外源质粒电击转入Pseudomonas putida的条件研究

中国生物工程杂志

2009, Vol. 29

Issue (08): 81-85

技术与方法

外源质粒电击转入Pseudomonas putida的条件研究

邓婷婷¹,李晖²,刘燕²,郑元元²,李春³

1. 石河子大学农学院/新疆兵团化工绿色过程重点实验室石河子 832003
2. 北京理工大学生命科学与技术学院生物工程系北京 100081

Study on Electrotransformation of vitro DNA into Pseudomonas putida

DENG Ting-Ting¹, LI Hui¹, LIU Yan², ZHENG Yuan-Yuan², LI Chun^1,2

1.Agricultural College of Shihezi University/Key Laboratory for Green Processing of Xinjiang Bingtuan,Shihezi832003,China
2.School of Life Science and Technology, Beijing Institute of Technology, Beijing100081,China

全文: PDF(619 KB) HTML

摘要：

以自行筛选的恶臭假单胞菌（Pseudomonas putida）(命名为Rs198，Genbank登录号为FJ788425）为受体菌，将具有卡那霉素抗性标记的大肠杆菌假单胞菌穿梭质粒PDSK519通过电转化法导入到受体菌中，对细胞生长状态、电转化温度、质粒DNA及感受态细胞浓度、电击电压及电转化介质给予转化效率的影响进行研究。结果表明，在细胞生长至OD600为0.5左右时收集菌体，在低温条件下制备浓度为 4.6×1012/ml 的感受态细胞，以0.3mol/L的蔗糖为电转化介质，在13kV/cm的场强下电击能获得较高的转化效率，最高可达1.3×107个转化子/μ g DNA。为构建恶臭假单胞的遗传转化系统，利用基因工程手段为该菌的进一步研究奠定了理论基础。

关键词： 恶臭假单胞菌;电转化;转化效率;转化条件;外源质粒

Abstract:

Pseudomonas putida isolated relieving salt stress and promoting plant growth was used as the recipient strain. Optimum conditions including growth stage of the strain, concentration of competent cell, electroshock voltage and the meduim were investigated for the electroporation of P.putidaE.coli shulter vector PDSK519 into P.putida Rs198. It was showed that the highest transformation efficiency was up to 1.3×107CFU/μ g DNA in the optimium condition, under which the competent cells were collected at logarithmic growth phase(OD600=0.5), the mixture of the competent cells(4.6×1012/ml) and PDSK519 plasmid DNA was electroporated at 13kV/cm. It will be very helpful for developing a genetic transformation system and studying the genomic function of P.putida.

Key words: Pseudomonas putida Electrotransformation Efficiency Condition Plasmid

收稿日期: 2009-03-10 出版日期: 2009-07-28

ZTFLH:

Q785

基金资助:

国家自然科学基金(20776017)、新疆维吾尔自治区高新技术重点项目（200811108)资助项目

通讯作者: 邓婷婷

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	邓婷婷
	李晖
	刘燕
	郑元元
	李春

引用本文:

邓婷婷,李晖,刘燕,郑元元,李春. 外源质粒电击转入Pseudomonas putida的条件研究[J]. 中国生物工程杂志, 2009, 29(08): 81-85.

DENG Ting-Ting, LI Hui, LIU Yan, ZHENG Yuan-Yuan, LI Chun. Study on Electrotransformation of vitro DNA into Pseudomonas putida. China Biotechnology, 2009, 29(08): 81-85.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2009/V29/I08/81

