CRISPR/Cas9介导靶向敲除拟南芥MS2基因突变体的鉴定
|
摘要
CRISPR/Cas9(Clustered regulatory interspaced short palindromic repeat/Cas9)基因编辑技术在生命科学领域掀起了一场重大的技术革命,它比锌指核酸酶(ZFNs)和转录激活因子效应物核酸酶(TALENs)技术更易于操作,而且更高效。CRISPR/Cas9系统已经被广泛应用到植物科学研究领域中。本实验选取拟南芥MS2为目的基因,构建植物基因编辑系统的表达载体,并通过农杆菌介导的方法转化拟南芥,从而利用CRISPR/Cas9系统靶向敲除拟南芥MS2基因。对转基因后代的MS2测序结果分析表明,在获得的12个阳性转化植株中,发现了5个阳性植株存在基因序列上的新碱基插入,由此形成CRISPR/Cas9介导的拟南芥MS2突变体。这一工作为后续分析其他物种中MS2同源基因的功能研究提供参考依据。
The clustered regulatory interspaced short palindromic repeat/Cas9(CRISPR/Cas9) genome editing technology launches a great technological revolution in the field of life sciences. Compared with zinc finger nucleases(ZFNs) and transcription activator-like effector nucleases(TALENs) technologies, the CRISPR/Cas9 technology is easier to operate and more efficient, CRISPR/Cas9 system has been widely applied in the field of plant science. MS2 gene of Arabidopsis thaliana was selected as the target gene in this study, and the expression vector of plant gene editing system was constructed. Then, the Arabidopsis thaliana was transferred by Agrobacterium-mediated method, and the MS2 gene of Arabidopsis thaliana was knocked out by CRISPR/Cas9 system. The results of MS2 sequencing of transgenic progeny showed that in the 12 positive transgenic plants, 5 positive plants were found to have new bases inserted in the gene sequence, thus forming a CRISPR/Cas9 mediated Arabidopsis MS2 mutant. This study provided a basis for subsequent functional analysis of MS2 homologous genes in other species.
The clustered regulatory interspaced short palindromic repeat/Cas9(CRISPR/Cas9) genome editing technology launches a great technological revolution in the field of life sciences. Compared with zinc finger nucleases(ZFNs) and transcription activator-like effector nucleases(TALENs) technologies, the CRISPR/Cas9 technology is easier to operate and more efficient, CRISPR/Cas9 system has been widely applied in the field of plant science. MS2 gene of Arabidopsis thaliana was selected as the target gene in this study, and the expression vector of plant gene editing system was constructed. Then, the Arabidopsis thaliana was transferred by Agrobacterium-mediated method, and the MS2 gene of Arabidopsis thaliana was knocked out by CRISPR/Cas9 system. The results of MS2 sequencing of transgenic progeny showed that in the 12 positive transgenic plants, 5 positive plants were found to have new bases inserted in the gene sequence, thus forming a CRISPR/Cas9 mediated Arabidopsis MS2 mutant. This study provided a basis for subsequent functional analysis of MS2 homologous genes in other species.
