利用CRISPR/Cas9技术的烟草NtLS基因敲除分析
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摘要
为了培育无腋芽或少腋芽烟草,基于CRISPR/Cas9系统,对烟草NtLS基因进行靶向基因组编辑,构建了2个特异靶向NtLS基因的CRISPR/Cas9载体cLS1和cLS2,并借助农杆菌介导的烟草转基因技术转化烤烟品种K326,获得20株抗卡那霉素的阳性烟株。10株阳性烟株经过PCR扩增、测序和比对,检测到2株发生蛋白翻译错误的T_0突变烟株。研究结果不仅为腋芽形成的分子机理研究创制了有价值的突变体,而且为培育无腋芽或少腋芽优质烤烟品系提供了遗传材料。
To breed new tobacco varieties with no or less axillary buds, based on CRISPR/Cas9 system, the DNA sequence of NtLS was chosen as the target site for designing two CRISPR/Cas9 vectors(cLS1 and cLS2).Agrobacterium tumefaciens-mediated transformation of cv. K326 was used and twenty kanamycin-resistance tobacco plants(T_0 generation) were obtained. Ten positively transformed tobacco plants were analyzed by PCR amplification, sequencing and alignment, and some mutations of NtLS protein were detected in two plants.These research results provide useful mutants to study the molecular mechanism of axillary bud formation and create genetic materials for new flue-cured tobacco varieties with no or less axillary buds.
To breed new tobacco varieties with no or less axillary buds, based on CRISPR/Cas9 system, the DNA sequence of NtLS was chosen as the target site for designing two CRISPR/Cas9 vectors(cLS1 and cLS2).Agrobacterium tumefaciens-mediated transformation of cv. K326 was used and twenty kanamycin-resistance tobacco plants(T_0 generation) were obtained. Ten positively transformed tobacco plants were analyzed by PCR amplification, sequencing and alignment, and some mutations of NtLS protein were detected in two plants.These research results provide useful mutants to study the molecular mechanism of axillary bud formation and create genetic materials for new flue-cured tobacco varieties with no or less axillary buds.
引文
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[21]Xie X D,Qin G Y,Si P,et al.Analysis of Nicotiana tabacum PIN genes identifies Nt PIN4 as a key regulator of axillary bud growth[J].Physiologia Plantarum,2017,160(2):222-239.
[22]聂梦云,高军平,罗培,等.基于CRISPR/Cas9技术的烟草Nt DXR基因敲除及功能分析[J].烟草科技,2016,49(6):1-7.NIE Mengyun,GAO Junping,LUO Pei,et al.CRISPR/Cas9-mediated targeted mutagenesis and function analysis of DXR in Nicotiana tabacum[J].Tobacco Science&Technology,2016,49(6):1-7.
[23]高军平.CRISPR/Cas9介导的烟草基因组编辑与基因功能研究[D].重庆:西南大学,2016.GAO Junping.Genome editing and gene function research in Nicotiana tabacum using CRISPR/Cas9[D].Chongqing:Southwest University,2016.
[24]Sander J D,Maeder M L,Reyon D,et al.Zi Fi T(Zinc Finger Targeter):an updated zinc finger engineering tool[J].Nucleic Acids Research,2010,38(S2):462-468.
[2]张文霞,刁志娟,吴为人.植物GRAS蛋白研究的新进展[J].分子植物育种,2016,14(5):1159-1165.ZHANG Wenxia,DIAO Zhijuan,WU Weiren.New advances in the study of plant GRAS proteins[J].Molecular Plant Breeding,2016,14(5):1159-1165.
[3]Schumacher K,Schmitt T,Rossberg M,et al.The Lateral suppressor(Ls)gene of tomato encodes a new member of the VHIID protein family[J].Proceedings of the National Academy of Sciences of the United States of America,1999,96(1):290-295.
[4]Liu X Y,Widmer A.Genome-wide comparative analysis of the GRAS gene family in Populus,Arabidopsis and rice[J].Plant Molecular Biology Reporter,2014,32(6):1129-1145.
[5]Lu J X,Wang T,Xu Z D,et al.Genome-wide analysis of the GRAS gene family in Prunus mume[J].Molecular Genetics and Genomics,2015,290(1):303-317.
[6]孙欣,王晨,房经贵,等.葡萄GRAS基因家族生物信息学分析[J].江西农业学报,2011,23(7):1-8.SUN Xin,WANG Chen,FANG Jinggui,et al.Bioinformatics analysis of GRAS gene family in grapevine[J].Acta Agriculturae Jiangxi,2011,23(7):1-8.
