红麻miR394基因的克隆及CRISPR/Cas9载体构建
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摘要
为探明miRNA在红麻中的功能,以红麻基因组DNA为模板,根据红麻microRNA(miRNA)高通量测序数据结果,克隆红麻miR394前体基因序列,并构建CRISPR/Cas9载体。结果表明红麻miR394前体基因序列大小为175bp,用Mfold在线软件分析,其前体序列能形成稳定的二级结构。根据红麻miR394前体基因序列,设计合适的CRISPR/Cas9靶标序列,利用改良的CRISPR/Cas9多靶点载体系统,以AtU3b和AtU6-29质粒为模板,使用Overlapping PCR法构建AtU3b-sgRNA和AtU6-29-sgRNA表达盒,使用Golden Gate Cloning方法成功把靶标AtU3b-sgRNA和AtU6-29-sgRNA表达盒构建到pYLCRISPR/Cas9载体中,并将构建好的载体命名为pCas9-miR394。分离红麻叶片原生质体,将pCas9-miR394载体转入红麻叶片原生质体中进行瞬时表达,测序结果表明:2个靶点Target site 1(T1)和Target site 2(T2)处都发生碱基突变,表明pCas9-miR394载体在红麻原生质体中具有靶向切割的活性。
In order to explore the function of miRNA in kenaf,based on the results of high throughput miRNA sequencing,miR394,a microRNA,precursor gene sequence of kenaf was cloned using kenaf genomic DNA as template.The result showed that the sequence size of pre-miR394 of kenaf was 175 bp,and the precursor sequence formed a stable secondary structure analyzed using Mfold software.A reliable CRISPR/Cas9 target sequence was designed according to the miR394 precursor gene sequence of kenaf.In this study,taking the AtU3 band AtU6-29 plasmids as templates and based on an improved CRISPR/Cas9 multi-target vector system,AtU3 b-sgRNA and AtU6-29-sgRNA expression cassettes were successfully constructed using overlapping PCR technique.The two expression cassettes were successfully constructed into pYLCRISPR/Cas9 vectors according to Golden Gate Cloning method.The constructed vector was named pCas9-miR394.pCas9-miR394 was then transiently expressed in the protoplasts of kenaf leaves.The sequencing result indicated that one base mutation occurred at Target site 1(T1)and Target site2(T2),demonstrating that the pCas9-miR394 vector had target cutting activity in kenaf protoplasts.This vector could be used for subsequent genetic transformation analysis in kenaf.
In order to explore the function of miRNA in kenaf,based on the results of high throughput miRNA sequencing,miR394,a microRNA,precursor gene sequence of kenaf was cloned using kenaf genomic DNA as template.The result showed that the sequence size of pre-miR394 of kenaf was 175 bp,and the precursor sequence formed a stable secondary structure analyzed using Mfold software.A reliable CRISPR/Cas9 target sequence was designed according to the miR394 precursor gene sequence of kenaf.In this study,taking the AtU3 band AtU6-29 plasmids as templates and based on an improved CRISPR/Cas9 multi-target vector system,AtU3 b-sgRNA and AtU6-29-sgRNA expression cassettes were successfully constructed using overlapping PCR technique.The two expression cassettes were successfully constructed into pYLCRISPR/Cas9 vectors according to Golden Gate Cloning method.The constructed vector was named pCas9-miR394.pCas9-miR394 was then transiently expressed in the protoplasts of kenaf leaves.The sequencing result indicated that one base mutation occurred at Target site 1(T1)and Target site2(T2),demonstrating that the pCas9-miR394 vector had target cutting activity in kenaf protoplasts.This vector could be used for subsequent genetic transformation analysis in kenaf.
