靶向马铃薯StProDH1基因的CRISPR/Cas9 sgRNA表达载体构建
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摘要
CRISPR/Cas9基因编辑系统已成为许多作物基因功能研究和品种改良的重要工具。脯氨酸(Pro)在植物响应干旱胁迫方面起着重要作用,脯氨酸脱氢酶ProDH是脯氨酸分解途径中的限速酶。为了研究StProDH1在马铃薯脯氨酸代谢中的作用,本研究以StProDH1为靶基因,构建了表达马铃薯脯氨酸脱氢酶g RNA的CRISPR/Cas9基因敲除系统,在StProDH1基因5'端第一个外显子上为靶标区域选择设计sgRNA,以p P1C.4为模板,克隆得到sgRNA克隆框,将得到的sgRNA克隆框通过同源重组技术连接到pP1C.4载体中,获得pP1C.4-Cas9-ProDH1-g RNA载体。通过设计引物特异性扩增以及测序进一步确定sgRNA准确地连入载体,这对马铃薯中脯氨酸累积及其培育抗旱马铃薯新品种具有重要意义。
CRISPR/Cas9 gene editing system has become an important tool for gene function study and varieties improvement in many crops. Proline plays an important role in plant response to drought stress, While Proline dehydrogenase(ProDH) is a rate-limit enzyme in proline decomposition pathway. In order to study the effects of StProDH1 on proline metabolism in potato, StProDH1 as a target gene was used to generate a CRISPR/Cas9 knockout system expressing the ProDH1-g RNA of potato. On the first exon of 5' end of the StProDH1 gene, the sgRNA was designed for the target region and pP1 C.4 as the template was used to clone sgRNA cloning box; the obtained sgRNA cloning box was connected to pP1 C.4 by homologous recombination technology, and the pP1 C.4-Cas9-ProDH1-g RNA vector was obtained. To further validated the sgRNA whether accurately connected to the vector by designing specific primers amplification and sequencing. It would of great significance for proline accumulation and cultivation of new drought-resistant varieties in potato.
CRISPR/Cas9 gene editing system has become an important tool for gene function study and varieties improvement in many crops. Proline plays an important role in plant response to drought stress, While Proline dehydrogenase(ProDH) is a rate-limit enzyme in proline decomposition pathway. In order to study the effects of StProDH1 on proline metabolism in potato, StProDH1 as a target gene was used to generate a CRISPR/Cas9 knockout system expressing the ProDH1-g RNA of potato. On the first exon of 5' end of the StProDH1 gene, the sgRNA was designed for the target region and pP1 C.4 as the template was used to clone sgRNA cloning box; the obtained sgRNA cloning box was connected to pP1 C.4 by homologous recombination technology, and the pP1 C.4-Cas9-ProDH1-g RNA vector was obtained. To further validated the sgRNA whether accurately connected to the vector by designing specific primers amplification and sequencing. It would of great significance for proline accumulation and cultivation of new drought-resistant varieties in potato.
引文
Andersson M.,Turesson H.,Nicolia A.,Falt A.S.,Samuelsson M.,and Hofvander P.,2017,Efficient targeted multiallelic mutagenesis in tetraploid potato(Solanum tuberosum)by transient Crispr-Cas9 expression in protoplasts,Plant Cell Rep.,36(1):117-128
Belhaj K.,Chaparro-Garcia A.,Kamoun S.,and Nekrasov V.,2013,Plant genome editing made easy:targeted mutagenesis in model and crop plants using the CRISPR/Cas system,Plant Methods,9(1):39
Bortesi L.,and Fischer R.,2015,The CRISPR/Cas9 system for plantgenome editing and beyond,Biotechnol.Adv.,33(1):41-52
Brooks C.,Nekrasov V.,Lippman Z.B.,and Van Eck J.,2014,Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system,Plant Physiol.,166(3):1292-1297
Butler N.M.,Atkins P.A.,Voytas D.F.,and Douches D.S.,2015,Generation and inheritance of targeted mutations in potato(Solanum tuberosum L.)using the CRISPR/Cas system,PLoSOne,10(12):e0144591