[7]Greb T,Clarenz O,Sch?fer E,et al.Molecular analysis of the LATERAL SUPPRESSOR gene in Arabidopsis reveals a conserved control mechanism for axillary meristem formation[J].Genes&Development,2003,17(9):1175-1187.
[8]Li X Y,Qian Q,Fu Z M,et al.Control of tillering in rice[J].Nature,2003,422(6932):618-621.
[9]周冀衡.烟草生理与生物化学[M].合肥:中国科学技术大学出版社,1996.ZHOU Jiheng.Tobacco physiology and biochemistry[M].Hefei:Press of University of Science and Technology of China,1996.
[10]林桂华,周冀衡,范启福,等.打顶技术对烤烟产质量和生物碱组成的影响[J].中国烟草科学,2002,23(4):8-12.LIN Guihua,ZHOU Jiheng,FAN Qifu,et al.Effects of topping technology on leaf yield,quality and alkaloid content of flue-cured tobacco[J].Chinese Tobacco Science,2002,23(4):8-12.
[11]中国农业科学院烟草研究所.中国烟草栽培学[M].上海:上海科学技术出版社,1987.Institute of Tobacco Research of CAAS.Tobacco cultivation in China[M].Shanghai:Shanghai Scientific&Technical Publishers,1987.
[12]陈德鑫,王凤龙,杨清林,等.烟草抑芽剂及其使用方法[J].烟草科技,2003(6):46-48.CHEN Dexin,WANG Fenglong,YANG Qinglin,et al.Tobacco suckercides and their applying methods[J].Tobacco Science&Technology,2003(6):46-48.
[13]陈雅琼.烟草腋芽发育相关基因的克隆与功能分析[D].北京:中国农业科学院,2015.CHEN Yaqiong.Cloning and functional analysis of axillary bud development-related genes in tobacco[D].Beijing:Chinese Academy of Agricultural Sciences,2015.
[14]陈兴江.烟草抑芽基因克隆及遗传转化研究[D].海口:华南热带农业大学,2005.CHEN Xingjiang.Cloning the gene inhabiting tobacco axillary bud and its genetic transformation on tobacco[D].Haikou:South China University of TropicalAgriculture,2005.
[15]吴华.基因工程创建无侧芽烟草的初步研究[D].海口:海南大学,2008.WU Hua.A preliminary study on tobacco without axillary buds created by genetic engineering[D].Haikou:Hainan University,2008.
[16]王卫锋,太帅帅,王鲁,等.烟草Nt LS基因RNA干扰表达载体的构建及遗传转化[J].中国烟草科学,2011,32(4):31-35.WANG Weifeng,TAI Shuaishuai,WANG Lu,et al.Construction of RNAi vector of Nt LS gene and its transformation in tobacco[J].Chinese Tobacco Science,2011,32(4):31-35.
[17]Pyott D E,Sheehan E,Molnar A.Engineering of CRISPR/Cas9-mediated potyvirus resistance in transgene-free Arabidopsis plants[J].Molecular Plant Pathology,2016,17(8):1276-1288.
[18]Zhang H,Zhang J S,Wei P L,et al.The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation[J].Plant Biotechnology Journal,2014,12(6):797-807.
[19]Shan Q W,Wang Y P,Li J,et al.Genome editing in rice and wheat using the CRISPR/Cas system[J].Nature Protocols,2014,9(10):2395-2410.
[20]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(1/2):99-110.
[21]Xie X D,Qin G Y,Si P,et al.Analysis of Nicotiana tabacum PIN genes identifies Nt PIN4 as a key regulator of axillary bud growth[J].Physiologia Plantarum,2017,160(2):222-239.
[22]聂梦云,高军平,罗培,等.基于CRISPR/Cas9技术的烟草Nt DXR基因敲除及功能分析[J].烟草科技,2016,49(6):1-7.NIE Mengyun,GAO Junping,LUO Pei,et al.CRISPR/Cas9-mediated targeted mutagenesis and function analysis of DXR in Nicotiana tabacum[J].Tobacco Science&Technology,2016,49(6):1-7.
[23]高军平.CRISPR/Cas9介导的烟草基因组编辑与基因功能研究[D].重庆:西南大学,2016.GAO Junping.Genome editing and gene function research in Nicotiana tabacum using CRISPR/Cas9[D].Chongqing:Southwest University,2016.
[24]Sander J D,Maeder M L,Reyon D,et al.Zi Fi T(Zinc Finger Targeter):an updated zinc finger engineering tool[J].Nucleic Acids Research,2010,38(S2):462-468.