高效严谨型大肠杆菌Targetron基因打靶系统的构建
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摘要
构建高效严谨型大肠杆菌Targetron基因打靶系统。将来源于pET28a的T7-lac操纵子与来源于pSY6的Ⅱ型内含子组装构建大肠杆菌IPTG诱导型Targetron质粒系统。以lacZ基因为例,选择lacZ-635s和lacZ-1063a两个位点为靶位点,利用构建的IPTG诱导型Targetron系统进行基因打靶,通过分析诱导前和诱导后Ⅱ型内含子在靶位点的插入效率,验证大肠杆菌IPTG诱导型Targetron系统严谨性和打靶效率。最后,通过优化诱导剂浓度及诱导时间,建立高效严谨的诱导型大肠杆菌Targetron基因打靶系统。在没有IPTG诱导时,Ⅱ型内含子在两个位点均不能插入,打靶效率均为0;当加入0.5 mmol/L IPTG诱导45 min时,其在lacZ-635s位点的打靶效率提高到90.8±5.5%,在lacZ-1063a位点的打靶效率提高到92.6±2.4%。成功建立高效严谨型大肠杆菌Targetron基因打靶系统,旨为Ⅱ型内含子的机理研究及应用奠定基础。
The objective is to construct a highly efficient and rigorous Targetron gene targeting system in Escherichia coli. Firstly,the T7-lac operon from pET28 a and the group Ⅱ intron from pSY6 were assembled to construct an IPTG-inducible Targetron plasmid system in E.coli. Then,taking the lacZ gene as an example,the efficiency and stringency of the IPTG-inducible Targetron targeting system were verified by analyzing the insertion efficiency of the group Ⅱ introns before and after induction. Finally,a highly efficient and rigorous inducible Targetron gene targeting system in E. coli was obtained after optimizing the IPTG concentration and induction time. As results,group Ⅱ introns were unable to be inserted into 2 target sites at all in the absence of IPTG induction,the targeting efficiency was 0. When induced for 45 min after adding 0.5 mmol/L IPTG,the targeting efficiency at lacZ-635 s site increased to 90.8±5.5%,and the targeting efficiency at lacZ-1063 a site increased to 92.6±2.4%. In conclusion,the IPTG-inducible Targetron gene targeting system in E. coli is successfully established,laying a foundation for the research and application of the group Ⅱ intron.
The objective is to construct a highly efficient and rigorous Targetron gene targeting system in Escherichia coli. Firstly,the T7-lac operon from pET28 a and the group Ⅱ intron from pSY6 were assembled to construct an IPTG-inducible Targetron plasmid system in E.coli. Then,taking the lacZ gene as an example,the efficiency and stringency of the IPTG-inducible Targetron targeting system were verified by analyzing the insertion efficiency of the group Ⅱ introns before and after induction. Finally,a highly efficient and rigorous inducible Targetron gene targeting system in E. coli was obtained after optimizing the IPTG concentration and induction time. As results,group Ⅱ introns were unable to be inserted into 2 target sites at all in the absence of IPTG induction,the targeting efficiency was 0. When induced for 45 min after adding 0.5 mmol/L IPTG,the targeting efficiency at lacZ-635 s site increased to 90.8±5.5%,and the targeting efficiency at lacZ-1063 a site increased to 92.6±2.4%. In conclusion,the IPTG-inducible Targetron gene targeting system in E. coli is successfully established,laying a foundation for the research and application of the group Ⅱ intron.
引文
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[2]Toro N, Molina-Sanchez MD, Nisa-Martinez R, et al. Bacterial group II introns:identification and mobility assay[J]. Methods in Molecular Biology, 2016, 1400:21-32.
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[4]Zhao C, Pyle AM. The maturase:a reverse transcriptase and splicing factor go hand in hand[J]. Curr Opin Struct Biol, 2017,47:30-39.
[5]Lambowitz AM, Zimmerly S. Group II introns:mobile ribozymes that invade DNA[J]. Cold Spring Harb Perspect Biol, 2011, 3(8):a003616.
[6]Enyeart PJ, Mohr G, Ellington AD, et al. Biotechnological applications of mobile group II introns and their reverse transcriptases:gene targeting, RNA-seq, and non-coding RNA analysis[J]. Mobile DNA, 2014, 5(1):2.