引文
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[13]孙现军.CRISPR/Cas9基因定点突变体系构建与大豆抗旱相关gma-miR160功能研究[D].杨凌:西北农林科技大学,2015.Sun X J. Construction of CRISPR/Cas9 site-directed mutagenesis system and fuctional research of droughtresponsive soybean gma-miR160[D].Yangling:Northwest Agriculture and Forestry University,2015
[14] Ma X L,Zhang Q N,Zhu Q L,Liu W,Chen Y,Qiu R,Wang B,Yang Z F,Li H Y,Lin Y R,Xie Y Y,Shen R X,Chen S F,Wang Z,Chen Y L,Guo J X,Chen L T,Zhao X C,Dong Z C,Liu Y G.A robust CRISPR/Cas9system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants[J].Molecular Plant,2015,8(8):1274-1284
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[16] 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
[17]王丽爽,张宗申,郑来久.红麻原生质体分离条件的优化[J].大连工业大学学报,2010,29(4):244-247Wang L S,Zhang Z S,Zheng L J. Optimization for protoplast isolation from the Hibiscus cannabinus L[J].Journal of Dalian Polytechnic University,2010,29(4):244-247
[18]牛英.红麻的器官培养及原生质体培养[D].武汉:华中农业大学,2004Niu Y.A study on organ culture and protoplast culture of kenaf[D].Wuhan:Huazhong Agricultural University,2004
[19]李荣华,夏岩石,刘顺枝,孙莉丽,郭培国,缪绅裕,陈建辉.改进的CTAB提取植物DNA方法.实验室研究与探索[J],2009,28(9):14-16Li R H,Xia Y S,Liu S Z,Sun L L,Guo P G,Miao S Y,Chen J H.The improved CTAB extraction plant DNA method[J].Research and Exploration in Laboratory.2009,28(9):14-16
[20] Bartel D P.MicroRNAs:Genomics,biogenesis,mechanism and function[J].Cell,2004,116(2):281-297
[21] Jones-RhoadesMW, BartelDP. Computational identification of plant microRNAs and their targerts,including a stress-induced miRNA[J].Molecular Cell,2004,14(6):787-799
[22] Barakat A,Wall K,Leebens-Mack J,Wang Y J,Carlson J E, DepamphilisCW. Large-scaleidentificationof microRNAs from a basal eudicot(Eschscholzia californica)and conservation in flowering plants[J].The Plant Journal,2007,51(6):991-1003
[23] Sunkar R,Jagadeeswaran G.In silico identification of conserved microRNAs in large number of diverse plant species[J].BMC Plant Biology.2008,16(8):37
[24] Chellappan P,Jin H.Discover of plant microRNAs and short interfering RNAs by deep parallel sequencing[J].Methodes Molecular Biology,2009,495:121-132
[25] Zhang H,Zhang J,Wei P,Zhang B T,Gou F,Feng Z Y,Mao Y F,Yang L,Zhang H,Xu N F,Zhu J K.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
[26] Gao J,Wang G,Ma S,Xie X D,Wu X W,Zhang X T,Wu Y Q,Zhao P,Xia Q Y.CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum[J].Plant molecular biology,2015,87(1/2):99-110
[27] Jiang W Z,Yang B,Weeks D P.Efficient CRISPR/Cas9-mediatedgene editing in Arabidopsis thaliana and inheritance of modified genes in the T2and T3generations[J].PloS One,(2014-09-06)DOI:10.1371/journal.pone.0099225
[28]单奇伟,高彩霞.植物基因组编辑及衍生技术最新研究进展[J].遗传,2015,37(10):953-973Shan Q W,Gao C X.Research progress of genome editing and derivative technologies in plants[J].Hereditas,2015,37(10):953-973
[29] Xing H L,Dong L,Wang Z P,Zhang H Y,Han C Y,Liu B, Wang X C,Chen Q J.A CRISPR/Cas9toolkit for multiplex genome editing in plants[J].BMC Plant Biology,2014,14(1):327
[2]陶爱芬,张晓琛,祁建民.红麻综合利用研究进展与产业化前景[J].中国麻业科学,2007,29(1):1-5Tao A F,Zhang X C,Qi J M.Research progress and industrialization prospect of comprehensive utilization on kenaf[J].Chinese Hemp Science,2007,29(1):1-5
[3] Voinnet O. Origin, biogenesis and activity of plant MicroRNAs[J].Cell,2009,136(4):669-687
[4] Llave C,Xie Z X,Kasschau K D,Carrington J C.Cleavage of scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA[J].Science,2002,297(5589):2053-2056
[5] Jover-Gil S,Candela H,Ponce M.Plant microRNAs and development[J].International Journal of Developmental Biology,2005,49(5/6):733
[6] Willmann M R,Poethig R S.Conservation and evolution of miRNA regulatory programs in plant development[J].Current Opinion in Plant Biology,2007,10(5):503-511
[7] Song J B,Shu X X,Shen Q,Li B W,Song J,Yang Z M.Altered fruit and seed development of transgenic Rapeseed(Brassica napus)overexpressing MicroRNA394[J].PLoS ONE,(2015-10-05)DOI:10.1371/journal.pone.0125427
[8]宋剑波.miRNA394调控拟南芥叶片形态、盐和干旱胁迫的反应[D].南京:南京农业大学,2013Song J B.Regulation of leaf morphology,salt and drought stress responses by microRNA394in arabidopsis[D].Nanjing:Nanjing Agricultural University,2013
