棉花U3和U6启动子在CRISPR/Cas9基因组编辑体系中的功能鉴定
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摘要
【目的】对已克隆的海岛棉U3和U6启动子进行功能鉴定,为构建棉花CRISPR/Cas9多位点基因编辑技术体系提供更多可用的U3和U6启动子。【方法】分别构建以GbU3-2P和GbU6-7P为启动子驱动sg RNA,以抗旱负调控基因GGB为靶序列的CRISPR/Cas9基因编辑载体,然后在新海16的棉花叶片原生质体中进行功能鉴定。通过Polymerase chain reaction方法富集构建好的CRISPR/Cas9基因编辑载体的核心片段,并利用PEG瞬时转化法将核心片段转入原生质体中。提取转化后的原生质体基因组DNA,采用酶切/Polymerase chain reaction法分析棉花GGB基因的突变情况并测序验证。最后绘制靶基因突变的频率分布图来计算该CRISPR/Cas9系统的编辑效率和确认突变的真实性。【结果】靶基因测序结果显示靶标位点序列突变的类型全部为碱基替换。【结论】以GbU3-2P和GbU6-7P为启动子的CRISPR/Cas9基因编辑体系可以成功地定点编辑棉花GGB基因的序列,引起基因突变。
[Objective] The function of U3 and U6 promoters that were cloned from sea-island cotton were identified in order to provide more available U3 and U6 promoters for the construction of cotton CRISPR/Cas9 multi-sites gene editing system.[Method] Two CRISPR/Cas9 gene editing vectors were constructed,in which sg RNA were driven by GbU6-7 P and GbU3-2 P,respectively,and GGB,a negative regulator in drought tolerance,was used as target gene.The function of the vector were identified in cotton leaf protoplast of Xinhai 16.The core fragment of CRISPR/Cas9 gene editing vector were enriched by Polymerase chain reaction method and were delivered into protoplast through PEG transient transformation.Then genomic DNA were extracted from protoplast.Gene mutations were analyzed using Enzyme digest/Polymerase chain reaction and sequenced.Finaly the mutation efficiencies of the CRISPR/Cas9 system were calculated and the frequency distribution of the mutation in target site were drawn in order to confirm the authenticity of the mutation.[Result] All type of mutation in target loci were base substitution.[Conclusion]Both CRISPR/Cas9 gene editing systems based on GbU3-2P and GbU6-7P promoters could successfully edit the sequence of cotton GGB gene and cause gene mutation.
[Objective] The function of U3 and U6 promoters that were cloned from sea-island cotton were identified in order to provide more available U3 and U6 promoters for the construction of cotton CRISPR/Cas9 multi-sites gene editing system.[Method] Two CRISPR/Cas9 gene editing vectors were constructed,in which sg RNA were driven by GbU6-7 P and GbU3-2 P,respectively,and GGB,a negative regulator in drought tolerance,was used as target gene.The function of the vector were identified in cotton leaf protoplast of Xinhai 16.The core fragment of CRISPR/Cas9 gene editing vector were enriched by Polymerase chain reaction method and were delivered into protoplast through PEG transient transformation.Then genomic DNA were extracted from protoplast.Gene mutations were analyzed using Enzyme digest/Polymerase chain reaction and sequenced.Finaly the mutation efficiencies of the CRISPR/Cas9 system were calculated and the frequency distribution of the mutation in target site were drawn in order to confirm the authenticity of the mutation.[Result] All type of mutation in target loci were base substitution.[Conclusion]Both CRISPR/Cas9 gene editing systems based on GbU3-2P and GbU6-7P promoters could successfully edit the sequence of cotton GGB gene and cause gene mutation.
引文
[1]胡高峰,张小勇,莫海涛,等.抗盐碱制剂对棉花幼苗生理生化特性的影响[J].西北农业学报,2009,18(2):169-172.Hu Gaofeng,Zhang Xiaoyong,Mo Haitao,et al.The effects of resistance salt-alkaline preparation on the characters of physiological and biochemical index of cotton seedling[J].Acta Agriculturae Boreali-Occidentalis Sinica,2009,18(2):169-172.
[2]徐立华.我国棉花高产、高效栽培技术研究现状与发展思路[J].中国棉花,2001,28(3):5-8.Xu Lihua.Research stat us and development ideas of high yield and high efficient cultivation technology of cotton in China[J].China Cotton,2001,28(3):5-8.
[3]林玲,张昕,邓晟,等.棉花黄萎病研究进展[J].棉花学报,2014,26(3):260-267.Lin Ling,Zhang Xin,Deng Sheng,et al.Research progress of cotton Verticillium wilt[J].Cotton Science,2014,26(3):260-267.
