苏云金杆菌cry7Ab7基因的克隆表达及融合基因和工程菌构建
|
摘要
苏云金杆菌(Bacillus thuringiensis,简称Bt)是目前研究最深入的杀虫微生物,其杀虫蛋白基因也是应用最广泛和最有发展潜力的抗虫基因,因此,深入发掘Bt新菌株、分离克隆新型的杀虫基因对害虫的防治具有重要意义。本研究利用温度筛选法,对采自河北保定周边的的182份土壤样品进行分离,得到9株野生型Bt菌株。利用40对通用引物对9株野生型菌株的基因型进行PCR鉴定,分离克隆了对瓢甲科害虫有活性的新型杀虫蛋白基因,构建了新型Bt融合基因,为构建高效广谱的Bt工程菌和培育转基因抗虫植物提供新基因资源。本研究的主要内容和结果如下:
1从保定周边采集的182份土壤分离出9株Bt菌株,Bt菌株的平均分出率为4.95%,主要为小菱形、大菱形、方形、不规则形等晶体形态。
2.利用40对通用引物鉴定9株野生型Bt菌株的基因型,选择仅含有cry7基因的GW6菌株设计特异性引物cry7Forword/cry7Reverse,将PCR产物连接到pGEM Easy载体进行克隆,由上海生工完成了该基因的全序列测定,该基因核苷酸序列已在NCBI和GenBank中登记(Accession number:FJ940776),并由国际Bt-δ-内毒素基因命名委员会正式命名为cry7Ab7。cry7Ab7基因的读码框(ORF)3414bp,编码1138个氨基酸,亮氨酸(Leu)、异亮氨酸(Ile)和天冬酰胺(Asn)含量最高,分别为8.87%,7.11%,7.11%,预测分子量为129.64kDa。Cry7Ab7蛋白等电点pI 4.97,为弱酸性蛋白。
3.将cry7Ab7全长基因插入表达载体pET21b,转化E.coli BL21得到高效表达;利用Bt-E.coli穿梭载体pSXY422b,构建了重组质粒pSXY422b-7Ab7,转化Bt无晶体突变株HD73(cry-),得到工程菌Bt HD7AB,工程菌Bt HD7AB蛋白表达量是野生型菌株的2.6倍。生物测定结果表明,在E.coli中表达的融合蛋白和HD73(cry-)的晶体蛋白对马铃薯瓢虫(Henosepilachna vigintioctomaculata)2龄幼虫的LC50分别为1.1664μg/μL和0.779μg/μL。
4.以苏云金杆菌野生型菌株GW6和Bt11总DNA作为PCR反应模板,利用特异性引物CRY7AbF/CRY1IaR融合cry7Ab7和cry1Ia14基因得到融合基因Bt7Ia,克隆得到的全长基因提交GenBank登录号为GU462130。Bt7Ia基因具有Cry7Ab7的DomainⅠ和Cry1Ia14的DomainⅡ和DomainⅢ。将该基因分别插入表达载体pET21b和Bt-E.coli穿梭载体pSXY422b构建了重组质粒pET21b-7Ia和pSXY422b-7Ia,转化E.coli BL21和Bt无晶体突变株HD73(cry-),分别得到E.coli EC7Ia和Bt HD7Ia。在E.coli BL21中的EC7Ia获得了高效表达,而含有cry1I沉默基因片段的融合基因Bt7Ia,在无晶体突变株HD73(cry-)中不表达。生物活性测定表明EC7Ia的表达产物对粉纹夜蛾(Trichoplusia ni)和小菜蛾(Plutella xylostella)的LC50分别为2.0953μg/mL和0.3927μg/mL。这些结果为扩大Bt基因的杀虫谱以及Cry7Ab7和Cry1Ia14各功能域功能的研究奠定了基础。
Bacillus thuringiensis(Bt) is the most in-depth study of microbial pathogens, and its insecticidal protein gene is the most widely and potentially used against pest insect, Therefore, exploring new Bt strains and isolating novel insecticidal gene to control pest are of great significance. Using the temperature screening method, 9 Bacillus thuringiensis isolates were obtained from 182 soil samples collected from some locations of Baoding, Hebei provinces, identification of cry gene-type of Bt strains through PCR system and cloning, expression and insecticidal activity assay of new protein gene that is toxic to Coccinellidae, and subsequently a novel Bt fusion gene gene was cloned by PCR technique. In order to provide new genetic resources for constructing high Bt engineered strain and cultivating genetically modified plant. The main contents of this study are as follows:
1. 9 Bt strains were isolated from 182 soil samples in Baoding, Hebei Province by temperature screening method. On an average, 4.95% Separate rate of Bt strain. Crystal shapes could be observed, such as bipyramidal, square and so on.
2. The DNA template from 9 Wild-type Bt strains were amplified by using 40 pairs universal primers, then a pair of special primer with cry7 gene from GW6 strain only for the identification by PCR techniques. The results of sequencing and structural analysis showed that the gene, named Cry7Ab7 (GenBank accession number: FJ940776) by International Nomenclature Committee ofδ-endotoxin was the open reading frame encoded a 129.64kDa 1138 amino acids.
3. The recombinant plasmids, pET21b-7Ab7 and pSXY422b-7Ab7 were constructed by insertion of the cry7Ab7 gene into E.coli expression vector pET21b and Bt-E.coli shuttle vector pSXY422b. These two plasmids were transformed into E.coli BL21 and Bt HD73(cry-) strains to acquire two engineering strains. E.coli EC7Ab7 and Bt HD73(cry-), respectively. The results of SDS-PAGE suggested that cry7Ab7 gene was expressed with a high level, and the expressed products from the two engineering strains were toxic to second instar larvae of Henosepilachna vigintioctomaculata with the LC50 values of 1.1664μg/μL and 0.779μg/μL, respectively.
4. The DNA template from Bacillus thuringiensis (Bt) strain GW6 and Bt11 was used for PCR amplification with one pairs special primers designed on the basis of the sequence of CRY7AbF/CRY1IaR gene from Bt cry7Ab7 and cry1Ia14 gene to obtain fusion gene Bt7Ia, the full-length gene was designated Bt7Ia in GenBank (Accession No. GU462130). Bt7Ia gene was inserted into the BamHⅠ/SalⅠsites of E.coli expression vector pET21b and Bt-E.coli shuttle vector pSXY422b to obtain the recombinant plasmid pET21b-7Ia and pSXY422b-7Ia, While E.coli EC7Ia and the engineering strains Bt HD7Ia were acquired by constructed plasmid transformed into E.coli BL21 and Bt HD73(cry-) respectively. EC7Ia were high level expressed in E.coli, cry1I silence gene fragment containing the fusion Bt7Ia gene, which was not expressed in the acrystalliferous mutant (HD73cry-). The results of bioassay showed that the expressed product of the EC7Ia was highly toxic to the first-instar larvae of Trichoplusia ni and Plutella xylostella with the LC50 values of 2.0953μg/mL and 0.3927μg/mL, An expression plasmid with the fusion protein was also created for further research relating to transgenic crops and engineered microorganism to control pest.
