction and expression of a Bacillus thuringiensis cryIIIA gene in protoplasts and potato plants. Plant Mol Biol, 1993,21: 1131-1145
10 Ali S and Taylor WC. Quantitative regulation of the Flaveria Mel gene is controlled by the 3'-untranslated region and sequences near the amino terminus. Plant Molecular Biology, 2001,46:251-261
11 Ali S and Taylor WC. The 3' non-coding region of a C4 photosynthesis gene increase transgene expression when combined with heterologous promoters. Plant Molecular Biology, 2001,46:325-333
12 Armstrong CL, Parker GB, Pershing JC, Brown SM, Sanders PR, Duncan DR, Stone T et al. Field evaluation of European corn borer control in progeny of 173 transgenie corn events expressing an insecticidal protein from Bacillus thuringiensis. Crop Sci, 1995, 35:550-557
13 Ballester V, Escriche B, Mensua jl, Riethmacher GW, Ferre J. Lack of cross-resistance to other Bacillus thuringiensis crystal proteins in a population of Plutella xylostella highly resistant to CryIA(b). Biocontrol Sci Technol, 1994, 4:437-443
14 Barton K, Whitely H., et al. Bacillus thuringensis δ-endotoxin in transgenic Nicotiana tabacum provides resistance to lepidopteran insets. Plant Physiol, 1987,85:1103-1109
15 Bergvinson D, Willcox M, Hoisington D. Efficacy and deployment of transgenic plants for stemborer management. Insect Sci Applic, 1997,(17) 1:157-167
16 Bohorova N, Frutos R, Royer M, Estaol P, Pacheco M, Rascón Q, McLean S, Hoisington D. Novel synthetic Bacillus thuringiensis cry1Bgene and the cry1B-cry1Abtranslational fusion confer resistance to southwestern corn borer, sugarcane borer and fall armyworm in transgenic tropical maize. Theor Appl Genet, 2001, 103:817-826
17 Bohorova N., Zhang W., Julstrum P., et al, Production of transgenic tropical maize with cry1A(b) and cry1A(c) genes via microprojectile bombardment of immatureembryos. Theor. Appl. Genet.,1999, 99:437-444
18 Brnke F, Hajirezaei M, Heineke D, Melzer M, Herbers K, Sonnewald U. High-level production of the noncariogenic sucrose isomer palatinose in transgenic tobacco plants strongly impairs development. Planta, 2002, 214:356-364
19 Bosch D., Schipper B, et al. Recombinant Bacillus thuringiensis crystal proteins with new properties: possibilities for resistance management. Bio/Technology, 12:915-918
20 Bruce E, et al. Global variation in the genetic and biochemical basis of diamondback moth resistance to bacillus thuringiensis, 1997, 94:12780-12785
21 Callis J, Fromm M, Walbot V. Introns increase gene expression in cultured maize cells. Genes. Devel., 1987,1: 1183-1200
22 Cao J, Zhao JZ, Tang JD, Shelton AM, Earle ED. Broccoli plants with pyramided crylAc and crylC Bt genes control diamondback moths resistant to Cry1A and Cry1C proteins. Theor. Appl. Genet, 2002,105:258-264
23 Cheng X Y, Sardana R, et al. Agrobacterium-transformed rice plants expressing synthetic CryIA(b) and CryIA(c) genes are highly toxic to striped stem borer and yellow stem borer. Proc. Natl. Acad. Sci. USA, 1998,95:2767-2772
24 Choi SK, Shin BS, Kong EM, Rho HM and Park SH. Cloning of a new Bacillus thuringiensis cry1I-type crystal protein gene. Crrrent Microbiology, 2000,41:65-69
25 Christensen AH, Sharrock RA, Quail PH. Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing and promoter activity following transfer to protoplasts by electroporation. Plant Mol Biol, 1992,18:675-689
26 Crickmore N. 2004 http://www.biols.susx.ac.uk/home/Neil_Crickmore/Bt/toxins2.html)
27 Dean C et al, Nuleic. Acids Research, 1986,14:2221-2240
