苏云金芽胞杆菌大质粒pBMB28的克隆

doi:10.3724/SP.J.1005.2011.01141

遗传 ›› 2011, Vol. 33 ›› Issue (10): 1141-1146.doi: 10.3724/SP.J.1005.2011.01141

苏云金芽胞杆菌大质粒pBMB28的克隆

戚军良, 朱义广, 尚卉, 纪芳, 朱倩, 孙明

华中农业大学生命科学技术学院, 农业微生物学国家重点实验室, 武汉 430070

收稿日期:2011-05-05 修回日期:2011-07-24 出版日期:2011-10-20 发布日期:2011-10-25
通讯作者: 孙明 E-mail:m98sun@mail.hzau.edu.cn
基金资助:
国家高技术研究发展计划项目(863计划)(编号：2011AA10A203), 国家重点基础研究发展规划(973计划)项目(编号：2009CB118902)和国家自然科学基金项目(编号：30870066)资助

Cloning of a large plasmid pBMB28 in Bacillus thuringiensis

QI Jun-Liang, ZHU Yi-Guang, SHANG Hui, JI Fang, ZHU Qian, SUN Ming

State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China

Received:2011-05-05 Revised:2011-07-24 Online:2011-10-20 Published:2011-10-25

摘要/Abstract

摘要： 苏云金芽胞杆菌幕虫亚种YBT-020具有典型的晶胞粘连表型。在前期的研究中, 通过质粒消除实验, 推测晶胞粘连现象与YBT-020内生质粒pBMB28有关。为了定位质粒pBMB28上控制晶胞粘连表型的基因, 首先对质粒pBMB28进行克隆。利用穿梭载体pEMB0557, 成功构建了苏云金芽胞杆菌YBT-020的基因组人工染色体(BAC)文库。前期的研究表明晶体蛋白基因cry28Aa定位在质粒pBMB28上, 根据cry28Aa基因序列设计引物, 从文库中筛选到含有cry28Aa的重组质粒pBMB231。镜检和SDS-PAGE证明质粒pBMB231转化无晶体突变株BMB171形成的重组子BMB231可以产生Cry28Aa晶体蛋白, 但不能恢复晶胞粘连表型。对重组质粒pBMB231的插入片段末端序列测定并设计引物筛选文库, 通过染色体步移方式得到4个可以重叠覆盖质粒pBMB28不同区域的克隆子, 从而克隆了该质粒。对这4个克隆子末端测序和酶切分析, 测算出该质粒的大小约为140 kb。进一步确定应用基因组BAC文库以及重叠片段筛选的方法, 可以快速有效的克隆苏云金芽胞杆菌大质粒。

关键词: 苏云金芽胞杆菌, 质粒, BAC文库, 染色体步移, 末端测序

Abstract: Bacillus thuringiensis serovar. finitimus strain YBT-020 is a typical strain with the spore-crystal association (SCA) phenotype. In our previous studies, plasmid curing experiment suggested that native plasmid pBMB28 of strain YBT-020 might contribute to the SCA phenotype. Thus, plasmid pBMB28 was cloned in order to isolate the genes related to SCA on pBMB28. Using shuttle vector pEMB0557, a shuttle genomic bacterial artificial chromosome (BAC) library of B. thuringiensis strain YBT-020 was constructed. The plasmid pBMB231 containing crystal protein gene cry28Aa, which was located on plasmid pBMB28, was screened out. By SDS-PAGE analysis and microscopic observation, we discovered the recombinant strain BMB231 that originated from the electrotransfer strain BMB171 with pBMB231 could produce Cry28Aa protein. With the chromosome walking strategy and terminal sequencing of pBMB231, four clones covering the full length of plasmid pBMB28 were screened out from this BAC library. With pulsed gel analysis of the four BAC clones and terminal sequencing, the size of the plasmid was calculated to be 140 kb. This study additionally revealed that we could clone a large plasmid from B. thuringiensis by genomic BAC library con-struction and overlaping fragment screening.

Key words: Bacillus thuringiensis, plasmid, BAC library, chromosome walking, terminal sequencing

戚军良，朱义广，尚卉，纪芳，朱倩，孙明. 苏云金芽胞杆菌大质粒pBMB28的克隆[J]. 遗传, 2011, 33(10): 1141-1146.