1从保定周边采集的182份土壤分离出9株Bt菌株,Bt菌株的平均分出率为4.95%,主要为小菱形、大菱形、方形、不规则形等晶体形态。
2.利用40对通用引物鉴定9株野生型Bt菌株的基因型,选择仅含有cry7基因的GW6菌株设计特异性引物cry7Forword/cry7Reverse,将PCR产物连接到pGEM Easy载体进行克隆,由上海生工完成了该基因的全序列测定,该基因核苷酸序列已在NCBI和GenBank中登记(Accession number:FJ940776),并由国际Bt-δ-内毒素基因命名委员会正式命名为cry7Ab7。cry7Ab7基因的读码框(ORF)3414bp,编码1138个氨基酸,亮氨酸(Leu)、异亮氨酸(Ile)和天冬酰胺(Asn)含量最高,分别为8.87%,7.11%,7.11%,预测分子量为129.64kDa。Cry7Ab7蛋白等电点pI 4.97,为弱酸性蛋白。
3.将cry7Ab7全长基因插入表达载体pET21b,转化E.coli BL21得到高效表达;利用Bt-E.coli穿梭载体pSXY422b,构建了重组质粒pSXY422b-7Ab7,转化Bt无晶体突变株HD73(cry-),得到工程菌Bt HD7AB,工程菌Bt HD7AB蛋白表达量是野生型菌株的2.6倍。生物测定结果表明,在E.coli中表达的融合蛋白和HD73(cry-)的晶体蛋白对马铃薯瓢虫(Henosepilachna vigintioctomaculata)2龄幼虫的LC50分别为1.1664μg/μL和0.779μg/μL。
4.以苏云金杆菌野生型菌株GW6和Bt11总DNA作为PCR反应模板,利用特异性引物CRY7AbF/CRY1IaR融合cry7Ab7和cry1Ia14基因得到融合基因Bt7Ia,克隆得到的全长基因提交GenBank登录号为GU462130。Bt7Ia基因具有Cry7Ab7的DomainⅠ和Cry1Ia14的DomainⅡ和DomainⅢ。将该基因分别插入表达载体pET21b和Bt-E.coli穿梭载体pSXY422b构建了重组质粒pET21b-7Ia和pSXY422b-7Ia,转化E.coli BL21和Bt无晶体突变株HD73(cry-),分别得到E.coli EC7Ia和Bt HD7Ia。在E.coli BL21中的EC7Ia获得了高效表达,而含有cry1I沉默基因片段的融合基因Bt7Ia,在无晶体突变株HD73(cry-)中不表达。生物活性测定表明EC7Ia的表达产物对粉纹夜蛾(Trichoplusia ni)和小菜蛾(Plutella xylostella)的LC50分别为2.0953μg/mL和0.3927μg/mL。这些结果为扩大Bt基因的杀虫谱以及Cry7Ab7和Cry1Ia14各功能域功能的研究奠定了基础。
Bacillus thuringiensis(Bt) is the most in-depth study of microbial pathogens, and its insecticidal protein gene is the most widely and potentially used against pest insect, Therefore, exploring new Bt strains and isolating novel insecticidal gene to control pest are of great significance. Using the temperature screening method, 9 Bacillus thuringiensis isolates were obtained from 182 soil samples collected from some locations of Baoding, Hebei provinces, identification of cry gene-type of Bt strains through PCR system and cloning, expression and insecticidal activity assay of new protein gene that is toxic to Coccinellidae, and subsequently a novel Bt fusion gene gene was cloned by PCR technique. In order to provide new genetic resources for constructing high Bt engineered strain and cultivating genetically modified plant. The main contents of this study are as follows:
1. 9 Bt strains were isolated from 182 soil samples in Baoding, Hebei Province by temperature screening method. On an average, 4.95% Separate rate of Bt strain. Crystal shapes could be observed, such as bipyramidal, square and so on.
2. The DNA template from 9 Wild-type Bt strains were amplified by using 40 pairs universal primers, then a pair of special primer with cry7 gene from GW6 strain only for the identification by PCR techniques. The results of sequencing and structural analysis showed that the gene, named Cry7Ab7 (GenBank accession number: FJ940776) by International Nomenclature Committee ofδ-endotoxin was the open reading frame encoded a 129.64kDa 1138 amino acids.
3. The recombinant plasmids, pET21b-7Ab7 and pSXY422b-7Ab7 were constructed by insertion of the cry7Ab7 gene into E.coli expression vector pET21b and Bt-E.coli shuttle vector pSXY422b. These two plasmids were transformed into E.coli BL21 and Bt HD73(cry-) strains to acquire two engineering strains. E.coli EC7Ab7 and Bt HD73(cry-), respectively. The results of SDS-PAGE suggested that cry7Ab7 gene was expressed with a high level, and the expressed products from the two engineering strains were toxic to second instar larvae of Henosepilachna vigintioctomaculata with the LC50 values of 1.1664μg/μL and 0.779μg/μL, respectively.
4. The DNA template from Bacillus thuringiensis (Bt) strain GW6 and Bt11 was used for PCR amplification with one pairs special primers designed on the basis of the sequence of CRY7AbF/CRY1IaR gene from Bt cry7Ab7 and cry1Ia14 gene to obtain fusion gene Bt7Ia, the full-length gene was designated Bt7Ia in GenBank (Accession No. GU462130). Bt7Ia gene was inserted into the BamHⅠ/SalⅠsites of E.coli expression vector pET21b and Bt-E.coli shuttle vector pSXY422b to obtain the recombinant plasmid pET21b-7Ia and pSXY422b-7Ia, While E.coli EC7Ia and the engineering strains Bt HD7Ia were acquired by constructed plasmid transformed into E.coli BL21 and Bt HD73(cry-) respectively. EC7Ia were high level expressed in E.coli, cry1I silence gene fragment containing the fusion Bt7Ia gene, which was not expressed in the acrystalliferous mutant (HD73cry-). The results of bioassay showed that the expressed product of the EC7Ia was highly toxic to the first-instar larvae of Trichoplusia ni and Plutella xylostella with the LC50 values of 2.0953μg/mL and 0.3927μg/mL, An expression plasmid with the fusion protein was also created for further research relating to transgenic crops and engineered microorganism to control pest.
引文
[1]Mycogen C, San D. Bacillus thuringiensis and pesticidal proteins[J]. Microbiology and Molecular Biology Reviews, 1998 (62):775-806.