引文
Aarts M.G.,Dirkse W.G.,Stiekema W.J.,and Pereira A.,1993Transposon tagging of a male sterility gene in Arabidopsis Nature,363(6431):715-717
Aarts M.G.,Hodge R.,Kalantidis K.,Florack D.,Wilson Z.A.Mulligan B.J.,Stiekema W.J.,Scott R.,and Pereira A.1997,The Aabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation/condensation complexes,Plant J.,12(3):615-623
Canales C.,Bhatt A.M.,Scott R.,and Dickinson H.,2002,EXSa putative LRR receptor kinase,regulates male germline cel number and tapetal identity and promotes seed developmen in Arabidopsis,Curr.Biol.,12(20):1718-1727
Cong L.,Ran F.A.,Cox D.,Lin S.,Barretto R.,Habib N.,Hsu PD.,Wu X.,Jiang W.,Marraffini L.A.,and Zhang F.,2013Multiplex genome engineering using CRISPR/Cas syste MSScience,339(6121):819-823
Di Carlo J.E.,Norville J.E.,Mali P.,Rios X,.Aach J.,and Church G.M.,2013,Genome engineering in Saccharomyces cerevisiaeusing CRISPR-Cas syste MS,Nucleic Acids Res.,41(7):4336-4343
Doan T.T.,Carlsson A.S.,Hamberg M.,Bülow L.,Stymne S.and Olsson P.,2009,Functional expression of five Arabidopsis fatty acyl-Co A reductase genes in Escherichia coli J.Plant Physiol.,166(8):787-796
Esvelt K.M.,and Wang H.H.,2013,Genome-scale engineering for syste MS and synthetic biology,Mol.Syst.Biol.,9(1)641
Fauser F.,Roth N.,Pacher M.,Ilg G.,Sánchezfernández R.Biesgen C.,and Puchta H.,2012,In planta gene targeting Proc.Natl.Acad.Sci.USA,109(19):7535-7540
Horvath P.,and Barrangou R.,2010,CRISPR/Cas,the immune system of bacteria and archaea,Science,327(5962):167-170
James G.M.,Michael R.P.,Lana A.,Thomas R.H.,and Michae W.L.,2000,Purification of jojoba embryo fatty acyl-coenzyme A reductase and expression of its c DNA in high erucic acid rapeseed,Plant Physiol.,122(3):635-644
Jiang W.Y.,Bikard D.,Cox D.,Zhang F.,and Marraffini L.A.2013,RNA-guided editing of bacterial genomes using CRI-SPR-Cas systems,Nat.Biotechnol.,31(3):233-239
Li J.,Zhang Y.,Chen K.L.,Shan Q.W.,Wang Y.P.,Liang Z.,and Gao C.X.,2013,CRISPR/Cas:a novel way of RNA-guided genome editing,Yichuan(Hereditas),35(11):1265-1273(李君,张毅,陈坤玲,单奇伟,王延鹏,梁振,高彩霞,2013CRISPR/Cas系统:RNA靶向的基因组定向编辑新技术遗传,35(11):1265-1273)
Li T.,Liu B.,Spalding M.H.,Weeks D.P.,and Yang B.,2012High-efficiency T ALEN-based gene editing produces disease-resistant rice,Nat.Biotechchnol.,30(5):390-392
Ma H.,2005,Molecular genetic analyses of microsporogenesis and microgametogenesis in flowering plants,Annu.Rev.Plant Biol.,56(1):393
Mizuno S.,Osakabe Y.,Maruyama K.,Ito T.,Osakabe K.,Sato T.,Shinozaki K.,and Yamaguchi S.K.,2007,Receptor-like protein kinase 2(RPK 2)is a novel factor controlling anther development in Arabidopsis thaliana,Plant J.,50(5):751-766
Schiefthaler U.,Balasubramanian S.,Sieber P.,Chevalier D.,Wisman E.,and Schneitz K.,1999,Molecular analysis of NOZZLE,a gene involved in pattern formation and early sporogenesis during sex organ development in Arabidopsis thaliana,Proc.Natl.Acad.Sci.USA,96(20):11664-11669