［1］ Ma Zhan, Zhang De chun. Development and prospects of genetic engineered Pseudomonas Application. Chinese Journal of Microecology, 2003, 15(3) : 186～187
［2］ Masato U, Brian R, Elizabeth A.Cd adsorption onto Pseudomonas putida in the presence and absence of extracellular polymeric substances. Geochemical et Cosmochimica Acta. 2008, 72: 5885～5895
［3］翁酥颖.环境微生物学.北京：科学出版社，1987
Wong S Y. Environmental Microbiology.Beijing: Science Press, 1987
［4］ Atkinson B W, Mudaly D D. Contribution of Pseudomonas to phosphorus uptake in the anoxic zone of an anaerobic anoxic aerobic continuous activated sludge system. Water Science and Technology, 2001, 43 ( 1 ):139～146
［5］ Lei Cai, MeiQing Yuan, Feng Liu,et al. Enhanced production of mediumchainlength polyhyhroxyalkanoates (PHA) by PHA depolymerase knockout mutant of Pseudomonas putida KT2442. Bioresource Technology, 2009, 100: 2265～2270
［6］ Olivera E R, Carnicero D, Jodra R. Genetically engineered Pseudomonas: a factory of new bioplastics with broad applications.Environ Microbiol, 2001, 3 (10): 612～618
［7］ Glick B R, Karaturovic D M, Newell P C. A novel procedure for rapid isolation of plant growth promoting pseudomonas.Canada Microbiol, 1995, 41: 533～536
［8］杨海君，谭周进，肖启明，等.假单胞菌的生物防治作用研究.中国生态农业学报，2004， 12（3）：158～161
Yang H J, Tan Z J, Xiao Q M,et al.Chinese Journal of EcoAgriculture,2004， 12（3）：158～161
［9］ Jackson W A,Pardue J H.Potential for enhancement of biodegradation of crude oil in Louisiana salt marshes using nutrient amendments. Water, Air, Soil and Pollution,1999, 109: 343～355
［10］陈晓斌，张炳欣，何勇.假单胞菌属生防菌株的遗传工程改良.微生物学通报，2000，27(4)：293～296
Chen X B, Zhang B X, He Y.Acta Microbiologica Sinica, 2000，27(4)：293～296
［11］ Shen H B, Han F, Lin Y Z,et al. A high efficient electroporation of Pseudomonas sp. QDA pretreated with alginate lyase. Enzyme and Microbial Technology,2006, 39: 677～682
［12］ Lennard S. Standard protocol for the construction of ScFv libraries. Methods in Molecular Biology, 2002, 178: 59～60
［13］郑元元，岳海涛，石在强，等.盐胁迫下解盐促生细菌Rs5和恶臭假单胞菌(P.putida) Rs198促进棉花种子发芽的机理探讨.中国农业科学，2008，41(5)：1326～1332
Zheng Y Y, Yue H T, Shi Z Q,et al.Scientia Agricultura Sinica, 2008,41(5):1326~1332
［14］ Shan Z Y, Xu H J, Shi X Q, Acta Genetic asinica, 2004, 31(3): 311～316
［15］彭惠，傅百灵，毛忠贵，等.嗜热厌氧乙醇菌 JW200转化条件的研究.中国生物工程杂志, 2006, 26(11): 59～61
Peng H, Fu B, Mao Z G,et al. China Biotechnology, 2006, 26(11): 59～61
［16］ Tu Z M, He G Y, Li K X. An improved system for competent cell preparation and high efficiency plasmid transformation using different E.coli strains.Electron Biotechnology, 2005, 8(1): 113～120
［17］令利军，朱艳，王红梅,等.保存时间及温度对大肠杆菌感受态转化效率的影响.生物技术通讯，2004, 15 (1): 51～52
Ling L J, Zhu Y, Wang H M,et al.Letters in Biotechnology，2004, 15 (1): 51～52
［18］ Kimoto H, Taketo A. Efficient electrotransformation system and gene targeting in phylogenic streptococci.Biosocial Biotechnology Biochemist, 2003, 67 (10): 2203～2209
［19］ Tackle E, Astumian R D, Chock P B. Selective and a symmetric molecular transport across electroporated cellmembranes.Proc Nat Acad Sci USA. 1994, 91(24): 1151～1152
［20］周莹冰，申晓冬，丛延广，等.铜绿假单胞菌噬菌体PaP3基因组电转化宿主菌的条件初探.医学研究生学报，2007，20 (11): 1134～1138
Zhou Y B, Shen X D, Cong Y G,Journal of Medical Postgraduates，2007，20 (11): 1134～1138

[1]	高瑞平, 程隆斌, 李振秋. 一种简便快速的单引物PCR定点突变方法[J]. 中国生物工程杂志, 2015, 35(5): 61-65.
[2]	刘培磊, 康定明, 李宁. 我国转基因技术风险交流分析[J]. 中国生物工程杂志, 2011, 31(8): 145-149.
[3]	宋修鹏, 武波, 申佩弘, 蒋承建, 田丹丹, 唐咸来. *甲基营养菌Methylobacterium* sp. MB200中部分一碳代谢相关基因的定位及mtdA和mtdB 基因的克隆研究**[J]. 中国生物工程杂志, 2011, 31(02): 50-55.
[4]	刘丽军, 宋敏, 苏颖异. 主要农作物转化事件的专利保护及对我国的启示[J]. 中国生物工程杂志, 2010, 30(11): 112-117.
[5]	许丽丽, 阎光宇, 王全喜, 吴双秀. 转基因衣藻lba及对照藻产氢培养条件的优化[J]. 中国生物工程杂志, 2010, 30(11): 44-49.
[6]	蔡文伟,王正鹏,张树珍,杨本鹏. 甘蔗茎杆特异表达基因启动子的克隆及初步分析[J]. 中国生物工程杂志, 2009, 29(08): 92-96.

Viewed

Full text

Abstract

Cited

Shared

Discussed