[2]http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/
[3]张杰,宋福平,李长友,等.对鞘翅目害虫高毒力Bt基因cry3Aa7的分离克隆及表达研究.中国农业科学. 2002,35(6): 650-653.
[4]王广君.高效广谱苏云金芽胞杆菌工程菌的构建及杀虫晶体蛋白的研究[D].中国农业科学院. 2005.
[5]乐超银,刘子铎,曾晓慧等.苏云金芽胞杆菌杀虫晶体蛋白基因cry3A在鳞翅目特异菌株中的表达及杀虫特性.微生物学报. 2000, 40(2): 139-142
[6]李荣森,陈涛.几种苏云金芽胞杆菌的毒力及形态结构[J].微生物学报,1981,21(3):311-317.
[7]Calabrese D M,Nickerson K W,Lane L C.A Comparison of Protein Cristal Subunit Size Bacillus thuringiensis [J].Can J Microbiol,1980,26:1006-1010.
[8]任改新,冯喜昌,冯维熊.苏云金芽胞杆菌伴胞晶体的形态及抗原特性[J].微生物学报,1983,23 (1): 57-62.
[9]Norris J R.The classification of Bacillus thuringiensis[J].Appl Bact,1964,27:439-447.
[10]Barjac de.Indentification of H-serotypes of Bacillus thuringiensis,In“Microbial Control of Pests and Plant Diseases 1970-1980”[M].Academic press,1981,34-44.
[11]张用梅,杨一平,陈宗胜.聚丙烯酰胺凝胶电泳对几个苏云金芽胞杆菌变种营养细胞酯酶图谱的分析[J].微生物学通报,1979,6 (1):6-8.
[12]喻子牛.苏云金芽胞杆菌的分类及生物活性蛋白基因[J].中国生物防治,1996,12(2):85-89.
[13]Hossain M A,Ahmed S,and Hoque S.Abundance and distribution of Bacillus thuringiensis in the agricultural soil of Bangladesh[J].Journal of Invertebrate Pathology,1997,70(3):221-225.
[14]Ichimatsu T,Mizuki E,Nishimura K,et al.Occurrence of Bacillus thuringiensis in fresh waters of Japan[J].Current Microbiology,2000,40(4):217-220.
[15]沉锦权.Bt 8010菌电镜观察生化反应及对家蚕的致病力测定[J].福建农业大学学报, 1996,25(1):61-65.
[16]Chaufaux J,archal M,Gilois N,et al.Investigation of natural strains of Bacillus thuringiensis in different biotopes throughout the world[J].Can J Microbiol,1997,43:337-343.
[17]冯书亮,任国栋.苏云金芽胞杆菌HBF-1及其在有害金龟治理中的应用[M].北京:中国农业科学技术出版社,2005.
[18]喻子牛.苏云金芽胞杆菌[M].北京:科学出版社,1990:34-37.
[19]SU Xu-dong, SHU Chang-long, ZHANG Jie, et.al. Identification and Distribution of Bacillus thuringiensis Isolates from Primeval Forests in Yunnan and Hainan Provinces and Northeast Region of China. Agricultural Sciences in China. 2007, 6(11): 1343-1351
[20]杨自文,吴宏文,王开梅,等.从土壤中高效分离苏云金芽胞杆菌的方法[J].中国生物防治,2000,16(1):6-3.
[21]Travers R S,M artin P A W,Reichelderfer C F.Selective process for efficient isolation of soil Bacillus spp[J].Applied and Environmental Microbiology,1987,53(6):1263-1266.
[22]中国科学院微生物研究所翻译.伯杰氏细菌鉴定手册[M].北京:科学出版社,1984.
[23]喻子牛,孙明,刘子铎,等.Bt的分类及生物活性蛋白基因[J].中国生物防治,1996,12 (2):85-89.
[24]Krieg A,Huger A M,Lagenbruch G A,et al.Bacillus thuringiensis subsp.tenebrionis:a new pathotype effective against larvae of Coleoptera [J].J Appl Entomol,1983,96:500-508.
[25]Arturo R R,Jorge E.Fingerprinting of Bacillus thuringiensis Type Strains and Isolates by Using Bacillus cereus Group-Specific Repetitive Extragenic Palindromic Sequence-Based PCR Analysis.Applied and Environmental Microbiology.2005,1346-1355.
[26]Fernando H V,Marliton R B.Bacillus thuringiensis survey in Brazil:geographical distribution and insecticidal activity against spodoptera frugiperda[J].Neotropical Entomology,2003,32 (4):639- 644.
[27]Barjac de.Indentification of H-serotypes of Bacillus thuringiensis,In“Microbial Control of Pests and Plant Diseases 1970-1980”[M].Academic press,1981,34-44.
[28]张用梅,陈宗胜.苏云金芽胞杆菌的酯酶分析[J].微生物学报[J],1981,21 (21):197-203.
[29]张用梅,陈宗胜,库竹,等.与苏云金芽胞杆菌各H-血清型相同但不产生晶体的芽胞杆菌的生化特性,酯酶型和毒力的比较[J].华中农学院学报,1983,2:42-47.
[30]喻子牛.苏云金芽胞杆菌的酯酶型[J].华中农学院学报,1983,4:55-63.
[31]姚江.高效广谱Bt Ly30株的分子生物学研究[D].中国农业科学院博士学位论文,2002.
[32]Heimpel A M.A critical review of Bacillus thuringiensis var.thuringiensis Berliner and other crystal liferous bacteria.Annual Review of Entomology,1967,12:287-322.
[33]Faust G M,Abe K,Held G A,et al.Evidence for Plasmid-Associated Crystal ToxinProduction in Bacillus thuringiensis subsp.Israelensis.Plasmid,1983,9:98-103.
[34]Edwards D L,Payne J,Soares G.Novel isolates of Bacillus thuringiensis having Activity against Nematodes.European Patent Application,1988,303-426.
[35]Baroy,Lecadet M M,Deleluse A.Cloning and Sequencing of Three New Putative Toxin Genes From Clostridium Bifermentans.Gene,1998,211:293-295.
[36]Drion G B.etal. Principless of Insect Pathology. Boston/Dordrecht/London: Kluwer Academic Publishers, 1998.
[37]Crickmore et al. Revision of the Nomenclature for the Bacillus thuringiensis Pesticidal Crystal Proteins. American Society for Microbiology, 1998, 62: 807–813
[38]Li J D, Carroll J, Ellar D J. Crystal structure of insecticidal delta-endotoxin from Bacillus thuringiensis at 2.5 A resolution[J]. Nature, 1991, 353: 815-821.