[9] Barrangou R,Fremaux C,Deveau H,Richards M,Boyaval P,Moineau S,Romero D A,Horvath P.CRISPR provides acquired resistance against viruses in prokaryotes[J].Science,2007,315(5819):1709-1712
[10]刘婷婷,范迪,冉玲玉,姜渊忠,刘瑞,罗克明.应用CRISPR/Cas9技术在杨树中高效敲除多个靶基因[J].遗传,2015,37(10):1044-1052Liu T T,Fan D,Ran L Y,Jiang Y Z,Liu R,Luo K M.Highly efficient CRISPR/Cas9-mediated targeted mutagenesis of multiple genes in populus[J].Hereditas,2015,37(10):1044-1052
[11] Chang H,Yi B,Ma R X,Zhang X G,Zhao H Y,Xi Y G.CRISPR/cas9,a novel genomic tool to knock down microRNA in vitro and in vivo[J].Scientific Reports,2016,6:22312
[12]刘宣,季青,李文,周利红,王璇,江海丽,陈静,畅立圣,李琦.应用CRISPR/Cas9基因编辑技术建立miRNA-301a敲除小鼠[J].第二军医大学学报,2015,35(3):256-260Liu X,Ji Q,Li W,Zhou L H,Wang X,Jing H L,Chen J,Chang L S, Li Q. Application of CRISPR/Cas9 gene targeting technology for establishing miRNA-301aknockout mouse model[J].Academic Journal of Second Military Medical University,2015,35(3):256-260
[13]孙现军.CRISPR/Cas9基因定点突变体系构建与大豆抗旱相关gma-miR160功能研究[D].杨凌:西北农林科技大学,2015.Sun X J. Construction of CRISPR/Cas9 site-directed mutagenesis system and fuctional research of droughtresponsive soybean gma-miR160[D].Yangling:Northwest Agriculture and Forestry University,2015
[14] Ma X L,Zhang Q N,Zhu Q L,Liu W,Chen Y,Qiu R,Wang B,Yang Z F,Li H Y,Lin Y R,Xie Y Y,Shen R X,Chen S F,Wang Z,Chen Y L,Guo J X,Chen L T,Zhao X C,Dong Z C,Liu Y G.A robust CRISPR/Cas9system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants[J].Molecular Plant,2015,8(8):1274-1284
[15] Engler C,Gruetzner R,Kandzia R,Marillonnet S.Golden gate shuffling:A one-pot DNA shuffling method based on type IIs restriction enzymes[J].PLoS One,(2009-04-05)DOI:10.1371/journal.pone.0005553
[16] 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
[17]王丽爽,张宗申,郑来久.红麻原生质体分离条件的优化[J].大连工业大学学报,2010,29(4):244-247Wang L S,Zhang Z S,Zheng L J. Optimization for protoplast isolation from the Hibiscus cannabinus L[J].Journal of Dalian Polytechnic University,2010,29(4):244-247
[18]牛英.红麻的器官培养及原生质体培养[D].武汉:华中农业大学,2004Niu Y.A study on organ culture and protoplast culture of kenaf[D].Wuhan:Huazhong Agricultural University,2004
[19]李荣华,夏岩石,刘顺枝,孙莉丽,郭培国,缪绅裕,陈建辉.改进的CTAB提取植物DNA方法.实验室研究与探索[J],2009,28(9):14-16Li R H,Xia Y S,Liu S Z,Sun L L,Guo P G,Miao S Y,Chen J H.The improved CTAB extraction plant DNA method[J].Research and Exploration in Laboratory.2009,28(9):14-16
[20] Bartel D P.MicroRNAs:Genomics,biogenesis,mechanism and function[J].Cell,2004,116(2):281-297
[21] Jones-RhoadesMW, BartelDP. Computational identification of plant microRNAs and their targerts,including a stress-induced miRNA[J].Molecular Cell,2004,14(6):787-799
[22] Barakat A,Wall K,Leebens-Mack J,Wang Y J,Carlson J E, DepamphilisCW. Large-scaleidentificationof microRNAs from a basal eudicot(Eschscholzia californica)and conservation in flowering plants[J].The Plant Journal,2007,51(6):991-1003
[23] Sunkar R,Jagadeeswaran G.In silico identification of conserved microRNAs in large number of diverse plant species[J].BMC Plant Biology.2008,16(8):37
[24] Chellappan P,Jin H.Discover of plant microRNAs and short interfering RNAs by deep parallel sequencing[J].Methodes Molecular Biology,2009,495:121-132
[25] Zhang H,Zhang J,Wei P,Zhang B T,Gou F,Feng Z Y,Mao Y F,Yang L,Zhang H,Xu N F,Zhu J K.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
[26] Gao J,Wang G,Ma S,Xie X D,Wu X W,Zhang X T,Wu Y Q,Zhao P,Xia Q Y.CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum[J].Plant molecular biology,2015,87(1/2):99-110
[27] Jiang W Z,Yang B,Weeks D P.Efficient CRISPR/Cas9-mediatedgene editing in Arabidopsis thaliana and inheritance of modified genes in the T2and T3generations[J].PloS One,(2014-09-06)DOI:10.1371/journal.pone.0099225
[28]单奇伟,高彩霞.植物基因组编辑及衍生技术最新研究进展[J].遗传,2015,37(10):953-973Shan Q W,Gao C X.Research progress of genome editing and derivative technologies in plants[J].Hereditas,2015,37(10):953-973
[29] Xing H L,Dong L,Wang Z P,Zhang H Y,Han C Y,Liu B, Wang X C,Chen Q J.A CRISPR/Cas9toolkit for multiplex genome editing in plants[J].BMC Plant Biology,2014,14(1):327
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