Shu L.Y.,Li F.X.,Hui Z.M.,Ching S.P.,Wei C.Y.,Li X.J.,Venkatesan S.,and De Y.,2003,TAPETUM DETERMI-NANT1 is required for cell specialization in the Arabidopsis anther,Plant Cell,15(12):2792-2804
Sieber P.,Schorderet M.,buchala A.,Kolattukudy P.,Metraux J.P.,and Nawrath C.,2000,Transgenic Arabidopsis plants expressing a fungal cutinase show alterations in the structure and properties of the cuticle and postgenital organ fusions,Plant Cell,12(5):721-738
Tang Y.W.,Liu L.P.,Wang R.X.,Chen Y.H.,Liu Z.H.,and Liu S.Q.,2016,Development of a CRISPR/Cas9 constructed for genome editing caffeine synthase in Camellia sinensis,Chashu Kexue(Journal of Tea Science),36(4):414-426(唐雨薇,刘丽萍,王若娴,陈宇宏,刘仲华,刘硕谦,2013,茶树咖啡碱合成酶CRISPR/Cas9基因组编辑载体的构建,茶树科学,36(4):414-426)
Unte U.S.,Sorensen A.M.,Pesaresi P.,Gandikota M.,Leister D.,Saedler H.,and Huijser P.,2003,SPL8,an SBP-box gene that affects pollen sac development in Arabidopsis,Plant Cell,15(4):1009-1019
Wang A.,Xia Q.,Xie W.,Dumonceaux T.,Zou J.,Datla R.,and Selvaraj G.,2002,Male gametophyte development in bread wheat(Triticum aestivum L.):molecular,cellular,and biochemical analyses of a sporophytic contribution to pollen wall ontogeny,Plant J.,30(6):613-623
Yang W.C.,Ye D.,Xu J.,and Sundaresan V.,1999,The SPORO-CYTELESS gene of Arabidopsis is required for initiation of sporogenesis and encodes a novel nuclear protein,Gene.Dev.,13(16):2108-2117
Zheng W.,and Gu F.,2015,Progress of application and off-target effects of CRISPR/Cas9,Yichuan(Hereditas),37(10):1003-1010(郑武,谷峰,2015,CRISPR/Cas9的应用及脱靶效应研究进展,遗传,37(10):1003-1010)
Aarts M.G.,Hodge R.,Kalantidis K.,Florack D.,Wilson Z.A.Mulligan B.J.,Stiekema W.J.,Scott R.,and Pereira A.1997,The Aabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation/condensation complexes,Plant J.,12(3):615-623
Canales C.,Bhatt A.M.,Scott R.,and Dickinson H.,2002,EXSa putative LRR receptor kinase,regulates male germline cel number and tapetal identity and promotes seed developmen in Arabidopsis,Curr.Biol.,12(20):1718-1727
Cong L.,Ran F.A.,Cox D.,Lin S.,Barretto R.,Habib N.,Hsu PD.,Wu X.,Jiang W.,Marraffini L.A.,and Zhang F.,2013Multiplex genome engineering using CRISPR/Cas syste MSScience,339(6121):819-823
Di Carlo J.E.,Norville J.E.,Mali P.,Rios X,.Aach J.,and Church G.M.,2013,Genome engineering in Saccharomyces cerevisiaeusing CRISPR-Cas syste MS,Nucleic Acids Res.,41(7):4336-4343
Doan T.T.,Carlsson A.S.,Hamberg M.,Bülow L.,Stymne S.and Olsson P.,2009,Functional expression of five Arabidopsis fatty acyl-Co A reductase genes in Escherichia coli J.Plant Physiol.,166(8):787-796
Esvelt K.M.,and Wang H.H.,2013,Genome-scale engineering for syste MS and synthetic biology,Mol.Syst.Biol.,9(1)641
Fauser F.,Roth N.,Pacher M.,Ilg G.,Sánchezfernández R.Biesgen C.,and Puchta H.,2012,In planta gene targeting Proc.Natl.Acad.Sci.USA,109(19):7535-7540
Horvath P.,and Barrangou R.,2010,CRISPR/Cas,the immune system of bacteria and archaea,Science,327(5962):167-170