[39]Grochulski P, Masson L, Borisova S, et al. Bacillus thuringiensis CryIA(a) insecticidal toxin: crystal structure and channel formation[J]. J Mol Biol, 1995, 254: 447-464.
[40]Morse R J, Yamamoto T, Stroud R M. Structure of Cry2Aa suggests an unexpected receptor binding epitope[J]. Structure, 2001, 9: 409-417.
[41]Boonserm P, Davis P, Ellar D J, et al. Crystal structure od the mosquito-larvicidal toxin Cry4Ba and its biological implications[J]. Journal of Molecular Biology, 2005, 348: 363-382.
[42]Craig R, Pigott, David J, et al. Role of Receptors in Bacillus thuringiensis Crystal Toxin Activity[J]. Microbiol Mol Biol Rev, 2007, 71: 255-281.
[43]Sankaranarayanan R, Sekar K, Banerjee R, et al. A novel mode of carbohydrate recognition in jacalin, a Moraceae plant lectin with aβ-prism fold[J]. Nature structural biology, 1996, 3: 596-603.
[44]Shimizu T, Morikawa K. Theβ-prism: a new folding motif[J]. Trends in biochemical sciences, 1996, 21: 3-6.
[45]Grochulski P, Masson L, Borisova S, et al. Bacillus thuringiensis CryIA(a) insecticidal toxin: crystal structure and channel formation[J]. J Mol Biol 1995, 254: 447-464.
[46]Gerber D, Shai Y. Insertion and organization within membranes of the delta-endotoxin pore- forming domain, helix 4-loop-helix 5, and inhibition of its activity by a mutant helix 4 peptide[J]. The Journal of Biological Chemistry, 2000, 275(31): 23602-23607.
[47]Jurat-Fuentes J L, Adang M J. Importance of Cry1 delta-endotoxin domain II loops for binding specificity in Heliothis virescens [J]. Appl Environ Microbiol, 2001, 67: 323-329.
[48]Choe S, Bennett M J, Fujii G, et al. The crystal structure of diphtheria toxin[J]. Nature, 1992, 357: 216-222.
[49]Chen X J, Lee M K, Dean D H. Site-directed mutations in a highly conserved region of Bacillus thuringiensis delta-endotoxin affect inhibition of short circuit current across Bombyx mori midguts[J]. Proc Natl Acad Sci USA, 1993, 90: 9041-9045.
[50]Schnepf E, Crickmore N, Van R J, et al. Bacillus thuringiensis and its pesticidal crystal proteins[J]. Microbiol Mol Biol Rev, 1998, 62: 775-806.
[51]Li J, Koni P A, Ellar D J. Structure of the mosquitocidalδ-endotoxin CytB from Bacillus thuringiensis sp. kyushuensis and implica-tions for membrane pore formation[J]. Journal. Molecular. Biology, 1996, 257: 129-152.
[52]Schnepf H E, Lee S, Dojillo J, et al. Characterization of Cry34/Cry35 Binary Insecticidal Proteins from Diverse Bacillus thuringiensis Strain Collections[J]. Appl Environ Microbiol, 2005, 71: 1765-1774.
[53]Toshihiko A, Higuchi K, Mizuki E, et al. Nontoxic crystal protein from Bacillus thuringiensis demonstrates a remarkable structural similarity toβ-pore-forming toxins[J]. Proteins: Structure, Function, and Bioinformatics, 2006, 63(1): 243-248.
[54]黄大昉,林敏.农业微生物基因工程[M].北京:科学出版社,2001.
[55]White-shang Kuo and Kin-Fu Chak. Identification of Novel cry-Type Genes from Bacillus thuringiensis Strains on the Basis of Restriction Fragment LengthPolymorphism of the PCR-Amplified DNA Appl Environ Microbiol, 1996, p. 1369-137
[56]Du C and Nickerson J W.The Bacillus thuringiensis insecticidal toxin bind biotincontaining proteins.Appl.Envion Microbiol.1996.62:2932-2939.
[57]Kuo W S, Chak K F.Identification of novel cry-type genes from Bacillus thuringiensis strains on thebasis of restriction fragment length polymorphism of the PCR-amplified DNA[J].Appl Environ Microbiol,1996,62(4):1369-1377.
[58]宋福平,张杰,黄大昉,等.苏云金芽胞杆菌cry基因PCR-RFLP鉴定体系的建[J].中国农业科学, 1998,31(3):13-18
[59]程家安唐振华主编昆虫分子科学科学出版社2001.
[60]中国医学科学院药用植物资源开发研究所.中国药用植物栽培学[M].北京:农业出版社, 1991 : 684.
[51]李含毅,魏信平,粟均平.马铃薯瓢虫发生规律及其防治研究[J ].陕西农业科学,1996 , (3) :24 - 25.
[62]云雷,丁华,赵发胜,杜建军,白于山区马铃薯二十八星瓢虫发生特点与防治对策。陕西农业科学;2008(3);28-29
[63]Krieg A,Huger A M,Lagenbruch G A,et al.Bacillus thuringiensis subsp.tenebrionis:a new pathotype effective against larvae of Coleoptera [J].J Appl Entomol,1983,96:500-508.
[64]Entwistle P F,Cory J S,Bailey M J.Bacillus thurigiensis,An Environmengtal Bio-pestcide:Theory and Practice.UK:wiley Chichester Press,1993:125-146.
[65]Nicholas D,Stephen E.Transgenic Plant and Insect Pests Biocontrol.USA:John Wiley & Sons Press,1997:1-18.
[66]Wasano N,Yasunaga A C,Sato R,Komato T.Spherical parasporal inclusions of the lepidoptera- specific and coleoptera-specific Bt stains:a comparative electron microscopic study. Curr Mocrobiol,2000,40 (2):128-131.
[67]Zhang J,Schaier H U,Lereclus D,Agaisse H.Cloning and analysis of the first cry gene from Bacillus popilliae.J Bacterial.1997,179:4336-4341.
[68]Crikmore N.Bacillus thuringiensis toxin nomenclature[BD].http://www.Boils.susx.ac. uk/home/ Neil_Crickmore/Bt/toxins2.htmL. 2008.4.
[69]Rang C, Vachon V, de Maagd RA, et al. Interaction between functional domains of Bacillus thuringiensis insecticidal crystal proteins. Appl Environ Microbiol,1999,65:2918-2925
[70]袁美妗,邓日强,胡晓晖等.苏云金芽胞杆菌Cry1Aa和Cry3A结构域编码区的融合.农业生物技术学报. 2002, 10(3): 287-290
[71]Bravo A, Sarabia S, et al. Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection. Appl & Environ M icrobiol, 1998, 64 ( 12 ) :4965-4972
[72]Bendov E, Zaritsky A, et al. Margalith Y.Extended screening by PCR for seven cry group genes from field -collected strains of Bacillus thuringiensis. Appl & Environ M icrobiol,1997, 63 (12) : 4883-4890
[73]苏旭东.苏云金芽胞杆菌菌株的分离和cry基因的鉴定[D].保定:河北农业大学硕士学位论文,2005
[74]M.A. Santana, et al. Bacillus thuringiensis improved isolation methodology from soil samples[J]. Journal of Microbiological Methods 2008,75(2);357-358
[75]中国科学院微生物研究所.伯杰氏细菌鉴定手册[M].北京:科学出版社,1984.