[4]李明桃.棉花枯萎病的研究[J].农业灾害研究,2012,2(4):13-16.Li Mingtao.Study on cotton wilt disease[J].Agricultural Disaster Research,2012,2(4):13-16.
[5]白岩,毛树春,田立文,等.新疆棉花高产简化栽培技术评述与展望[J].中国农业科学,2017,50(1):38-50.Bai Yan,Mao Shuchun,Tian Liwen,et al.Review and prospect of simplified cultivation techniques for high yield of cotton in Xinjiang[J].Scientia Agricultura Sinica,2017,50(1):38-50.
[6]Sun Yongwei,Li Jingying,Xia Lanqin.Precise genome modification via sequence-specific nucleases-mediated gene targeting for crop improvement[J].Frontiers in Plant Science,2016,7(1045):1928.
[7]Chen K,Gao C.Targeted genome modification technologies and their applications in crop improvements[J].Plant Cell Reports,2014,33(4):575-583.
[8]周想春,邢永忠.基因组编辑技术在植物基因功能鉴定及作物育种中的应用[J].遗传,2016,38(3):227-242.Zhou Xiangchun,Xing Yongzhong.Application of genome editing technology in plant gene function identification and crop breeding[J].Genetics,2016,38(3):227-242.
[9]方锐,畅飞,孙照霖,等.CRISPR/Cas9介导的基因组定点编辑技术[J].生物化学与生物物理进展,2013,40(8):691-702.Fang Rui,Chang Fei,Sun Zhaolin,et al.CRISPR/Cas9-mediated genome-based editing technology[J].Progress in Biochemistry and Biophysics,2013,40(8):691-702.
[10]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.
[11]Chen X,Lu X,Shu N,et al.Targeted mutagenesis in cotton(Gossypium hirsutum L.)using the CRISPR/Cas9 system[J].Scientific Reports,2017,7:44304.
[12]Shan Q,Wang Y,Li J,et al.Genome editing in rice and wheat using the CRISPR/Cas system[J].Nature Protocols,2014,9(10):2395.
[13]Jiang W,Zhou H,Bi 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.
[14]Jacobs T,La Fayette B P R,Schmitz R J,et al.Targeted genome modifications in soybean with CRISPR/Cas9[J].BMCBiotechnol,2015,15(1):16.
[15]金双侠,张献龙,聂以春,等.启动子诱捕在棉花基因组中的功能分析[J].遗传学报,2005,32(12):1266-1274.Jin Shuangxia,Zhang Xianlong,Nie Yichun,et al.Functional analysis of promoter trapping in cotton genome[J].Acta Genetica Sinica,2005(12):1266-1274.
[16]雷建峰,徐新霞,代培红,等.不同截短U3启动子在棉花中的功能分析[J].棉花学报,2016,28(3):307-314.Lei Jianfeng,Xu Xinxia,Dai Peihong,et al.Functional analysis of different truncated U3 promoters in cotton[J].Cotton Science,2016,28(3):307-314.
[17]雷建峰,李月,徐新霞,等.棉花不同GbU6启动子截短克隆及功能鉴定[J].作物学报,2016,42(5):675-683.Lei Jianfeng,Li Yue,Xu Xinxia,et al.Truncated clones and functional identification of different GbU6 promoters in cotton[J].Acta Agronomica Sinica,2016,42(5):675-683.
[18]雷建峰,伍娟,陈晓俊,等.棉花花粉中高效转录U6启动子的克隆及功能分析[J].中国农业科学,2015,48(19):3794-3802.Lei Jianfeng,Wu Juan,Chen Xiaojun,et al.Cloning and functional analysis of highly efficient transcriptional U6 promoter in cotton pollen[J].Scientia Agricultura Sinica,2015,48(19):3794-3802.
[19]李继洋,雷建峰,代培红,等.基于棉花U6启动子的海岛棉CRISPR/Cas9基因组编辑体系的建立[J].作物学报,2018,44(2):227-235.Li Jiyang,Lei Jianfeng,Dai Peihong,et al.Establishment of the CRISPR/Cas9 genome editing system based on cotton U6 promoter[J].Acta Agronomica Sinica,2018,44(2):227-235.
[20]Ma X,Zhu Q,Chen Y,et al.CRISPR/Cas9 platforms for genome editing in plants:developments and applications[J]Mol Plant,2016,9(7):961-974.