CU Jun-Liang, SHU Xi-An, CHANG Hui, JI Fang, SHU Qian, SUN Meng. Cloning of a large plasmid pBMB28 in Bacillus thuringiensis[J]. HEREDITAS, 2011, 33(10): 1141-1146.

参考文献

[1] 张宏宇, 邓望喜, 喻子牛. 苏云金芽胞杆菌的遗传多样性II. 杀虫晶体蛋白及其基因型. 遗传, 2000, 22(2): 125-128.
[2] Schnepf E, Crickmore N, Van-Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev, 1998, 62(3): 775-806.
[3] Debro L, Fitz-James PC, Aronson A. Two different parasporal inclusions are produced by Bacillus thuringiensis subsp. finitimus. J Bacteriol, 1986, 165(1): 258-268.
[4] Ji F, Zhu YG, Ju SY, Zhang R, Yu ZN, Sun M. Promoters of crystal protein genes do not control crystal formation inside exosporium of Bacillus thuringiensis ssp. finitimus strain YBT-020. FEMS Microbiol Lett, 2009, 300(1): 11-17.
[5] Wojciechowska JA, Lewitin E, Revina LP, Zalunin IA, Chestukhina GG. Two novel delta-endotoxin gene families cry26 and cry28 from Bacillus thuringiensis ssp. finitimus. FEBS Lett, 1999, 453(1-2): 46-48.
[6] Lopez-Meza JE, Ibarra JE. Characterization of a novel strain of Bacillus thuringiensis. Appl Environ Microbiol, 1996, 62(4): 1306-1310.
[7] Carlton BC, Gonzalez JM. Plasmid and delta-endotoxin production in different subspecies of Bacillus thuringiensis. In: Hoch JA, Setlow P, eds. Molecular biology of microbial differentiation. Washington DC: American Society for Microbiology, 1985: 246-252.
[8] 孙明, 魏芳, 刘子铎, 喻子牛. 苏云金芽胞杆菌质粒pBMB2062的克隆及遗传稳定载体的构建. 遗传学报, 2000, 27(10): 932-938.
[9] Baum JA, Coyle DM, Gilbert MP, Jany CS, Gawron-Burke C. Novel cloning vectors for Bacillus thuringiensis. Appl Environ Microbiol, 1990, 56(11): 3420-3428.
[10] Baum JA, Gonzalez JM Jr. Mode of replication, size and distribution of naturally occurring plasmids in Bacillus thuringiensis. FEMS Microbiol Lett, 1992, 75(2-3): 143-148.
[11] Zhong CY, Peng DH, Ye WX, Chai LJ, Qi JL, Yu ZN, Ruan LF, Sun M. Determination of plasmid copy number reveals the total plasmid DNA amount is greater than the chromosomal DNA amount in Bacillus thuringien-sis YBT-1520. PLoS One, 2011, 6(1): e16025.
[12] 王莉, 郭素霞, 黄军艳, 喻子牛, 孙明. 苏云金芽胞杆菌大质粒pBMB165的克隆与分析. 微生物学报, 2008, 48(1): 15-20.
[13] 鞠守勇, 纪芳, 朱自敏, 喻子牛, 孙明, 汤治国, 张睿. 苏云金芽胞杆菌幕虫亚种晶胞粘连现象与晶体蛋白基因cry26Aa所在质粒有关. 微生物学报, 2007, 47(1): 88-91.
[14] 李林, 杨超, 刘子铎, 李阜棣, 喻子牛. 苏云金芽孢杆菌无晶体突变株的逐级升温筛选及其转化性能. 微生物学报, 2000, 40(1): 85-90.
[15] Liu XY, Peng DH, Luo Y, Ruan LF, Yu ZN, Sun M. Con-struction of an Escherichia coli to Bacillus thuringiensis shuttle vector for large DNA fragments. Appl Microbiol Biotechnol, 2009, 82(4): 765-772.
[16] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A laboratory manual. 2nd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory, 1989.
[17] Peng D, Luo Y, Guo S, Zeng H, Ju S, Yu Z, Sun M. Elaboration of an electroporation protocol for large plas-mids and wild-type strains of Bacillus thuringiensis. J Appl Microbiol, 2009, 106(6): 1849-1858.
[18] Luo MZ, Wing RA. An improved method for plant BAC library construction. Methods Mol Biol, 2003, 236: 3-19.
[19] 齐义鹏. 基因及其操作原理. 武汉: 武汉大学出版社, 1998.
[20] 孙明, 吴岚, 刘子铎, 李阜棣, 喻子牛, Dean DH. 苏云金芽胞杆菌杀虫晶体蛋白基因cry218的克隆和表达. 农业生物技术学报, 1996, 4(3): 293-297.
[21] 朱晨光, 孙明, 喻子牛. 苏云金芽胞杆菌CTC菌株的S-层蛋白可以形成伴胞晶体. 微生物学报, 2002, 42(6): 670-674.
[22] Clarke L, Carbon J. Biochemical construction and selection of hybrid plasmids containing specific segments of the Escherichia coli genome. Proc Natl Acad Sci, 1975, 72(11): 4361-4365.
[23] Zhu YG, Shang H, Zhu Q, Ji F, Wang PX, Fu JJ, Deng Y, Xu CC, Ye WX, Zheng JS, Zhu L, Ruan LF, Peng DH, Sun M. Complete genome sequence of Bacillus thuringiensis serovar. finitimus Strain YBT-020. J Bacteriol, 2011, 193(9): 2379-2380.