Clasen B.M.,Stoddard T.J.,Luo S.,Demorest Z.L.,Li J.,Cedrone F.,Tibebu R.,Davison S.,Ray E.E.,Daulhac A.,Coffman A.,Yabandith A.,Retterath A.,Haun W.,Baltes N.J.,Mathis L.,Voytas D.F.,and Zhang F.,2016,Improving cold storage and processing traits in potato through targeted gene knockout,Plant Biotechnol.J.,14(1):169-176
Curtin S.J.,Voytas D.F.,a nd Stupar R.M.,2012,Genome engineering of crops with designer nucleases,Plant Genome5(2):42-50
Feng Z.,Mao Y.,Xu N.,Zhang B.,Wei P.,Yang D.L.,Wang Z.Zhang Z.,Zheng R.,Yang L.,Zeng L.,Liu X.,and Zhu J.K.,2014,Multigeneration analysis reveals the inheritance,specificity,and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis,Proc.Natl.Acad.Sci.USA,111(12):4632-4637
Funck D.,Eckard S.,and Muller G.,2010,Non-redundant functions of two proline dehydrogenase isoforms in Arabidopsis BMC Plant Biology,10(1):70
Gaj T.,Gersbach C.A.,and Barbas C.F.,2013,ZFN,TALENand CRISPR/Cas-based methods for genome engineering Trends Biotechnol.,31(7):397-405
Gasiunas G.,Barrangou R.,Horvath P.,and Siksnys V.,2012Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria,Proc.Natl.Acad.Sci.USA,109(39):2579-2586
Huang Y.J.,Deng R.Y.,Gao F.,Luo X.P.,and Wu Qi.,2015Cloning and expression analysis of transcription factor gene FtMYB21 from tartary buckwheat under abiotic stress Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(9):1939-1945(黄云吉,邓仁榆,高飞雒晓鹏,吴琦,2015,苦荞转录因子基因FtMYB21的克隆及其非生物胁迫下的表达分析,基因组学与应用生物学35(9):1939-1945)
Jiang W.,Zhou H.,Bi H.,Fromm M.,Yang B.,and Weeks D.P.2013,Demonstration of CRISPR/Cas9/sg RNA-mediated targeted gene modification in Arabidopsis,tobacco,sorghum and rice,Nucleic Acids Res.,41(20):e188
Konieczny A.,and Ausubel F.M.,1993,A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers,Plant J.,4(2):403-410
Li J.F.,Norville J.E.,Aach J.,McCormack M.,Zhang D.,Bush J.Church G.M.,and Sheen J.,2013,Multiplex and homologous recombination mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9,Nat Biotechnol.,31(8):688-691
Liu C.,Ma L.,Li Y.,Dai P.H.,Yao Z.P.,and Zhang H.,2015Cloning and expression analysis of HaDREB2A gene from Haloxylon ammodendron,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(9):1928-1933(刘超,马丽,李月,代培红,姚正培,张桦,2015,梭梭中HaDREB2A的克隆与表达分析,基因组学与应用生物学35(9):1928-1933)
Makarova K.S.,Haft D.H.,Barrangou R.,Brouns S.J.,Charpentier E.,Horvath P.,Moineau S.,Mojica F.J.,Wolf Y.I.,Yakunin A.F.,van der Oost J.,and Koonin E.V.,2011,Evolution and classification of the CRISPR-Cas systems,Nat.Rev.Microbiol.,9(6):467-477
Mani S.,Cotte B.V.D.,Montagu M.V.,and Verbruggen N.,2002,Altered levels of proline dehydrogenase cause hypersensitivity to proline and its analogs in Arabidopsis,Plant Physiol.,128(1):73-83
Miao J.,Guo D.,Zhang J.,Huang Q.,Qin G.,Zhang X.,Wan J.,Gu H.,and Qu L.J.,2013,Targeted mutagenesis in rice using CRISPR-Cas system,Cell Research,23(10):1233-1236
Muthoni J.,Kabira J.,Shimelis H.,and Melis R.,2015,Tetrasomic inheritance in cultivated potato and implications in conventional breeding,Aust.J.Crop Sci.,(3):185-190
Nanjo T.,Kobayashi M.,Yoshiba Y.,Kakubari Y.,YamaguchiShinozaki K.,and Shinozaki K.,1999,Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana,FEBS Lett.,461(3):205-210
Nekrasov V.,Staskawicz B.,Weigel D.,Jones J.D.G.,and Kamoun S.,2013,Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease,Nat.Biotechnol.,31(8):691-693