[21]Feng Z,Zhang B,Ding W,et al.Efficient genome editing in plants using a CRISPR/Cas system[J].Cell Research,2013,23(10):1229-1232.
[22]Yoo S D,Cho Y H,Sheen J.Arabidopsis mesophyll protoplasts:a versatile cell system for transient gene expression analysis[J].Nature Protocols,2007,2(7):1565-1572.
[23]Belhaj K,Chaparro-garcia A,Kamous S,et al.Editing plant genomes with CRISPR/Cas9[J].Current Opinion in Biotechnology,2015,32:76-84.
[24]李妮娜,丁林云,张志远,等.棉花叶肉原生质体分离及目标基因瞬时表达体系的建立[J].作物学报,2014,40(2):231-239.Li Nina,Ding Linyun,Zhang Zhiyuan,et al.Protoplast isolation of cotton mesophyll and establishment of transient expression system of target genes[J].Acta Agronomica Sinica,2014,40(2):231-239.
[25]Zlotorynski E.Plant cell biology:CRISPR-Cas protection from plant viruses[J].Nature Reviews Molecular Cell Biology,2015,16(11):642.
[26]Li C,Unver T,Zhang B.A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in cotton(Gossypium hirsutum L.)[J].Scientific Reports,2017,7:43902.
[27]杨勇,田新权,刘会利,等.陆地棉NAC转录因子基因GhNAC6的克隆、表达和耐盐性分析[J].棉花学报,2017,29(2):138-146.Yang Yong,Tian Xinquan,Liu Huili,et al.Cloning,expression and salt-tolerance analysis of the NAC transcription factor gene GhNAC6 in upland cotton[J].Cotton Science,2017,29(2):138-146.
[28]苏莹,甄军波,张曦,等.陆地棉转录因子基因GhC2H2的克隆与功能分析[J].棉花学报,2016,28(6):555-564.Su Ying,Zhen Junbo,Zhang Xi,et al.Cloning and functional analysis of a transcription factor gene,GhC2H2,in upland cotton(Gossypium hirsutum L.)[J].Cotton Science,2016,28(6):555-564.
[2]徐立华.我国棉花高产、高效栽培技术研究现状与发展思路[J].中国棉花,2001,28(3):5-8.Xu Lihua.Research stat us and development ideas of high yield and high efficient cultivation technology of cotton in China[J].China Cotton,2001,28(3):5-8.
[3]林玲,张昕,邓晟,等.棉花黄萎病研究进展[J].棉花学报,2014,26(3):260-267.Lin Ling,Zhang Xin,Deng Sheng,et al.Research progress of cotton Verticillium wilt[J].Cotton Science,2014,26(3):260-267.
[4]李明桃.棉花枯萎病的研究[J].农业灾害研究,2012,2(4):13-16.Li Mingtao.Study on cotton wilt disease[J].Agricultural Disaster Research,2012,2(4):13-16.
[5]白岩,毛树春,田立文,等.新疆棉花高产简化栽培技术评述与展望[J].中国农业科学,2017,50(1):38-50.Bai Yan,Mao Shuchun,Tian Liwen,et al.Review and prospect of simplified cultivation techniques for high yield of cotton in Xinjiang[J].Scientia Agricultura Sinica,2017,50(1):38-50.
[6]Sun Yongwei,Li Jingying,Xia Lanqin.Precise genome modification via sequence-specific nucleases-mediated gene targeting for crop improvement[J].Frontiers in Plant Science,2016,7(1045):1928.
[7]Chen K,Gao C.Targeted genome modification technologies and their applications in crop improvements[J].Plant Cell Reports,2014,33(4):575-583.
[8]周想春,邢永忠.基因组编辑技术在植物基因功能鉴定及作物育种中的应用[J].遗传,2016,38(3):227-242.Zhou Xiangchun,Xing Yongzhong.Application of genome editing technology in plant gene function identification and crop breeding[J].Genetics,2016,38(3):227-242.
[9]方锐,畅飞,孙照霖,等.CRISPR/Cas9介导的基因组定点编辑技术[J].生物化学与生物物理进展,2013,40(8):691-702.Fang Rui,Chang Fei,Sun Zhaolin,et al.CRISPR/Cas9-mediated genome-based editing technology[J].Progress in Biochemistry and Biophysics,2013,40(8):691-702.
[10]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.
[11]Chen X,Lu X,Shu N,et al.Targeted mutagenesis in cotton(Gossypium hirsutum L.)using the CRISPR/Cas9 system[J].Scientific Reports,2017,7:44304.