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

苏云金芽胞杆菌大质粒pBMB28的克隆

Cloning of a large plasmid pBMB28 in Bacillus thuringiensis

PDF (PC)

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

[1]	徐纪明, 朱建树, 李梦真, 胡晗, 毛传澡. 侧翼序列获取技术研究进展[J]. 遗传, 2022, 44(4): 313-321.
[2]	家琳达, 高坦坦, 彭琦, 吕静, 张杰, 陈敏, 宋福平. yhcZ基因在苏云金芽胞杆菌生长中的功能研究[J]. 遗传, 2018, 40(5): 415-424.
[3]	易灵娴,刘艺云,吴仁杰,梁梓森,刘健华. 质粒介导的黏菌素耐药基因mcr-1研究进展[J]. 遗传, 2017, 39(2): 110-126.
[4]	朱军莉，李德葆，余旭平. 阴沟肠杆菌B8拮抗活性基因‘admA’及上游调控序列的克隆与功能鉴定[J]. 遗传, 2012, 34(4): 495-502.
[5]	张巨永，黄盛丰，王蔚，徐安龙，王义权. 白氏文昌鱼单个体基因组BAC文库的构建[J]. 遗传, 2010, 32(1): 67-72.
[6]	刘博，苏乔，汤敏谦，袁晓东，安利佳. 应用于染色体步移的PCR扩增技术的研究进展[J]. 遗传, 2006, 28(5): 587-595.
[7]	汪宗桂，郑文岭，崔东，马文丽. 哺乳动物中基因持续表达的策略[J]. 遗传, 2004, 26(5): 745-748.
[8]	严世荣，龚坚，严洁，邱云城. pET28a-TAT-LacZ重组子的构建[J]. 遗传, 2003, 25(2): 141-144.
[9]	钟万芳，蔡平钟，阎文昭，裴炎. 苏云金芽孢杆菌大型质粒DNA的小量提取[J]. 遗传, 2003, 25(1): 71-72.
[10]	刘成君，Walter Klingmüller. 体内遗传标记成团肠杆菌固氮质粒pEA9[J]. 遗传, 2002, 24(4): 455-100.
[11]	张利莉，Aronson IArthur，喻子牛. 碳源对苏云金芽胞杆菌融合基因cry1D-lacZ表达的影响[J]. 遗传, 2002, 24(3): 301-304.
[12]	陈琪，陈向东，谢志雄，沈萍. 遗传工程微生物细胞间发生的自然遗传转化[J]. 遗传, 2000, 22(3): 140-143.
[13]	张宏宇，邓望喜，喻子牛. 苏云金芽胞杆菌的遗传多样性II. 杀虫晶体蛋白及其基因型[J]. 遗传, 2000, 22(2): 125-128.
[14]	魏东，周俊初. 用电脉冲法将外源质粒DNA导入花生根瘤菌的研究[J]. 遗传, 1996, 18(3): 30-33.
[15]	刘巍峰，高东. 酿酒酵母嗜杀质粒(dsRNA)的提取与分离纯化[J]. 遗传, 1995, 17(5): 41-44.