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[10]刘亚君,崔古贞,洪伟,等.典型产纤维小体梭菌的遗传改造及其在纤维素乙醇中的应用研究进展[J].生物加工过程,2014, 12(1):55-62.
[11]Cui GZ, Zhang J, Hong W, et al. Improvement of ClosTron for successive gene disruption in Clostridium cellulolyticum using a pyrF-based screening system[J]. Appl Microbiol Biotechnol,2014, 98(1):313-323.
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[13]Heap JT, Pennington OJ, Cartman ST, et al. A modular system for Clostridium shuttle plasmids[J]. Journal of Microbiological Methods, 2009, 78(1):79-85.
[14]Shao L, Hu S, Yang Y, et al. Targeted gene disruption by use of a group II intron(targetron)vector in Clostridium acetobutylicum[J]. Cell Research, 2007, 17(11):963-965.
[15]Heap JT, Pennington OJ, Cartman ST, et al. The ClosTron:a universal gene knock-out system for the genus Clostridium[J].Journal of Microbiological Methods, 2007, 70(3):452-464.
[16]Yao J, Zhong J, Fang Y, et al. Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll. LtrB group II intron splicing[J].RNA, 2006, 12(7):1271-1281.
[17]Yao J, Lambowitz AM. Gene targeting in gram-negative bacteria by use of a mobile group II intron(“Targetron”)expressed from a broad-host-range vector[J]. Applied and Environmental Microbiology, 2007, 73(8):2735-2743.
[18]Zhang J, Liu YJ, Cui GZ, et al. A novel arabinose-inducible genetic operation system developed for Clostridium cellulolyticum[J].Biotechnology for Biofuels, 2015, 8:36.
[19]Alonzo F, 3rd, Port GC, Cao M, et al. The posttranslocation chaperone PrsA2 contributes to multiple facets of Listeria monocytogenes pathogenesis[J]. Infection and Immunity, 2009,77(7):2612-2623.
[20]Sayeed S, Uzal FA, Fisher DJ, et al. Beta toxin is essential for the intestinal virulence of Clostridium perfringens type C disease isolate CN3685 in a rabbit ileal loop model[J]. Molecular Microbiology,2008, 67(1):15-30.
[21]Carter GP, Awad MM, Hao Y, et al. TcsL is an essential virulence factor in Clostridium sordellii ATCC 9714[J]. Infection and Immunity, 2011, 79(3):1025-1032.
[22]Zoraghi R, See RH, Gong H, et al. Functional analysis,overexpression, and kinetic characterization of pyruvate kinase from methicillin-resistant Staphylococcus aureus[J]. Biochemistry,2010, 49(35):7733-7747.
[23]Zoraghi R, Worrall L, See RH, et al. Methicillin-resistant Staphylococcus aureus(MRSA)pyruvate kinase as a target for bis-indole alkaloids with antibacterial activities[J]. The Journal of Biological Chemistry, 2011, 286(52):44716-44725.
[24]张雪,温廷益. Red重组系统用于大肠杆菌基因修饰研究进展[J].中国生物工程杂志, 2008, 28(12):89-93.
[25]吴璐,王磊,任远,等.基因组编辑技术研究进展[J].生物技术通报, 2014(11):84-90.
[2]Toro N, Molina-Sanchez MD, Nisa-Martinez R, et al. Bacterial group II introns:identification and mobility assay[J]. Methods in Molecular Biology, 2016, 1400:21-32.
[3]Pyle AM. Group II intron self-splicing[J]. Annu Rev Biophys,2016, 45:183-205.
[4]Zhao C, Pyle AM. The maturase:a reverse transcriptase and splicing factor go hand in hand[J]. Curr Opin Struct Biol, 2017,47:30-39.
[5]Lambowitz AM, Zimmerly S. Group II introns:mobile ribozymes that invade DNA[J]. Cold Spring Harb Perspect Biol, 2011, 3(8):a003616.