[76]郭巍.粉纹夜蛾对苏云金芽胞杆菌Cry1Ac的抗性及其防御体系的研究[D].广州:中山大学, 2005.
[77]ZHAO J Z, CAO J, LI Y X, et al. Transgenic Plants Expressing Two Bacillus thuringiensis Toxins Delay Insect Resistance Evolution [J]. Nature Biotechnology, 2003, 21(12): 1493-1497.
[78]ABBOTT, W S. A Method of Computing the Effectiveness of an Insectcide [J]. J Econ Entomol, 1925, 18: 265-267.
[79]苏旭东,檀建新,张伟,等.苏云金芽胞杆菌的分离与鉴定[J].安徽农业科学,2006,34 (19):4835-4836.
[80]杨自文,吴宏文,王开梅,等.从土壤中高效分离苏云金芽胞杆菌的方法[J].中国生物防治, 2000,16(1):6-3.
[81]Travers R S,M artin P A W,Reichelderfer C F.Selective process for efficient isolation of soil Bacillus spp[J].Applied and Environmental Microbiology,1987,53(6):1263-1266.
[82]Wang JH (王津红) , Wu W H (吴卫辉) , Chen YH (陈月华) , Ren GX (任改新) . The distribution of Bacillus thuringiensis and cry gene diversity in China. BullM icrobiol (微生物学通报) , 2001, 28 ( 3) : 50-55
[83]Narva K E, Payne J M, Schwab G E, et al. European Patent Office no. EP 0462721, 1991.
[84]萨姆布鲁克J,弗里奇EF,曼尼阿蒂斯T.(金冬雁,黎孟枫,等译).分子克隆实验指南[M].第2版,北京:科学出版社, 1992.
[85]陈建武,唐丽霞,宋少云,等.苏云金芽胞杆菌vip3A基因的检测及保守性分析[J].生物工程报, 2003, 19(5): 538-543.
[86]宋萍,王勤英,等.苏云金芽胞杆菌WZ-9菌株对马铃薯瓢虫的毒力及其杀虫基因的研究农业生物技术学报(Journal of Agricultural Biotechnology). 2008,16 (3): 515-520
[87]Jurat-Fuentes J L, Adang M J. Importance of Cry1 delta-endotoxin domain II loops for binding specificity in Heliothis virescens. Appl Environ Microbiol, 2001, 67: 323-329.
[88]刘廷辉.苏云金芽胞杆菌菌株Bt1和GS8的研究[D].河北农业大学, 2009.
[89]中国科学院微生物研究所.伯杰氏细菌鉴定手册[M].北京:科学出版社,1984.
[90]丁之铨.杀虫遗传工程荧光假单孢菌的构建及生物学特性研究[D].北京:中国农业大学, 1997.
[91]Nickerson K W, Julian St G, Bulla L A. Physiology of sporeforming bacteria associated with insects: radiorespirometric survey of carbohydrate metabolism in the 12 serotypes of Bacillus thuringiensis[J]. Appl. Microbiol, 1974, 28: 129-132.
[92]胡晓婷,宋健,王容燕等.对铜绿丽金龟幼虫有活性的苏云金芽胞杆菌菌株的分离及鉴定.华北农学报,2008,23 ( 6 ) : 81-83
[93]Navon A. Control of lepidopteran pests with Bacillus thuringiensis. In: Bscillus thuringiensis, an Environmental Biopesticide: Theory and practice (eds. Entwistle P F, Cory J S, Bailey M J. Higgs S.), John Wiley and Sons, Chichester, 1993, 125-146.
[94]Herve A, dider L. How Does Bacillus thuringiensis produce so much insecticidal crystal protein, J Bacteriol,1995,21:6027-6032.
[95]Tabashnik B E. Delaying insect resistance to transgenic crops [J]. Proc Natl Acad Sci USA. 2008, 105:19029–19030.
[96]谭芙蓉,朱军等,李云艳,等.苏云金芽胞杆菌Rpp39杀虫晶体蛋白基因的鉴定及cry2Aa12基因的克隆表达.微生物学报, 2008; 48(5): 684-689.
[97]FUCHS R L, et al., Quantification of Bacillus thuringiensis insect control protein as expressed in transgenic plants ACS symposium series No. 432[R]. Analytical Chemistry of Bacillus thuringiensis, 1990, 105-113.
[98]BARTON K A, et al. Bacillus thuringiensisδ-Endotoxin Expressed in Transgenic Nicotiana tabacum Provides resistance to Lepidopteran insects[J ]. Plant Physiol, 1987, 85: 1103-1109.
[99]伍宁丰.苏云金芽胞杆菌HD-1δ-内毒素蛋白的纯化及其单克隆抗体细胞株的建立[J].中国农业科学, 1988, 21(6): 27-30.
[100]陈松.转基因抗虫棉组织中Bt毒蛋白表达的ELISA测定[J].江苏农业科学, 1997, 13 (3) : 154-156.
[101]王保民.转Bt基因植物毒蛋白免疫检测方法的研究[J].棉花学报, 1998, 10 (4) : 220-221.
[102]Jurat-Fuentes J L, Adang M J. Importance of Cry1 delta-endotoxin domain II loops for binding specificity in Heliothis virescens. Appl Environ Microbiol, 2001, 67: 323-329.
[103]Shin B S, Park S H, Choi S K, et al. Distribution of cryV-type insecticidal protein genes in Bacillus thuringiensis and cloning of cryV-type genes from Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. Entomocidus[J]. Appl Environ Microbiol, 1995, 61 (6): 2402-2407.
[104]宋福平,张杰,顾爱星等.苏云金芽胞杆菌cry1I基因沉默的研究[J].自然科学进展, 2002, 12: 934-938.
[105]Tailor R, Tippett J, Gibb G, et al. Identification and characterization of a novel Bacillus thuringiensis delta-endotoxin entomocidal to coleopteran and lepidopteran larvae[J]. Molecular Microbiology, 1992, 6:1211-1217
[106]Tounsi S, Jaoua S. Identification of promotor for the crystal protein-encoding gene cry1Ia from Bacillus thuringiensis in Diamonback moth (Lepidoplera:Plutellidac)[J]. Journal of Economic Entomelogy, 2007, 83:1671-1676.