28 Dowd PF. Indirect reduction of ear molds and associated mycotoxins in Bacillus thuringiensis corn under controlled and open field conditions: utility and limitation. Journal of Economic Entomology, 2000,93(6): 1669-1679
29 Estruch J. J., Warren G. W., Muillins M. A., et al. Vip3A, a novel bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidoteran insects, 1996,93:5389-5394
30 Farmer EE, Ryan CA. Octadecanoid precursors of jasmonie acid activate the synthesis of wound-inducible proteinase inhibitors. Plant Cell, 1992,4:371-382
31 Farran I, Snchez-Serrano JJ, Medina JF, Prieto J and Mingo-Castel AM. Targeted expression of human serum albumin to potato tubers. Transgenic Research, 2002, 11:337-346
32 Fischhoof et al. Insect tolerant transgenic tomato plants. Bio/Technology, 1987,5:807-812
33 Frutos R, Rang C, Royer M. Managing insect resistance to plants producing Bacillus thuringiensis toxins. Crit Rev Biotechnol, 1999, 19:227-276
34 Gahan LJ, Gould F, Heckel DG. Identification of a gene associated with Bt resistance in Heliothis virescens. Science, 2001,293:857-860
35 Gleave AP, Mitra DS, Markwick NP, et al. Enhanced expression of the Bacillus thuringiensis cry9Aa2 gene in transgenic plants by nucleotide sequence modification confers resistance to potato rubber moth. Molecular Breeding, 1998,4:459-472
36 Greenlate JT, Penn SR, Shappley Z, Oppenhuizen M, Mann J, Reich B, Osborn J. Bollgard Ⅱ efficacy: quantification of total lepidopteran activity in a two-gene product. Proc Beltwide Cotton Conf, 2000,vol2:1041-1043
37 Griffitts JS, Whitacre JL, Stevens DE, Aroian RV. Bt toxin resistance from loss of a prtative carbohydrate-modifying enzyme. Science, 2001,293:860-863
38 Grochuske P., Masson L, Borisova S., et al. Bacillus thuringensis cry1A(a) insecticidal toxin: cyustal structure and channel formation. Journal of Molecular Biology, 1995,254(3):447-464
39 Herbers K, Wilke I, Sonnewaid U. A thermostable xylanase from clostridium thermocellum expressed at high levels in the apoplast of transgenic tobacco has no detrimental effects and is easily purified. Bio/Technology,1995,13:63-66
40 Hilder V A et al. A novel mechanism of insect resistance engineered into tobacco. Nature, 1987,330:160-163
41 Honee G, Vriezen W, Visser B., et al. A translation fusion product of two different insecticidal crystal protein genes of Bacillus thuringiensis exhibits an enlarger insecticidal spectrum. Appl. And Envi. Mico, 1991,56(3):923-925
42 Hood EE, Murphy JM, Pendleton RC. Molecular characterization of maize extensin expression. Plant Molecular Biology, 1993,23:685-695
43 Huang E, Buschman L L, Higgins R.A., et al. Inheritance of resistance to bacillus thuringiensis toxin in the European corn borer. Science, 1999,284:965-967
44 Hudspeth R. Structure and expression of the maize gene encoding the phosphoenolpyruvate carboxylaase isozyme involved in C4 photosynthesis. Plant Molecular Biology, 1989,12:579-589
45 Jansens S, van Vliet A, Dickburt C, Buysse L, Piens C, Saey B, Wulf AD et al. Transgenic corn expressing a Cry9C insecticidal protein from Bacillus thuringiensis protected from European corn bore damage. Crop Sci, 1997, 37:1616-1624
46 Joachim W., Andreas K., et al. Transgenic Indica rice breeding line IR58 expressing a synthetic CryIA(b) gene from Bacillus thuringiensis provides effective insect pest control. Bio/Technology, 1996,14:171-176
47 Juan F., et al. Resistance to the bacillus thuringiensis bioinsecticide in a field population of plutella xylostella is due to a change in a midgut membrane receptor. Proc. Natl. Acad. Sci. USA, 1991, 88:5119-5123