[6]Enyeart PJ, Mohr G, Ellington AD, et al. Biotechnological applications of mobile group II introns and their reverse transcriptases:gene targeting, RNA-seq, and non-coding RNA analysis[J]. Mobile DNA, 2014, 5(1):2.
[7]Lambowitz AM, Belfort M. Mobile Bacterial Group II introns at the crux of eukaryotic evolution[J]. Microbiology Spectrum, 2015, 3(1):MDNA3-0050-2014.
[8]McNeil BA, Semper C, Zimmerly S. Group II introns:versatile ribozymes and retroelements[J]. Wiley interdisciplinary reviews RNA, 2016, 7(3):341-355.
[9]Qu G, Kaushal PS, Wang J, et al. Structure of a group II intron in complex with its reverse transcriptase[J]. Nat Struct Mol Biol,2016, 23(6):549-557.
[10]刘亚君,崔古贞,洪伟,等.典型产纤维小体梭菌的遗传改造及其在纤维素乙醇中的应用研究进展[J].生物加工过程,2014, 12(1):55-62.
[11]Cui GZ, Zhang J, Hong W, et al. Improvement of ClosTron for successive gene disruption in Clostridium cellulolyticum using a pyrF-based screening system[J]. Appl Microbiol Biotechnol,2014, 98(1):313-323.
[12]Cui GZ, Hong W, Zhang J, et al. Targeted gene engineering in Clostridium cellulolyticum H10 without methylation[J]. Journal of Microbiological Methods, 2012, 89(3):201-208.
[13]Heap JT, Pennington OJ, Cartman ST, et al. A modular system for Clostridium shuttle plasmids[J]. Journal of Microbiological Methods, 2009, 78(1):79-85.
[14]Shao L, Hu S, Yang Y, et al. Targeted gene disruption by use of a group II intron(targetron)vector in Clostridium acetobutylicum[J]. Cell Research, 2007, 17(11):963-965.
[15]Heap JT, Pennington OJ, Cartman ST, et al. The ClosTron:a universal gene knock-out system for the genus Clostridium[J].Journal of Microbiological Methods, 2007, 70(3):452-464.
[16]Yao J, Zhong J, Fang Y, et al. Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll. LtrB group II intron splicing[J].RNA, 2006, 12(7):1271-1281.
[17]Yao J, Lambowitz AM. Gene targeting in gram-negative bacteria by use of a mobile group II intron(“Targetron”)expressed from a broad-host-range vector[J]. Applied and Environmental Microbiology, 2007, 73(8):2735-2743.
[18]Zhang J, Liu YJ, Cui GZ, et al. A novel arabinose-inducible genetic operation system developed for Clostridium cellulolyticum[J].Biotechnology for Biofuels, 2015, 8:36.
[19]Alonzo F, 3rd, Port GC, Cao M, et al. The posttranslocation chaperone PrsA2 contributes to multiple facets of Listeria monocytogenes pathogenesis[J]. Infection and Immunity, 2009,77(7):2612-2623.
[20]Sayeed S, Uzal FA, Fisher DJ, et al. Beta toxin is essential for the intestinal virulence of Clostridium perfringens type C disease isolate CN3685 in a rabbit ileal loop model[J]. Molecular Microbiology,2008, 67(1):15-30.
[21]Carter GP, Awad MM, Hao Y, et al. TcsL is an essential virulence factor in Clostridium sordellii ATCC 9714[J]. Infection and Immunity, 2011, 79(3):1025-1032.
[22]Zoraghi R, See RH, Gong H, et al. Functional analysis,overexpression, and kinetic characterization of pyruvate kinase from methicillin-resistant Staphylococcus aureus[J]. Biochemistry,2010, 49(35):7733-7747.
[23]Zoraghi R, Worrall L, See RH, et al. Methicillin-resistant Staphylococcus aureus(MRSA)pyruvate kinase as a target for bis-indole alkaloids with antibacterial activities[J]. The Journal of Biological Chemistry, 2011, 286(52):44716-44725.
[24]张雪,温廷益. Red重组系统用于大肠杆菌基因修饰研究进展[J].中国生物工程杂志, 2008, 28(12):89-93.
[25]吴璐,王磊,任远,等.基因组编辑技术研究进展[J].生物技术通报, 2014(11):84-90.