[2]http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/
[3]张杰,宋福平,李长友,等.对鞘翅目害虫高毒力Bt基因cry3Aa7的分离克隆及表达研究.中国农业科学. 2002,35(6): 650-653.
[4]王广君.高效广谱苏云金芽胞杆菌工程菌的构建及杀虫晶体蛋白的研究[D].中国农业科学院. 2005.
[5]乐超银,刘子铎,曾晓慧等.苏云金芽胞杆菌杀虫晶体蛋白基因cry3A在鳞翅目特异菌株中的表达及杀虫特性.微生物学报. 2000, 40(2): 139-142
[6]李荣森,陈涛.几种苏云金芽胞杆菌的毒力及形态结构[J].微生物学报,1981,21(3):311-317.
[7]Calabrese D M,Nickerson K W,Lane L C.A Comparison of Protein Cristal Subunit Size Bacillus thuringiensis [J].Can J Microbiol,1980,26:1006-1010.
[8]任改新,冯喜昌,冯维熊.苏云金芽胞杆菌伴胞晶体的形态及抗原特性[J].微生物学报,1983,23 (1): 57-62.
[9]Norris J R.The classification of Bacillus thuringiensis[J].Appl Bact,1964,27:439-447.
[10]Barjac de.Indentification of H-serotypes of Bacillus thuringiensis,In“Microbial Control of Pests and Plant Diseases 1970-1980”[M].Academic press,1981,34-44.
[11]张用梅,杨一平,陈宗胜.聚丙烯酰胺凝胶电泳对几个苏云金芽胞杆菌变种营养细胞酯酶图谱的分析[J].微生物学通报,1979,6 (1):6-8.
[12]喻子牛.苏云金芽胞杆菌的分类及生物活性蛋白基因[J].中国生物防治,1996,12(2):85-89.
[13]Hossain M A,Ahmed S,and Hoque S.Abundance and distribution of Bacillus thuringiensis in the agricultural soil of Bangladesh[J].Journal of Invertebrate Pathology,1997,70(3):221-225.
[14]Ichimatsu T,Mizuki E,Nishimura K,et al.Occurrence of Bacillus thuringiensis in fresh waters of Japan[J].Current Microbiology,2000,40(4):217-220.
[15]沉锦权.Bt 8010菌电镜观察生化反应及对家蚕的致病力测定[J].福建农业大学学报, 1996,25(1):61-65.
[16]Chaufaux J,archal M,Gilois N,et al.Investigation of natural strains of Bacillus thuringiensis in different biotopes throughout the world[J].Can J Microbiol,1997,43:337-343.
[17]冯书亮,任国栋.苏云金芽胞杆菌HBF-1及其在有害金龟治理中的应用[M].北京:中国农业科学技术出版社,2005.
[18]喻子牛.苏云金芽胞杆菌[M].北京:科学出版社,1990:34-37.
[19]SU Xu-dong, SHU Chang-long, ZHANG Jie, et.al. Identification and Distribution of Bacillus thuringiensis Isolates from Primeval Forests in Yunnan and Hainan Provinces and Northeast Region of China. Agricultural Sciences in China. 2007, 6(11): 1343-1351
[20]杨自文,吴宏文,王开梅,等.从土壤中高效分离苏云金芽胞杆菌的方法[J].中国生物防治,2000,16(1):6-3.
[21]Travers R S,M artin P A W,Reichelderfer C F.Selective process for efficient isolation of soil Bacillus spp[J].Applied and Environmental Microbiology,1987,53(6):1263-1266.
[22]中国科学院微生物研究所翻译.伯杰氏细菌鉴定手册[M].北京:科学出版社,1984.
[23]喻子牛,孙明,刘子铎,等.Bt的分类及生物活性蛋白基因[J].中国生物防治,1996,12 (2):85-89.
[24]Krieg A,Huger A M,Lagenbruch G A,et al.Bacillus thuringiensis subsp.tenebrionis:a new pathotype effective against larvae of Coleoptera [J].J Appl Entomol,1983,96:500-508.
[25]Arturo R R,Jorge E.Fingerprinting of Bacillus thuringiensis Type Strains and Isolates by Using Bacillus cereus Group-Specific Repetitive Extragenic Palindromic Sequence-Based PCR Analysis.Applied and Environmental Microbiology.2005,1346-1355.
[26]Fernando H V,Marliton R B.Bacillus thuringiensis survey in Brazil:geographical distribution and insecticidal activity against spodoptera frugiperda[J].Neotropical Entomology,2003,32 (4):639- 644.
[27]Barjac de.Indentification of H-serotypes of Bacillus thuringiensis,In“Microbial Control of Pests and Plant Diseases 1970-1980”[M].Academic press,1981,34-44.
[28]张用梅,陈宗胜.苏云金芽胞杆菌的酯酶分析[J].微生物学报[J],1981,21 (21):197-203.
[29]张用梅,陈宗胜,库竹,等.与苏云金芽胞杆菌各H-血清型相同但不产生晶体的芽胞杆菌的生化特性,酯酶型和毒力的比较[J].华中农学院学报,1983,2:42-47.
[30]喻子牛.苏云金芽胞杆菌的酯酶型[J].华中农学院学报,1983,4:55-63.
[31]姚江.高效广谱Bt Ly30株的分子生物学研究[D].中国农业科学院博士学位论文,2002.
[32]Heimpel A M.A critical review of Bacillus thuringiensis var.thuringiensis Berliner and other crystal liferous bacteria.Annual Review of Entomology,1967,12:287-322.
[33]Faust G M,Abe K,Held G A,et al.Evidence for Plasmid-Associated Crystal ToxinProduction in Bacillus thuringiensis subsp.Israelensis.Plasmid,1983,9:98-103.
[34]Edwards D L,Payne J,Soares G.Novel isolates of Bacillus thuringiensis having Activity against Nematodes.European Patent Application,1988,303-426.
[35]Baroy,Lecadet M M,Deleluse A.Cloning and Sequencing of Three New Putative Toxin Genes From Clostridium Bifermentans.Gene,1998,211:293-295.
[36]Drion G B.etal. Principless of Insect Pathology. Boston/Dordrecht/London: Kluwer Academic Publishers, 1998.
[37]Crickmore et al. Revision of the Nomenclature for the Bacillus thuringiensis Pesticidal Crystal Proteins. American Society for Microbiology, 1998, 62: 807–813
[38]Li J D, Carroll J, Ellar D J. Crystal structure of insecticidal delta-endotoxin from Bacillus thuringiensis at 2.5 A resolution[J]. Nature, 1991, 353: 815-821.
[39]Grochulski P, Masson L, Borisova S, et al. Bacillus thuringiensis CryIA(a) insecticidal toxin: crystal structure and channel formation[J]. J Mol Biol, 1995, 254: 447-464.