Ribarits A.,Abdullaev A.,Tashpulatov A.,Richter A.,HeberleBors E.,and Ouraev A.,2007,Two tobacco proline dehydrogenases are differentially regulated and play a role in early plant development,Planta,225(5):1313-1324
Servet C.,Ghelis T.,Richard L.,Zilberstein A.,and Savoure A.,2012,Proline dehydrogenase:a key enzyme in controlling cellular homeostasis,Front.Biosci.,17(1):607-620
Shan Q.,Wang Y.,Li J.,Zhang Y.,Chen K.,Liang Z.,Zhang K.,Liu J.,Xi J.J.,Qiu J.L.,and Gao C.,2013,Targeted genome modification of crop plants using a CRISPR-Cas system,Nat.Biotechnol.,31(8):686-688
Van Eck J.,2017,Genome editing and plant transformation of solanaceous food crops,Curr.Opin.Biotechnol.,49:35-41
Wang S.,Zhang S.,Wang W.,Xiong X.,Meng F.,and Cui X.,2015,Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system,Plant Cell Rep.,34(9):1473-1476
Weeks D.P.,2017,Gene editing in polyploid crops:wheat,Camelina,canola,potato,cotton,peanut,sugar cane,and citrus,Prog.Mol.Biol.Transl.Sci.,149:66-77
Wittwer C.T.,Reed G.H.,Gundry C.N.,Vandersteen J.G.,and Pryor R.J.,200 3,High-resolution genotyping by amplicon melting analysis using LCGreen,Clin.Chem.,49:853-860
Yang Z.,Steentoft C.,Hauge C.,Hansen L.,Thomsen AL.,Niola F.,Vester-Christensen M.B.,Frodin M.,Clausen H.,Wandall H.H.,and Bennett E.P.,2015,Fast and sensitive detection of indels induced by precise gene targeting,Nucleic Acids Res.,43(9):e59
Belhaj K.,Chaparro-Garcia A.,Kamoun S.,and Nekrasov V.,2013,Plant genome editing made easy:targeted mutagenesis in model and crop plants using the CRISPR/Cas system,Plant Methods,9(1):39
Bortesi L.,and Fischer R.,2015,The CRISPR/Cas9 system for plantgenome editing and beyond,Biotechnol.Adv.,33(1):41-52
Brooks C.,Nekrasov V.,Lippman Z.B.,and Van Eck J.,2014,Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system,Plant Physiol.,166(3):1292-1297
Butler N.M.,Atkins P.A.,Voytas D.F.,and Douches D.S.,2015,Generation and inheritance of targeted mutations in potato(Solanum tuberosum L.)using the CRISPR/Cas system,PLoSOne,10(12):e0144591
Clasen B.M.,Stoddard T.J.,Luo S.,Demorest Z.L.,Li J.,Cedrone F.,Tibebu R.,Davison S.,Ray E.E.,Daulhac A.,Coffman A.,Yabandith A.,Retterath A.,Haun W.,Baltes N.J.,Mathis L.,Voytas D.F.,and Zhang F.,2016,Improving cold storage and processing traits in potato through targeted gene knockout,Plant Biotechnol.J.,14(1):169-176
Curtin S.J.,Voytas D.F.,a nd Stupar R.M.,2012,Genome engineering of crops with designer nucleases,Plant Genome5(2):42-50
Feng Z.,Mao Y.,Xu N.,Zhang B.,Wei P.,Yang D.L.,Wang Z.Zhang Z.,Zheng R.,Yang L.,Zeng L.,Liu X.,and Zhu J.K.,2014,Multigeneration analysis reveals the inheritance,specificity,and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis,Proc.Natl.Acad.Sci.USA,111(12):4632-4637
Funck D.,Eckard S.,and Muller G.,2010,Non-redundant functions of two proline dehydrogenase isoforms in Arabidopsis BMC Plant Biology,10(1):70
Gaj T.,Gersbach C.A.,and Barbas C.F.,2013,ZFN,TALENand CRISPR/Cas-based methods for genome engineering Trends Biotechnol.,31(7):397-405
Gasiunas G.,Barrangou R.,Horvath P.,and Siksnys V.,2012Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria,Proc.Natl.Acad.Sci.USA,109(39):2579-2586
Huang Y.J.,Deng R.Y.,Gao F.,Luo X.P.,and Wu Qi.,2015Cloning and expression analysis of transcription factor gene FtMYB21 from tartary buckwheat under abiotic stress Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(9):1939-1945(黄云吉,邓仁榆,高飞雒晓鹏,吴琦,2015,苦荞转录因子基因FtMYB21的克隆及其非生物胁迫下的表达分析,基因组学与应用生物学35(9):1939-1945)
