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应用“酶切连接”克隆法构建TALEs 被引量：1

Construction of TALEs by′Digestion-Ligation′Cloning
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摘要 TALEs是在植物病原菌黄单胞杆菌(Xanthomonas)上发现的一类天然的DNA结合蛋白,其重复域已经成为模块化构建单元,用于人工DNA结合蛋白的构建。人工TALEs能够用于多种基因工程操作,包括转录调节和基因组编辑。为了在常规分子生物学实验室稳定、经济地构建TALE-TFs和TALENs,本文采用TALE Toolbox试剂盒,运用PCR扩增和"酶切-连接"克隆法构建人工TALEs。结果表明,该构建策略是一种快速高效构建TALEs的方法,能够在1周内完成TALEs构建。 TALEs （Transcription activator like effectors） are a class of naturally DNA-binding proteins found in the plant pathogen Xanthornonas. Engineered repeat domains of TALE have been used as modular building units for construction of customized DNA-binding proteins. Artificial TALEs can be used for a wide variety of gene engineering applications,including transcriptional modulation and genome editing. The article describes process for reliable and affordable construction of TALE-TFs and TALENs by the combination of PCR amplification and ＇digestion-ligation＇ cloning based on the TALE Toolbox kit. The resuits show that the construction strategy is fast and efficient,and TALEs construction could be completed within 1 week.
作者 梁龙 杨华 杨永林 石国庆 刘守仁 皮文辉
机构地区 石河子大学动物科技学院 新疆农垦科学院畜牧兽医研究所 新疆兵团绵羊繁育生物技术重点实验室
出处 《石河子大学学报（自然科学版）》 CAS 2013年第3期306-312,共7页 Journal of Shihezi University(Natural Science)
基金 国家自然科学基金项目(31060159) 新疆兵团博士资金项目(2009JC05) 国家留学基金委"西部地区人才培养特别项目"
关键词 转录激活因子样效应子 “酶切-连接”克隆法 TALEs digestion-ligation cloning
分类号 Q78 [生物学—分子生物学] Q503 [生物学—生物化学]
  • 相关文献

参考文献25

  • 1Boch J,Bonas U. Xanthomonas AvrBs3 Family-Type III Effectors:Diseovery and Function[J]. Annu Rev Phytopathol, 2010,48 : 419-436.
  • 2Boch J,Scholze H,Sehornack S,et al. Breaking the code of DNA binding specificity of TAL-type III effectors [J]. Science,2009,326(5959) : 1509-1512.
  • 3Moscou M J, Bogdanove A J. A simple cipher governs DNA recognition by TAL effectors[J]. Science, 2009, 326(5959) : 1501.
  • 4Miller J C,Tan S,Qiao G,et al. A TALE nuclease archi- tecture for efficient genome editing[J]. Nature Biotech- nology,2011,29(2) ： 143-148.
  • 5Morbitzer R, R6mer P, Boeh J,et al. Regulation of se- lected genome loci using de novo-engineered transcrip- tion activator-like effector ( TALE )-type transcription factors[J]. Proc Natl Acad Sci USA, 2010, 107(50): 21617-21622.
  • 6Cong L,Zhou R H,Kuo Y C,et al. Comprehensive inter- rogation of natural TALE DNA-binding modules and transcriptional repressor domains[J]. Nature Communi- cation, 2012,3:968.
  • 7Deng D, Yan C, Pan X, eta[. Structural basis for se- quence-specific recognition of DNA by TAL effectors [J]. Science,2012,335(6069) : 720-723.
  • 8Mak A N, Bradley P, Cernadas R A, et al. The crystal structure of TAL effector PthXol bound to its DNA target[J]. Science, 2012,335 (6069) ： 716-719.
  • 9Sanjana N E,Cong L,Zhou Y,et al. A transcription acti- vator-like effector toolbox for genome engineering[J]. Nature Protocols,2012,7(1) ： 171-192.
  • 10Cermak T,Doyle E L,Christian M, et al. Efficient de- sign and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting[J]. Nucle- ic Acids Research,2011,39(12) :e82.

二级参考文献21

  • 1Boch J,Bonas U.Xanthomonas AvrBs3 family-type III effectors:discovery and function[J].Annu Rev Phytopathol,2010,48:419-436.
  • 2Boch J,Scholze H,Schornack S,et al.Breaking the code ofDNA binding specificity of TAL-type III effectors[J].Science,2009,326(5959):1509-1512.
  • 3Morbitzer R,Romer P,Boch J,et al.Regulation of selectedgenome loci using de novo-engineered transcription activator-likeeffector(TALE)-type transcription factors[J].Proc Natl AcadSci U S A,2010,107(50):21617-21622.
  • 4Moscou M J,Bogdanove A J.A simple cipher governs DNArecognition by TAL effectors[J].Science,2009,326(5959):1501.
  • 5Li T,Huang S,Jiang W Z,et al.TAL nucleases(TALNs):hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain[J].Nucleic Acids Res,2011,39(1):359-372.
  • 6Mussolino C,Morbitzer R,Lutge F,et al.A novel TALEnuclease scaffold enables high genome editing activity incombination with low toxicity[J].Nucleic Acids Res,2011,39(21):9283-9293.
  • 7Clark K J,Voytas D F,Ekker S C.A TALE of two nucleases:gene targeting for the masses?[J]Zebrafish,2011,8(3):147-149.
  • 8Mahfouz M M,Li L.TALE nucleases and next generation GMcrops[J].GM Crops,2011,2(2):99-103.
  • 9Bogdanove A J,Voytas D F.TAL effectors:customizable proteinsfor DNA targeting[J].Science,2011,333(6051):1843-1846.
  • 10Zhang F,Cong L,Lodato S,et al.Efficient construction ofsequence-specific TAL effectors for modulating mammaliantranscription[J].Nat Biotechnol,2011,29(2):149-153.

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石河子大学学报（自然科学版）
2013年 第3期

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