48 Kato T, Ttoh E, Whittien R F, et al. Increase of foreign gene expression in monocot and dicot cells by and intron in the 5' untranslated region of a soybean phosphoenolyruvate carboxylase gene. Bioscience Biotechnology Biochemistry, 1998,62(1): 152-1-153
49 Knight PJ, Crickmore N, Ellar DJ. The receptor for Bacillus thuringiensis Cry1A delta-endotoxin in the brush border membrane of the lepidopteran Manduca Sexta is aminopeptidase. N. Mol Microbiol, 1994,11:429-436
50 Knowles B and Dow J A T. The crystal δ-endotoxin of bacillus thuringiensis: models for their mechanism of action on the insect gut. Bioassays, 1993,15:469-476
51 Knowles BH. Mechanism of action of Bacillus thuringiensis insecticidal endotoxins. Adv Insect Physiol, 1994:24:275-308
52 Kostichka K, GW Warren, Mullins M, Mullins AD, Craig JA, Koziel MG and Estruch JJ. Cloning of a cryV-type insecticidal protein gene from Bacillus thuringiensis: the cryV-encoded protein is expressed early in stationary phase. J. Bacteriol. 1996,178:2141-2144
53 Kota M, Varma H, et al. Overexpression of the bacillus thuringiensis cry2A protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects. Proc. Natl. Acad. Sci. USA, 1999,96:1840-1845
54 Kozak M. Point mutations define a sequence flanding the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell, 1986,44:283-292
55 Krattiger A F. Insect resistance in crops: a case study of bacillus thuringiensis and its transfer to developing countries. ISAAA Briefs, 1997, No.2. ISAAA. Ithaca, New York
56 Leroy T, Henry A M, Royer M, Altosaar I, Frutos T, Duris D, Philippe R. Genetically modified coffee plants expressing the Bacillus thuringiensis cry1Ac gene for resistance to leaf miner. Plant Cell Reports, 2000, 19:382-389
57 Li J. D, Koni P. A, Ellar D J. Structure of the mosquitocidal δ-endotoxin CytB from Bacillus thuringensis SP. Kyushuensis and implications for membrane poration. Journal of Molecular Biology, 1996,257(1): 129-152
58 Li J. D., Carroll J., Ellar D.J. Crystal structure of insecticidal δ-endotoxin from Bacillus thuringensis at 2.5 A resolution, Nature,1991,353(31):815-821
59 Liu Y. B., Tabashnik B.E., Dennehy T.J., et al. Development time and resistance to Bt crops. Nature, 1999,400:519
60 Liu YJ, Wang GY. The inheritance and expression of Cry1A gene in transgenie maize. Acta Botanica Sinica, 2003, 45:253-256
61 Luehrsen KR, Walbot V. The impact of AUG start codon context on maize gene expression in vivo. Mol. Gen. Genet., 1994,13:454-458
62 Luehrsen KR, Waibot V. Inntron enhancement of gene expression and the splicing efficiency of introns in maize cells. Mol. Gen. Genet., 1991,225:81-93
63 Maas C, Laufs J, Grant S, et al. The combination of a novel stimulatory element in the first exon of the maize shrunken-1 gene with the following intron 1 enhance reporter gene expression up to 1000-fold. Plant Molecular Biology, 1991,16:199-207
64 Maqbool SB, Riazuddin S, Loc NT, Gatehouse AMR, Gatehouse JA, Christou E Expression of multiple insecticidal genes confers broad resistance against a range of different rice pests. Mol Breeding, 2001,7:85-93
65 Marnix P., Progress and prospects for field use of Bt genes in crops, 1997,15(5):173-177
66 Mascerenhas D, Mettler IJ, Pierce DA, et al. Intron mediated enhancement of heterologous gene expression in maize. Plant Mol. Biol., 1990,15:913-920
67 McBride K. E., Suab I., Schaaf D. E, et al. Amplification of and chimeric bacillus gene in chloroplasts leads to an extraordinary level of an insecticide protein in tobacco. Biotechnology, 1995, 13:362-365
