大肠杆菌Targetron基因打靶载体构建及应用
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摘要
目的:构建基于细菌Ⅱ型内含子的Targetron基因打靶载体,并在大肠杆菌中验证其功能。方法:以构建的p SY7载体为基础、以大肠杆菌lac Z基因为例,选择lac Z-1683a和lac Z-1874s位点为靶位点,利用在线设计软件设计打靶引物,通过重叠延伸PCR方法获得特异性打靶序列,并将其克隆到p SY7载体,获得p SY7-lacZ-1683a和p SY7-lacZ-1874s打靶载体,转化大肠杆菌后利用异丙基硫代半乳糖苷(IPTG)诱导插入型内含子的表达,最后利用菌落PCR和蓝白斑计数法验证其功能及打靶效率。结果:成功构建了p SY7-lacZ-1683a和p SY7-lac Z-1874s两种Targetron打靶载体,通过0.5 mmol/L IPTG诱导45 min后,菌落PCR显示Ⅱ型内含子能特异性插入到lac Z-1683a、lac Z-1874s位点,蓝白斑计数显示lac Z-1683a位点的打靶效率为(14.299±1.271)%,lac Z-1874s位点的打靶效率为(9.217±1.024)%。结论:构建的Targetron打靶载体能够特异性的插入大肠杆菌染色体靶位点。
Objective:To construct Targetron targeting vector based on bacteria group Ⅱ and verify its function in E.coli.Methods:Based on the constructed pSY7 vector and taking lacZ gene of E.coli as an example,lacZ-1683 a and lac Z-1874 s loci were selected as target sites.Targeting primers were designed by on-line design software.Specific targeting sequences were obtained by overlapping extended PCR and then cloned into p SY7 vectors.The pSY7-lacZ-1683 a and pSY7-lacZ-1874 s targeting vectors were obtained and transformed into E.coli.IPTG was used to induce the expression of insertional group Ⅱ introns.Finally,colony PCR and blue-white spot counting were used to verify the function and targeting efficiency.Results:Two Targetron vectors,pSY7-lac Z-1683 a and pSY7-lacZ-1874 s,were successfully constructed.After 45 minutes of induction by 0.5 mmol/L IPTG,colony PCR showed that Group Ⅱ intron could be specifically inserted into lac Z-1683 a and lacZ-1874 s sites.The targeting efficiency of lacZ-1683 a site was(14.299 ± 1.271) % and that of lac Z-1874 s site was(9.217 ±1.024) %.Conclusion:Targetron vectors constructed in this paper can specifically insert into the target site in E.coli.
Objective:To construct Targetron targeting vector based on bacteria group Ⅱ and verify its function in E.coli.Methods:Based on the constructed pSY7 vector and taking lacZ gene of E.coli as an example,lacZ-1683 a and lac Z-1874 s loci were selected as target sites.Targeting primers were designed by on-line design software.Specific targeting sequences were obtained by overlapping extended PCR and then cloned into p SY7 vectors.The pSY7-lacZ-1683 a and pSY7-lacZ-1874 s targeting vectors were obtained and transformed into E.coli.IPTG was used to induce the expression of insertional group Ⅱ introns.Finally,colony PCR and blue-white spot counting were used to verify the function and targeting efficiency.Results:Two Targetron vectors,pSY7-lac Z-1683 a and pSY7-lacZ-1874 s,were successfully constructed.After 45 minutes of induction by 0.5 mmol/L IPTG,colony PCR showed that Group Ⅱ intron could be specifically inserted into lac Z-1683 a and lacZ-1874 s sites.The targeting efficiency of lacZ-1683 a site was(14.299 ± 1.271) % and that of lac Z-1874 s site was(9.217 ±1.024) %.Conclusion:Targetron vectors constructed in this paper can specifically insert into the target site in E.coli.
引文
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[13]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 Ltr B group II intron splicing[J]. RNA,2006,12(7):1271-1281.
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[16]LAMBOWITZ A M,ZIMMERLY S. Group II introns:mobile ribozymes that invade DNA[J]. Cold Spring Harb Perspect Biol,2011,3(8):a003616.
[17]FRAZIER C L,SAN FILIPPO J,LAMBOWITZ A M,et al. Genetic manipulation of lactococcus lactis by using targeted group II introns:generation of stable insertions without selection[J]. Appl Environ Microbiol,2003,69(2):1121-1128.
[18]YAO J,ZHONG J,LAMBOWITZ A M. Gene targeting using randomly inserted group II introns(targetrons)recovered from an escherichia coli gene disruption library[J]. Nucleic Acids Res,2005,33(10):3351-3362.