[40]Morse R J, Yamamoto T, Stroud R M. Structure of Cry2Aa suggests an unexpected receptor binding epitope[J]. Structure, 2001, 9: 409-417.
[41]Boonserm P, Davis P, Ellar D J, et al. Crystal structure od the mosquito-larvicidal toxin Cry4Ba and its biological implications[J]. Journal of Molecular Biology, 2005, 348: 363-382.
[42]Craig R, Pigott, David J, et al. Role of Receptors in Bacillus thuringiensis Crystal Toxin Activity[J]. Microbiol Mol Biol Rev, 2007, 71: 255-281.
[43]Sankaranarayanan R, Sekar K, Banerjee R, et al. A novel mode of carbohydrate recognition in jacalin, a Moraceae plant lectin with aβ-prism fold[J]. Nature structural biology, 1996, 3: 596-603.
[44]Shimizu T, Morikawa K. Theβ-prism: a new folding motif[J]. Trends in biochemical sciences, 1996, 21: 3-6.
[45]Grochulski P, Masson L, Borisova S, et al. Bacillus thuringiensis CryIA(a) insecticidal toxin: crystal structure and channel formation[J]. J Mol Biol 1995, 254: 447-464.
[46]Gerber D, Shai Y. Insertion and organization within membranes of the delta-endotoxin pore- forming domain, helix 4-loop-helix 5, and inhibition of its activity by a mutant helix 4 peptide[J]. The Journal of Biological Chemistry, 2000, 275(31): 23602-23607.
[47]Jurat-Fuentes J L, Adang M J. Importance of Cry1 delta-endotoxin domain II loops for binding specificity in Heliothis virescens [J]. Appl Environ Microbiol, 2001, 67: 323-329.
[48]Choe S, Bennett M J, Fujii G, et al. The crystal structure of diphtheria toxin[J]. Nature, 1992, 357: 216-222.
[49]Chen X J, Lee M K, Dean D H. Site-directed mutations in a highly conserved region of Bacillus thuringiensis delta-endotoxin affect inhibition of short circuit current across Bombyx mori midguts[J]. Proc Natl Acad Sci USA, 1993, 90: 9041-9045.
[50]Schnepf E, Crickmore N, Van R J, et al. Bacillus thuringiensis and its pesticidal crystal proteins[J]. Microbiol Mol Biol Rev, 1998, 62: 775-806.
[51]Li J, Koni P A, Ellar D J. Structure of the mosquitocidalδ-endotoxin CytB from Bacillus thuringiensis sp. kyushuensis and implica-tions for membrane pore formation[J]. Journal. Molecular. Biology, 1996, 257: 129-152.
[52]Schnepf H E, Lee S, Dojillo J, et al. Characterization of Cry34/Cry35 Binary Insecticidal Proteins from Diverse Bacillus thuringiensis Strain Collections[J]. Appl Environ Microbiol, 2005, 71: 1765-1774.
[53]Toshihiko A, Higuchi K, Mizuki E, et al. Nontoxic crystal protein from Bacillus thuringiensis demonstrates a remarkable structural similarity toβ-pore-forming toxins[J]. Proteins: Structure, Function, and Bioinformatics, 2006, 63(1): 243-248.
[54]黄大昉,林敏.农业微生物基因工程[M].北京:科学出版社,2001.
[55]White-shang Kuo and Kin-Fu Chak. Identification of Novel cry-Type Genes from Bacillus thuringiensis Strains on the Basis of Restriction Fragment LengthPolymorphism of the PCR-Amplified DNA Appl Environ Microbiol, 1996, p. 1369-137
[56]Du C and Nickerson J W.The Bacillus thuringiensis insecticidal toxin bind biotincontaining proteins.Appl.Envion Microbiol.1996.62:2932-2939.
[57]Kuo W S, Chak K F.Identification of novel cry-type genes from Bacillus thuringiensis strains on thebasis of restriction fragment length polymorphism of the PCR-amplified DNA[J].Appl Environ Microbiol,1996,62(4):1369-1377.
[58]宋福平,张杰,黄大昉,等.苏云金芽胞杆菌cry基因PCR-RFLP鉴定体系的建[J].中国农业科学, 1998,31(3):13-18
[59]程家安唐振华主编昆虫分子科学科学出版社2001.
[60]中国医学科学院药用植物资源开发研究所.中国药用植物栽培学[M].北京:农业出版社, 1991 : 684.
[51]李含毅,魏信平,粟均平.马铃薯瓢虫发生规律及其防治研究[J ].陕西农业科学,1996 , (3) :24 - 25.
[62]云雷,丁华,赵发胜,杜建军,白于山区马铃薯二十八星瓢虫发生特点与防治对策。陕西农业科学;2008(3);28-29
[63]Krieg A,Huger A M,Lagenbruch G A,et al.Bacillus thuringiensis subsp.tenebrionis:a new pathotype effective against larvae of Coleoptera [J].J Appl Entomol,1983,96:500-508.
[64]Entwistle P F,Cory J S,Bailey M J.Bacillus thurigiensis,An Environmengtal Bio-pestcide:Theory and Practice.UK:wiley Chichester Press,1993:125-146.
[65]Nicholas D,Stephen E.Transgenic Plant and Insect Pests Biocontrol.USA:John Wiley & Sons Press,1997:1-18.
[66]Wasano N,Yasunaga A C,Sato R,Komato T.Spherical parasporal inclusions of the lepidoptera- specific and coleoptera-specific Bt stains:a comparative electron microscopic study. Curr Mocrobiol,2000,40 (2):128-131.
[67]Zhang J,Schaier H U,Lereclus D,Agaisse H.Cloning and analysis of the first cry gene from Bacillus popilliae.J Bacterial.1997,179:4336-4341.
[68]Crikmore N.Bacillus thuringiensis toxin nomenclature[BD].http://www.Boils.susx.ac. uk/home/ Neil_Crickmore/Bt/toxins2.htmL. 2008.4.
[69]Rang C, Vachon V, de Maagd RA, et al. Interaction between functional domains of Bacillus thuringiensis insecticidal crystal proteins. Appl Environ Microbiol,1999,65:2918-2925
[70]袁美妗,邓日强,胡晓晖等.苏云金芽胞杆菌Cry1Aa和Cry3A结构域编码区的融合.农业生物技术学报. 2002, 10(3): 287-290
[71]Bravo A, Sarabia S, et al. Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection. Appl & Environ M icrobiol, 1998, 64 ( 12 ) :4965-4972
[72]Bendov E, Zaritsky A, et al. Margalith Y.Extended screening by PCR for seven cry group genes from field -collected strains of Bacillus thuringiensis. Appl & Environ M icrobiol,1997, 63 (12) : 4883-4890
[73]苏旭东.苏云金芽胞杆菌菌株的分离和cry基因的鉴定[D].保定:河北农业大学硕士学位论文,2005
[74]M.A. Santana, et al. Bacillus thuringiensis improved isolation methodology from soil samples[J]. Journal of Microbiological Methods 2008,75(2);357-358
[75]中国科学院微生物研究所.伯杰氏细菌鉴定手册[M].北京:科学出版社,1984.