[12]Shan Q,Wang Y,Li J,et al.Genome editing in rice and wheat using the CRISPR/Cas system[J].Nature Protocols,2014,9(10):2395.
[13]Jiang W,Zhou H,Bi 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.
[14]Jacobs T,La Fayette B P R,Schmitz R J,et al.Targeted genome modifications in soybean with CRISPR/Cas9[J].BMCBiotechnol,2015,15(1):16.
[15]金双侠,张献龙,聂以春,等.启动子诱捕在棉花基因组中的功能分析[J].遗传学报,2005,32(12):1266-1274.Jin Shuangxia,Zhang Xianlong,Nie Yichun,et al.Functional analysis of promoter trapping in cotton genome[J].Acta Genetica Sinica,2005(12):1266-1274.
[16]雷建峰,徐新霞,代培红,等.不同截短U3启动子在棉花中的功能分析[J].棉花学报,2016,28(3):307-314.Lei Jianfeng,Xu Xinxia,Dai Peihong,et al.Functional analysis of different truncated U3 promoters in cotton[J].Cotton Science,2016,28(3):307-314.
[17]雷建峰,李月,徐新霞,等.棉花不同GbU6启动子截短克隆及功能鉴定[J].作物学报,2016,42(5):675-683.Lei Jianfeng,Li Yue,Xu Xinxia,et al.Truncated clones and functional identification of different GbU6 promoters in cotton[J].Acta Agronomica Sinica,2016,42(5):675-683.
[18]雷建峰,伍娟,陈晓俊,等.棉花花粉中高效转录U6启动子的克隆及功能分析[J].中国农业科学,2015,48(19):3794-3802.Lei Jianfeng,Wu Juan,Chen Xiaojun,et al.Cloning and functional analysis of highly efficient transcriptional U6 promoter in cotton pollen[J].Scientia Agricultura Sinica,2015,48(19):3794-3802.
[19]李继洋,雷建峰,代培红,等.基于棉花U6启动子的海岛棉CRISPR/Cas9基因组编辑体系的建立[J].作物学报,2018,44(2):227-235.Li Jiyang,Lei Jianfeng,Dai Peihong,et al.Establishment of the CRISPR/Cas9 genome editing system based on cotton U6 promoter[J].Acta Agronomica Sinica,2018,44(2):227-235.
[20]Ma X,Zhu Q,Chen Y,et al.CRISPR/Cas9 platforms for genome editing in plants:developments and applications[J]Mol Plant,2016,9(7):961-974.
[21]Feng Z,Zhang B,Ding W,et al.Efficient genome editing in plants using a CRISPR/Cas system[J].Cell Research,2013,23(10):1229-1232.
[22]Yoo S D,Cho Y H,Sheen J.Arabidopsis mesophyll protoplasts:a versatile cell system for transient gene expression analysis[J].Nature Protocols,2007,2(7):1565-1572.
[23]Belhaj K,Chaparro-garcia A,Kamous S,et al.Editing plant genomes with CRISPR/Cas9[J].Current Opinion in Biotechnology,2015,32:76-84.
[24]李妮娜,丁林云,张志远,等.棉花叶肉原生质体分离及目标基因瞬时表达体系的建立[J].作物学报,2014,40(2):231-239.Li Nina,Ding Linyun,Zhang Zhiyuan,et al.Protoplast isolation of cotton mesophyll and establishment of transient expression system of target genes[J].Acta Agronomica Sinica,2014,40(2):231-239.
[25]Zlotorynski E.Plant cell biology:CRISPR-Cas protection from plant viruses[J].Nature Reviews Molecular Cell Biology,2015,16(11):642.
[26]Li C,Unver T,Zhang B.A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in cotton(Gossypium hirsutum L.)[J].Scientific Reports,2017,7:43902.
[27]杨勇,田新权,刘会利,等.陆地棉NAC转录因子基因GhNAC6的克隆、表达和耐盐性分析[J].棉花学报,2017,29(2):138-146.Yang Yong,Tian Xinquan,Liu Huili,et al.Cloning,expression and salt-tolerance analysis of the NAC transcription factor gene GhNAC6 in upland cotton[J].Cotton Science,2017,29(2):138-146.
[28]苏莹,甄军波,张曦,等.陆地棉转录因子基因GhC2H2的克隆与功能分析[J].棉花学报,2016,28(6):555-564.Su Ying,Zhen Junbo,Zhang Xi,et al.Cloning and functional analysis of a transcription factor gene,GhC2H2,in upland cotton(Gossypium hirsutum L.)[J].Cotton Science,2016,28(6):555-564.
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