[19]CHEN Y,MCCLANE B A,FISHER D J,et al. Construction of an alpha toxin gene knockout mutant of clostridium perfringens type a by use of a mobile group II intron[J]. Appl Environ Microbiol,2005,71(11):7542-7547.
[20]MOHR G,HONG W,ZHANG J,et al. A targetron system for gene targeting in thermophiles and its application in clostridium thermocellum[J]. PLo S One,2013,8(7):e69032.
[21]ZHONG J,KARBERG M,LAMBOWITZ A M. Targeted and random bacterial gene disruption using a group II intron(targetron)vector containing a retrotransposition-activated selectable marker[J]. Nucleic Acids Res,2003,31(6):1656-1664.
[2]MCNEIL B A,SEMPER C,ZIMMERLY S. Group II introns:versatile ribozymes and retroelements[J]. Wiley Interdiscip Rev RNA,2016,7(3):341-355.
[3]QU G,KAUSHAL P S,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.
[4]TORO N,JIMENEZ-ZURDO J I,GARCIA-RODRIGUEZ F M. Bacterial group II introns:not just splicing[J].FEMS Microbiol Rev,2007,31(3):342-358.
[5]ZIMMERLY S,SEMPER C. Evolution of group II introns[J]. Mob DNA,2015,6:7.
[6]NOVIKOVA O,BELFORT M. Mobile group II introns as ancestral eukaryotic elements[J]. Trends Genet,2017,33(11):773-783.
[7]ZIMMERLY S,GUO H,PERLMAN P S,et al. Group II intron mobility occurs by target DNA-primed reverse transcription[J]. Cell,1995,82(4):545-554.
[8]LIU Y J,ZHANG J,CUI G Z,et al. Current progress of targetron technology:development,improvement and application in metabolic engineering[J]. Biotechnol J,2015,10(6):855-865.
[9]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 Res,2007,17(11):963-965.
[10]ENYEART P J,MOHR G,ELLINGTON A D,et al.Biotechnological applications of mobile group II introns and their reverse transcriptases:gene targeting,RNAseq,and non-coding RNA analysis[J]. Mob DNA,2014,5(1):2.
[11]SASIKUMAR P,PAUL E,GOMATHI S,et al. Mobile group II intron based gene targeting in lactobacillus plantarum WCFS1[J]. J Basic Microbiol,2016,56(10):1107-1116.
[12]KARBERG M,GUO H,ZHONG J,et al. Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria[J]. Nat Biotechnol,2001,19(12):1162-1167.
[13]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 Ltr B group II intron splicing[J]. RNA,2006,12(7):1271-1281.
[14]YAO J,LAMBOWITZ A M. Gene targeting in gram-negative bacteria by use of a mobile group II intron("Targetron")expressed from a broad-host-range vector[J].Appl Environ Microbiol,2007,73(8):2735-2743.
[15]KEY C E,FISHER D J. Use of group II intron technology for targeted mutagenesis in chlamydia trachomatis[J].Methods Mol Biol,2017,1498:163-177.
[16]LAMBOWITZ A M,ZIMMERLY S. Group II introns:mobile ribozymes that invade DNA[J]. Cold Spring Harb Perspect Biol,2011,3(8):a003616.
[17]FRAZIER C L,SAN FILIPPO J,LAMBOWITZ A M,et al. Genetic manipulation of lactococcus lactis by using targeted group II introns:generation of stable insertions without selection[J]. Appl Environ Microbiol,2003,69(2):1121-1128.
[18]YAO J,ZHONG J,LAMBOWITZ A M. Gene targeting using randomly inserted group II introns(targetrons)recovered from an escherichia coli gene disruption library[J]. Nucleic Acids Res,2005,33(10):3351-3362.
[19]CHEN Y,MCCLANE B A,FISHER D J,et al. Construction of an alpha toxin gene knockout mutant of clostridium perfringens type a by use of a mobile group II intron[J]. Appl Environ Microbiol,2005,71(11):7542-7547.
[20]MOHR G,HONG W,ZHANG J,et al. A targetron system for gene targeting in thermophiles and its application in clostridium thermocellum[J]. PLo S One,2013,8(7):e69032.
[21]ZHONG J,KARBERG M,LAMBOWITZ A M. Targeted and random bacterial gene disruption using a group II intron(targetron)vector containing a retrotransposition-activated selectable marker[J]. Nucleic Acids Res,2003,31(6):1656-1664.