[76]郭巍.粉纹夜蛾对苏云金芽胞杆菌Cry1Ac的抗性及其防御体系的研究[D].广州:中山大学, 2005.
[77]ZHAO J Z, CAO J, LI Y X, et al. Transgenic Plants Expressing Two Bacillus thuringiensis Toxins Delay Insect Resistance Evolution [J]. Nature Biotechnology, 2003, 21(12): 1493-1497.
[78]ABBOTT, W S. A Method of Computing the Effectiveness of an Insectcide [J]. J Econ Entomol, 1925, 18: 265-267.
[79]苏旭东,檀建新,张伟,等.苏云金芽胞杆菌的分离与鉴定[J].安徽农业科学,2006,34 (19):4835-4836.
[80]杨自文,吴宏文,王开梅,等.从土壤中高效分离苏云金芽胞杆菌的方法[J].中国生物防治, 2000,16(1):6-3.
[81]Travers R S,M artin P A W,Reichelderfer C F.Selective process for efficient isolation of soil Bacillus spp[J].Applied and Environmental Microbiology,1987,53(6):1263-1266.
[82]Wang JH (王津红) , Wu W H (吴卫辉) , Chen YH (陈月华) , Ren GX (任改新) . The distribution of Bacillus thuringiensis and cry gene diversity in China. BullM icrobiol (微生物学通报) , 2001, 28 ( 3) : 50-55
[83]Narva K E, Payne J M, Schwab G E, et al. European Patent Office no. EP 0462721, 1991.
[84]萨姆布鲁克J,弗里奇EF,曼尼阿蒂斯T.(金冬雁,黎孟枫,等译).分子克隆实验指南[M].第2版,北京:科学出版社, 1992.
[85]陈建武,唐丽霞,宋少云,等.苏云金芽胞杆菌vip3A基因的检测及保守性分析[J].生物工程报, 2003, 19(5): 538-543.
[86]宋萍,王勤英,等.苏云金芽胞杆菌WZ-9菌株对马铃薯瓢虫的毒力及其杀虫基因的研究农业生物技术学报(Journal of Agricultural Biotechnology). 2008,16 (3): 515-520
[87]Jurat-Fuentes J L, Adang M J. Importance of Cry1 delta-endotoxin domain II loops for binding specificity in Heliothis virescens. Appl Environ Microbiol, 2001, 67: 323-329.
[88]刘廷辉.苏云金芽胞杆菌菌株Bt1和GS8的研究[D].河北农业大学, 2009.
[89]中国科学院微生物研究所.伯杰氏细菌鉴定手册[M].北京:科学出版社,1984.
[90]丁之铨.杀虫遗传工程荧光假单孢菌的构建及生物学特性研究[D].北京:中国农业大学, 1997.
[91]Nickerson K W, Julian St G, Bulla L A. Physiology of sporeforming bacteria associated with insects: radiorespirometric survey of carbohydrate metabolism in the 12 serotypes of Bacillus thuringiensis[J]. Appl. Microbiol, 1974, 28: 129-132.
[92]胡晓婷,宋健,王容燕等.对铜绿丽金龟幼虫有活性的苏云金芽胞杆菌菌株的分离及鉴定.华北农学报,2008,23 ( 6 ) : 81-83
[93]Navon A. Control of lepidopteran pests with Bacillus thuringiensis. In: Bscillus thuringiensis, an Environmental Biopesticide: Theory and practice (eds. Entwistle P F, Cory J S, Bailey M J. Higgs S.), John Wiley and Sons, Chichester, 1993, 125-146.
[94]Herve A, dider L. How Does Bacillus thuringiensis produce so much insecticidal crystal protein, J Bacteriol,1995,21:6027-6032.
[95]Tabashnik B E. Delaying insect resistance to transgenic crops [J]. Proc Natl Acad Sci USA. 2008, 105:19029–19030.
[96]谭芙蓉,朱军等,李云艳,等.苏云金芽胞杆菌Rpp39杀虫晶体蛋白基因的鉴定及cry2Aa12基因的克隆表达.微生物学报, 2008; 48(5): 684-689.
[97]FUCHS R L, et al., Quantification of Bacillus thuringiensis insect control protein as expressed in transgenic plants ACS symposium series No. 432[R]. Analytical Chemistry of Bacillus thuringiensis, 1990, 105-113.
[98]BARTON K A, et al. Bacillus thuringiensisδ-Endotoxin Expressed in Transgenic Nicotiana tabacum Provides resistance to Lepidopteran insects[J ]. Plant Physiol, 1987, 85: 1103-1109.
[99]伍宁丰.苏云金芽胞杆菌HD-1δ-内毒素蛋白的纯化及其单克隆抗体细胞株的建立[J].中国农业科学, 1988, 21(6): 27-30.
[100]陈松.转基因抗虫棉组织中Bt毒蛋白表达的ELISA测定[J].江苏农业科学, 1997, 13 (3) : 154-156.
[101]王保民.转Bt基因植物毒蛋白免疫检测方法的研究[J].棉花学报, 1998, 10 (4) : 220-221.
[102]Jurat-Fuentes J L, Adang M J. Importance of Cry1 delta-endotoxin domain II loops for binding specificity in Heliothis virescens. Appl Environ Microbiol, 2001, 67: 323-329.
[103]Shin B S, Park S H, Choi S K, et al. Distribution of cryV-type insecticidal protein genes in Bacillus thuringiensis and cloning of cryV-type genes from Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. Entomocidus[J]. Appl Environ Microbiol, 1995, 61 (6): 2402-2407.
[104]宋福平,张杰,顾爱星等.苏云金芽胞杆菌cry1I基因沉默的研究[J].自然科学进展, 2002, 12: 934-938.
[105]Tailor R, Tippett J, Gibb G, et al. Identification and characterization of a novel Bacillus thuringiensis delta-endotoxin entomocidal to coleopteran and lepidopteran larvae[J]. Molecular Microbiology, 1992, 6:1211-1217
[106]Tounsi S, Jaoua S. Identification of promotor for the crystal protein-encoding gene cry1Ia from Bacillus thuringiensis in Diamonback moth (Lepidoplera:Plutellidac)[J]. Journal of Economic Entomelogy, 2007, 83:1671-1676.