Jiang W.,Zhou H.,Bi H.,Fromm M.,Yang B.,and Weeks D.P.2013,Demonstration of CRISPR/Cas9/sg RNA-mediated targeted gene modification in Arabidopsis,tobacco,sorghum and rice,Nucleic Acids Res.,41(20):e188
Konieczny A.,and Ausubel F.M.,1993,A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers,Plant J.,4(2):403-410
Li J.F.,Norville J.E.,Aach J.,McCormack M.,Zhang D.,Bush J.Church G.M.,and Sheen J.,2013,Multiplex and homologous recombination mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9,Nat Biotechnol.,31(8):688-691
Liu C.,Ma L.,Li Y.,Dai P.H.,Yao Z.P.,and Zhang H.,2015Cloning and expression analysis of HaDREB2A gene from Haloxylon ammodendron,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(9):1928-1933(刘超,马丽,李月,代培红,姚正培,张桦,2015,梭梭中HaDREB2A的克隆与表达分析,基因组学与应用生物学35(9):1928-1933)
Makarova K.S.,Haft D.H.,Barrangou R.,Brouns S.J.,Charpentier E.,Horvath P.,Moineau S.,Mojica F.J.,Wolf Y.I.,Yakunin A.F.,van der Oost J.,and Koonin E.V.,2011,Evolution and classification of the CRISPR-Cas systems,Nat.Rev.Microbiol.,9(6):467-477
Mani S.,Cotte B.V.D.,Montagu M.V.,and Verbruggen N.,2002,Altered levels of proline dehydrogenase cause hypersensitivity to proline and its analogs in Arabidopsis,Plant Physiol.,128(1):73-83
Miao J.,Guo D.,Zhang J.,Huang Q.,Qin G.,Zhang X.,Wan J.,Gu H.,and Qu L.J.,2013,Targeted mutagenesis in rice using CRISPR-Cas system,Cell Research,23(10):1233-1236
Muthoni J.,Kabira J.,Shimelis H.,and Melis R.,2015,Tetrasomic inheritance in cultivated potato and implications in conventional breeding,Aust.J.Crop Sci.,(3):185-190
Nanjo T.,Kobayashi M.,Yoshiba Y.,Kakubari Y.,YamaguchiShinozaki K.,and Shinozaki K.,1999,Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana,FEBS Lett.,461(3):205-210
Nekrasov V.,Staskawicz B.,Weigel D.,Jones J.D.G.,and Kamoun S.,2013,Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease,Nat.Biotechnol.,31(8):691-693
Ribarits A.,Abdullaev A.,Tashpulatov A.,Richter A.,HeberleBors E.,and Ouraev A.,2007,Two tobacco proline dehydrogenases are differentially regulated and play a role in early plant development,Planta,225(5):1313-1324
Servet C.,Ghelis T.,Richard L.,Zilberstein A.,and Savoure A.,2012,Proline dehydrogenase:a key enzyme in controlling cellular homeostasis,Front.Biosci.,17(1):607-620
Shan Q.,Wang Y.,Li J.,Zhang Y.,Chen K.,Liang Z.,Zhang K.,Liu J.,Xi J.J.,Qiu J.L.,and Gao C.,2013,Targeted genome modification of crop plants using a CRISPR-Cas system,Nat.Biotechnol.,31(8):686-688
Van Eck J.,2017,Genome editing and plant transformation of solanaceous food crops,Curr.Opin.Biotechnol.,49:35-41
Wang S.,Zhang S.,Wang W.,Xiong X.,Meng F.,and Cui X.,2015,Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system,Plant Cell Rep.,34(9):1473-1476
Weeks D.P.,2017,Gene editing in polyploid crops:wheat,Camelina,canola,potato,cotton,peanut,sugar cane,and citrus,Prog.Mol.Biol.Transl.Sci.,149:66-77
Wittwer C.T.,Reed G.H.,Gundry C.N.,Vandersteen J.G.,and Pryor R.J.,200 3,High-resolution genotyping by amplicon melting analysis using LCGreen,Clin.Chem.,49:853-860
Yang Z.,Steentoft C.,Hauge C.,Hansen L.,Thomsen AL.,Niola F.,Vester-Christensen M.B.,Frodin M.,Clausen H.,Wandall H.H.,and Bennett E.P.,2015,Fast and sensitive detection of indels induced by precise gene targeting,Nucleic Acids Res.,43(9):e59
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