基于CRISPR/Cas9技术的烟草NtDXR基因敲除及功能分析
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- 英文论文题名：CRISPR/Cas9-mediated targeted mutagenesis and function analysis of DXR in Nicotiana tabacum
- 论文作者：聂梦云 ; 高军平 ; 罗培 ; 陈晓乐 ; 易小慧 ; 王根洪 ; 夏庆友
- 英文论文作者：NIE Mengyun ; GAO Junping ; LUO Pei ; CHEN Xiaole ; YI Xiaohui ; WANG Genhong ; XIA Qingyou ; State Key Laboratory of Silkworm Genome Biology ; Southwest University
- 年：2016
- 作者机构：家蚕基因组生物学国家重点实验室西南大学;
- 论文关键词：CRISPR/Cas9 ; 烟草 ; 5-磷酸脱氧木酮糖还原异构酶 ; 荧光定量PCR ; 基因敲除
- 英文论文关键词：CRISPR/Cas9 ; Tobacco ; 1-Deoxy-D-xylulose-5-phosphate reductoisomerase(DXR) ; qRT-PCR ; Gene knocking out
- 会议召开时间：2016-12-01
- 会议录名称：中国烟草学会2016年度优秀论文汇编——烟草农业主题
- 语种：中文
- 分类号：S572;Q943.2
- 学会代码：ZGYG
- 学会名称：中国烟草学会
- 页数：7
- 文件大小：1452k
- 原文格式：D
摘要
为研究烟草5-磷酸脱氧木酮糖还原异构酶(1-Deoxy-D-xylulose-5-phosphate reductoisomerase,DXR)基因的表达模式和功能,从普通烟草(Nicotiana tabacum)品种红花大金元中克隆了NtDXR基因,并通过荧光定量PCR技术分析了NtDXR在红花大金元不同发育时期、不同组织中的表达模式。同时利用CRISPR/Cas9技术构建了NtDXR敲除载体,获得了DXR基因突变的植株。结果表明:NtDXR基因在花中的表达量最高,在雌蕊和叶中次之;利用CRISPR/Cas9系统定点敲除NtDXR基因后,检测目的基因靶位点序列发现有碱基的插入、缺失与置换,并且部分T0代转基因阳性植株呈现白化表型,暗示NtDXR基因在烟草叶绿素等色素合成过程中起重要作用,推测NtDXR基因的突变能阻断烟草质体色素的合成。
In order to study the expression pattern and function of 1-deoxy-D-xylulose-5-phosphate reductoisomerase(DXR) in tobacco, NtDXR gene was cloned from tobacco cultivar Hongda(Nicotiana tabacum)and the expression level of NtDXR was also analysed in different tissues and organs. The CRISPR/Cas9 plant expression vector was constructed and the NtDXR knockout mutations were obtained. The results showed that NtDXR had the highest expression level in flower, higher in pistil and leaves. DNA insertion, deletion and displacement had been found in the target sites of NtDXR by targeted mutagenesis using the type II CRISPR/Cas9 system[Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated 9]. Partial T0 mutant plants showed obviously photo-bleaching phenotypes, which implied that NtDXR played an important role in chlorophyll synthesis, and it suggested that the biosynthesis of plastid pigment in tobacco might be blocked by the disfunction of NtDXR.
In order to study the expression pattern and function of 1-deoxy-D-xylulose-5-phosphate reductoisomerase(DXR) in tobacco, NtDXR gene was cloned from tobacco cultivar Hongda(Nicotiana tabacum)and the expression level of NtDXR was also analysed in different tissues and organs. The CRISPR/Cas9 plant expression vector was constructed and the NtDXR knockout mutations were obtained. The results showed that NtDXR had the highest expression level in flower, higher in pistil and leaves. DNA insertion, deletion and displacement had been found in the target sites of NtDXR by targeted mutagenesis using the type II CRISPR/Cas9 system[Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated 9]. Partial T0 mutant plants showed obviously photo-bleaching phenotypes, which implied that NtDXR played an important role in chlorophyll synthesis, and it suggested that the biosynthesis of plastid pigment in tobacco might be blocked by the disfunction of NtDXR.
引文
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[15]Jia H G,Wang N.Targeted genome editing of sweet orange using Cas9/sg RNA[J/OL].PLo S ONE,2014,9(4):e93806[2015-10-09].http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0093806.
[16]Li J F,Norville J E,Aach J,et al.Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9[J].Nature Biotechnology,2013,31(8):688-691.
[17]朱晓宇,王景,赵二卫,等.烟草5-磷酸脱氧木酮糖还原异构酶基因(dxr)的克隆和表达分析[J].农业生物技术学报,2011,19(2):263-269.ZHU Xiaoyu,WANG Jing,ZHAO Erwei,et al.Cloning and expression analysis of 1-deoxy-Dxylulose-5-phosphate reductoisomerases gene(dxr)in Nicotiana tabacum[J].Journal of Agricultural Biotechnology,2011,19(2):263-269.