68 Mcelroy D, Zhang W, Wu R. Isolation of an efficient promoter for use in rice transformation. Plant Cell, 1990,2:163-171
69 McElroy D., Blowers A., Jenes R, et al. Construction of expression vectors based on the rice actinl 5' region for use in monocot transformation. Mol. Gen. Genet., 1991,231:150-160
70 McGaughey W H and Whalon M E. Managing insect resistance to Bacillus thuringiensis toxins. 1992,258:1451
71 McGaughey W H. Insect resistance to the biological insecticide bacillus thuringiensis. Science, 1985,229:193-195
72 McGaughey WH, Gould F, Gelernter W. Bt resistance management. Nature Biotechnology, 1998, 16: 144-146
73 Mett V., Lochhead, et al. Copper-controllable gene expression system for whole plants. Proc. Natl. Acad. Sci. USA, 1993,90:4567-4571
74 Michael G, et al. Field performance of elite transgenie maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. 1993,11: 194-200
75 Murray C, Sutherland PW, Phung MM, Lester MT, Marshall RK and Christeller JT. Expression of biotin-binding proteins, avidin and streptavidin, in plant tissues using plant vacuolar targeting sequences. Transgenic Research, 2002, 11:199-214
76 Murray EE, Lotler J and Eberle M, Codon usage in plant genes, Nuleic Acids Research, 1989,17(2):477-491
77 Murray et al. Analysis of unstable RNA transcripts of insecticidal cyustal protein genes of bacillus thuringiensis in transgenic plants, and electroporated protoplasts. Plant Molecular Biology, 1991,16:1559-1564
78 Nayak P, Basu D, Das S, Basu A, Dipankar G, Ramakrishnan NA, Ghosh M, et al. Transgenic elite indica rice plants expressing CryIAc δ-endotoxin of Bacillus thuringiensis are resistant against yellow stem borer (Scirpophaga incertulas). Proc Natl Acad Sci USA, 1997, 94:2111-2116
79 Nicole CG, et al. Tissue-dependent enhancement of transgene expression by introns of replacement histone H3 henes of arabidopsis. Plant Molecular Biology, 2001,45:17-30
80 Ponstein AS, Bade JB, Verwoerd TC, Molendijk L, Storms J, Beudeker RF, Pen J. Stable expression of Phytase(phyA) in canola (Brassica napus) seeds: towards a commercial product. Mol. Breeding, 2002, 10:31-44
81 Rose AB and Beliakoff JA. Intron-mediated enhancement of gene expression independent of unique intron sequences and splicing. Plant Physiology, 2000,122:535-542
82 Rott R., Liveanu V, Drager RG, et al. The sequence and structure of the 3'-untranslated regions of chloroplast transcripts are important determinants of mRNA accumulation and stability. Plant Molecular Biology, 1998,36:307-314
83 Roush RT. Bt-transgenic crops: just another pretty insecticide or a chance for a new start in resistance management. Pesticide Science, 1997,51:328-334
84 Schaewen AV, Stitt M, Schmidt R, et al. Expression of a yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photosynthesis and strongly influences growth and phenotype of transgenic tobacco plants. The EMBO Journal, 1990,9(10):3033-3044
85 Song FP, Zhang J, Gu AX, Wu Y, Han LL. Identification of cry11-Type Genes from Bacillus thuringiensis Strains and Characterization of a Novel cry11-Type Gene. Applied Environmental Microbiology, 2003, 69:5207-5211
86 Spiker S and Thompson WF. Nuclear Matrix attachment regions and transgene expression in plants. Plant Physiology, 1996,110:15-21
87 Stewart SD, Adamczyk JJ, Knighten KS, Advis FM. Impact of Bt cottons expressing one or two insecticidal proteins of Bacillus thuringiensis Berliner on growth and survival of noctuid larvae. J Econ Entomol, 2001, 94:752-760