James G.M.,Michael R.P.,Lana A.,Thomas R.H.,and Michae W.L.,2000,Purification of jojoba embryo fatty acyl-coenzyme A reductase and expression of its c DNA in high erucic acid rapeseed,Plant Physiol.,122(3):635-644
Jiang W.Y.,Bikard D.,Cox D.,Zhang F.,and Marraffini L.A.2013,RNA-guided editing of bacterial genomes using CRI-SPR-Cas systems,Nat.Biotechnol.,31(3):233-239
Li J.,Zhang Y.,Chen K.L.,Shan Q.W.,Wang Y.P.,Liang Z.,and Gao C.X.,2013,CRISPR/Cas:a novel way of RNA-guided genome editing,Yichuan(Hereditas),35(11):1265-1273(李君,张毅,陈坤玲,单奇伟,王延鹏,梁振,高彩霞,2013CRISPR/Cas系统:RNA靶向的基因组定向编辑新技术遗传,35(11):1265-1273)
Li T.,Liu B.,Spalding M.H.,Weeks D.P.,and Yang B.,2012High-efficiency T ALEN-based gene editing produces disease-resistant rice,Nat.Biotechchnol.,30(5):390-392
Ma H.,2005,Molecular genetic analyses of microsporogenesis and microgametogenesis in flowering plants,Annu.Rev.Plant Biol.,56(1):393
Mizuno S.,Osakabe Y.,Maruyama K.,Ito T.,Osakabe K.,Sato T.,Shinozaki K.,and Yamaguchi S.K.,2007,Receptor-like protein kinase 2(RPK 2)is a novel factor controlling anther development in Arabidopsis thaliana,Plant J.,50(5):751-766
Schiefthaler U.,Balasubramanian S.,Sieber P.,Chevalier D.,Wisman E.,and Schneitz K.,1999,Molecular analysis of NOZZLE,a gene involved in pattern formation and early sporogenesis during sex organ development in Arabidopsis thaliana,Proc.Natl.Acad.Sci.USA,96(20):11664-11669
Shu L.Y.,Li F.X.,Hui Z.M.,Ching S.P.,Wei C.Y.,Li X.J.,Venkatesan S.,and De Y.,2003,TAPETUM DETERMI-NANT1 is required for cell specialization in the Arabidopsis anther,Plant Cell,15(12):2792-2804
Sieber P.,Schorderet M.,buchala A.,Kolattukudy P.,Metraux J.P.,and Nawrath C.,2000,Transgenic Arabidopsis plants expressing a fungal cutinase show alterations in the structure and properties of the cuticle and postgenital organ fusions,Plant Cell,12(5):721-738
Tang Y.W.,Liu L.P.,Wang R.X.,Chen Y.H.,Liu Z.H.,and Liu S.Q.,2016,Development of a CRISPR/Cas9 constructed for genome editing caffeine synthase in Camellia sinensis,Chashu Kexue(Journal of Tea Science),36(4):414-426(唐雨薇,刘丽萍,王若娴,陈宇宏,刘仲华,刘硕谦,2013,茶树咖啡碱合成酶CRISPR/Cas9基因组编辑载体的构建,茶树科学,36(4):414-426)
Unte U.S.,Sorensen A.M.,Pesaresi P.,Gandikota M.,Leister D.,Saedler H.,and Huijser P.,2003,SPL8,an SBP-box gene that affects pollen sac development in Arabidopsis,Plant Cell,15(4):1009-1019
Wang A.,Xia Q.,Xie W.,Dumonceaux T.,Zou J.,Datla R.,and Selvaraj G.,2002,Male gametophyte development in bread wheat(Triticum aestivum L.):molecular,cellular,and biochemical analyses of a sporophytic contribution to pollen wall ontogeny,Plant J.,30(6):613-623
Yang W.C.,Ye D.,Xu J.,and Sundaresan V.,1999,The SPORO-CYTELESS gene of Arabidopsis is required for initiation of sporogenesis and encodes a novel nuclear protein,Gene.Dev.,13(16):2108-2117
Zheng W.,and Gu F.,2015,Progress of application and off-target effects of CRISPR/Cas9,Yichuan(Hereditas),37(10):1003-1010(郑武,谷峰,2015,CRISPR/Cas9的应用及脱靶效应研究进展,遗传,37(10):1003-1010)