[18]Jiang W Z,Zhou H B,Bi H H,et al.Demonstration of CRISPR/Cas9/sg RNA-mediated targeted gene modification in Arabidopsis,tobacco,sorghum and rice[J].Nucleic Acids Research,2013,41(20):e188.
[19]崔红,宋志红.类萜代谢工程研究进展及在烟草品种改良中的应用前景[J].中国烟草学报,2003,9(2):35-38,42.CUI Hong,SONG Zhihong.Recent advances in terpenoid metabolic engineering research and its prospective application in variety improvement in tobacco[J].Acta Tabacaria Sinica,2003,9(2):35-38,42.
[20]杨铁钊,李钦奎,李伟.植物次生代谢与烟草香味物质[J].中国烟草科学,2005,26(4):23-26.YANG Tiezhao,LI Qinkui,LI Wei.Plant secondary metabolism and aroma substances of tobacco[J].Chinese Tobacco Science,2005,26(4):23-26.
[2]Daum M,Herrmann S,Wilkinson B,et al.Genes and enzymes involved in bacterial isoprenoid biosynthesis[J].Current Opinion in Chemical Biology,2009,13(2):180-188.
[3]Kuzuyama T,Seto H.Diversity of the biosynthesis of the isoprene units[J].Natural Product Reports,2003,20(2):171-183.
[4]Sacchettini J C,Poulter C D.Creating isoprenoid diversity[J].Science,1997,277(5333):1788-1789.
[5]Mc Garvey D J,Croteau R.Terpenoid metabolism[J].The Plant Cell,1995,7(7):1015-1026.
[6]Dudareva N,Andersson S,Orlova I,et al.The nonmevalonate pathway supports both monoterpene and sesquiterpene formation in snapdragon flowers[J].Proceedings of the National Academy of Science of the United States of America,2005,102(3):933-938.
[7]Hasunuma T,Takeno S,Hayashi S,et al.Overexpression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene in chloroplast contributes to increment of isoprenoid production[J].Journal of Bioscience and Bioengineering,2008,105(5):518-526.
[8]Mahmoud S S,Croteau R B.Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase[J].Proceedings of the National Academy of Science of the United States of America,2001,98(15):8915-8920.
[9]Carretero-Paulet L,CairóA,Botella-Pavía P,et al.Enhanced flux through the methylerythritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxyxylulose 5-phosphate reductoisomerase[J].Plant Molecular Biology,2006,62(4):683-695.
[10]Shen B,Zhang J,Wu H Y,et al.Generation of gene-modified mice via Cas9/RNA-mediated gene targeting[J].Cell Research,2013,23(5):720-723.
[11]Jinek M,Chylinski K,Fonfara I,et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J].Science,2012,337(6096):816-821.
[12]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods,2001,25(4):402-408.
[13]Sander J D,Maeder M L,Reyon D,et al.Zi Fi T(Zinc Finger Targeter):an updated zinc finger engineering tool[J/OL].Nucleic Acids Research,2010,38(Suppl 2):W462-W468.
[14]Gao J P,Wang G H,Ma S Y,et al.CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum[J].Plant Molecular Biology,2015,87(1):99-110.
[15]Jia H G,Wang N.Targeted genome editing of sweet orange using Cas9/sg RNA[J/OL].PLo S ONE,2014,9(4):e93806[2015-10-09].http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0093806.
[16]Li J F,Norville J E,Aach J,et al.Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9[J].Nature Biotechnology,2013,31(8):688-691.
[17]朱晓宇,王景,赵二卫,等.烟草5-磷酸脱氧木酮糖还原异构酶基因(dxr)的克隆和表达分析[J].农业生物技术学报,2011,19(2):263-269.ZHU Xiaoyu,WANG Jing,ZHAO Erwei,et al.Cloning and expression analysis of 1-deoxy-Dxylulose-5-phosphate reductoisomerases gene(dxr)in Nicotiana tabacum[J].Journal of Agricultural Biotechnology,2011,19(2):263-269.
[18]Jiang W Z,Zhou H B,Bi H H,et al.Demonstration of CRISPR/Cas9/sg RNA-mediated targeted gene modification in Arabidopsis,tobacco,sorghum and rice[J].Nucleic Acids Research,2013,41(20):e188.
[19]崔红,宋志红.类萜代谢工程研究进展及在烟草品种改良中的应用前景[J].中国烟草学报,2003,9(2):35-38,42.CUI Hong,SONG Zhihong.Recent advances in terpenoid metabolic engineering research and its prospective application in variety improvement in tobacco[J].Acta Tabacaria Sinica,2003,9(2):35-38,42.
[20]杨铁钊,李钦奎,李伟.植物次生代谢与烟草香味物质[J].中国烟草科学,2005,26(4):23-26.YANG Tiezhao,LI Qinkui,LI Wei.Plant secondary metabolism and aroma substances of tobacco[J].Chinese Tobacco Science,2005,26(4):23-26.
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