88 Stdzhov N, Keller M, Mathur J, et al, A synthetic crylc gene, encoding a Bacillus thuringiensis δ-endotoxin, confers spodoptera resistance in alfalfa and tobacco, Proc. Natl. Acad. Sci. USA, 1996,93:15012-15017
89 Sullivan TD, Christensen AH, Quail PH. Isolation and characterization of a maize chlorophyll a/b binding protein gene that produces high levels of mRNA in the dark. Mol Gen Genet,
1989,215:431-440
90 Tabashnik BE, Finson N, Johnson MW, Moar WJ. Resistance to toxins from Bacillus thuringiensis subsp. Kurstaki causes minimal cross-resistance to B. thuringiensis subsp. Aizawai in the diamondback moth (Lepidoptera: Plutellida). Appl Environ Microbiol, 1993, 59:1332-1335
91 Tabashnik BE, Liu YB, Finson N, Masson L, Heckel DG. One gene in diamondback moth confers resistance to four Bacillus thuringiensis toxins. Proc Natl Acad Sci USA, 1997, 94:1640-1644
92 Tabe L. M., Higgins C.M., McNabb W. C., et al. Genetic engineering of grain and pasture legumes for improved nutritive valve. Genetical, 1993,90:181-200
93 Tailor R, Tippett J, Gibb G, Pells S, Pike D, Jordon L and S. Ely. Identification and characterization of a novel Bacillus thuringiensis delta-endotoxin entomocidai to coleopteran ans lepidopteran larvae. Mol. Microbiol. 1992,6; 1211-1217
94 Takagi MO, Taylor CB, Newman TC, et al, The effect of sequences with high AU content on mRNA stability in tobacco, Proc. Natl. Acad. Sci., 1993,90:11811-11815
95 Tang JD, Smadar G, Roush RT, Shelton AM. Inheritance, stability, and lack-of-fitness costs of field-selected resistance to bacillus thuringiensis in diamondback moth (lepidoptera: Plutellidae) from Florida. J Econ Entomol, 1997, 90:732-741
96 Taylor JL, Jones JDG, Sandier S et al. Optimizing the expression of chimeric genes in plant cells. Mol. Gen. Genet., 1987,210:572-577
97 Vadlamudi RK, Weber E, et al. Cloning and expression of a rcceptor for an insecticidal toxin of Bacillus thuringiensis. J Biol Chem, 1995,270:5490-5494
98 Vaeck M, Reynaerts A, Hofte H, et al. Transgenic plants protected from insect attack. Nature, 1987,328:33-37
99 Van Rie J, McGaughey W H, Johnson D E, et al. Mechanism of insect resistance to the microbial insecticide bacillus thuringiensis. Science, 1990,247:72-74
100 Vankan P, EMBO J, 1988,7:791-799
101 Ward ER et al. The chemical regulation of plant transgene. Plant Molecular Biology, 1993,22:361-366
102 Wklliams S, Friedrich L, Dincher S, et al. Chemical Regulation of Bacillus thuringiensis δ-endotoxin expression in transgenic plants. Bio/Technology, 1992,10:540-543
103 Wong EY, Hironaka CM, Fischhoff DA. Arabidopsis thaliana small subunit leader and transit peptide enhance the expression of Bacillus thruingiensis proteins in transgenic plants. Plant Molecular Biology, 1992,20:81-93
104 Zhao JZ, Collins H, Tang JD, Cao J, Earle ED, et al. Development and characterization of diamondback moth resistance to transgenie broccoli expressing high levels of Cry1C. Appl Environ Microbiol, 2000, 66:3784-3789
105 Zhao JZ, Fan YL, Fan XL, Shi XP, Lu MG. Evaluation of transgenic tobacco expressing two insecticidal genes to delay resistance development of Helicoverpa armigera. Chinese Sci Bull, 1999,44:1871-1874
106 Zhao JZ, Li YX, Collins H, Cao J, Earle ED, et al. Different cross-resistance patterns in the diamondback moth (lepidoptera: Plutellidae) resistant to bacillus thuringiensis toxin Cry1C. J Econ Entomol, 2001, 94:1547-155
107 Zhou FY, Wang GY, Xie YJ, Cui HZ, Guo SD, Dai JR. Establishment of a genetic transformation system for maize inbred P9-10. Chinese Sci Bull, 1998, 44:624-627