大麻THCA合成酶基因(CsTHCA)RNA干扰载体的构建及遗传转化
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摘要
大麻THCA合成酶基因CsTHCA在调控大麻THC含量方面起着重要的作用。通过RT-PCR方法从大麻中克隆了CsTHCA,该基因开放阅读框全长1638 bp,共编码545个氨基酸。通过pSKint中间载体,构建CsTHCA RNA干扰植物转化载体并转化大麻茎尖,获得转基因植株,通过分析大麻中THCA合成酶基因的表达并结合薄层色谱法和SPE-HPLC法发现,转基因植株中基因表达量降低且THC含量降低,这表明CsTHCA通过某种机理正调控THC的含量。
CsTHCA plays important roles in regulating THC contents.CsTHCA was isolated from Cannabis sativa L.by RT-PCR.The open reading frame of this gene is 1638 bp.The predicted CsTHCA protein has 545 amino acids.CsTHCA RNA interference vector was constructed through pSKint intermediate vector and transferred to the shoot tips of hemp to obtain transgenic plants.The expression of THCA synthase gene was analyzed by semi-quantitative RT-PCR,and combing with the method of thin-layer chromatography and SPEHPLC,the expression of CsTHCA and THC content were decreased.These results suggested that CsTHCApositively regulates THC content in hemp.
CsTHCA plays important roles in regulating THC contents.CsTHCA was isolated from Cannabis sativa L.by RT-PCR.The open reading frame of this gene is 1638 bp.The predicted CsTHCA protein has 545 amino acids.CsTHCA RNA interference vector was constructed through pSKint intermediate vector and transferred to the shoot tips of hemp to obtain transgenic plants.The expression of THCA synthase gene was analyzed by semi-quantitative RT-PCR,and combing with the method of thin-layer chromatography and SPEHPLC,the expression of CsTHCA and THC content were decreased.These results suggested that CsTHCApositively regulates THC content in hemp.
引文
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[15]杨飞,朱睿,林娜,刘飞虎.苎麻雄性不育相关基因atp6和atp9 RNA干扰载体的构建.中国农学通报,2013,29(21):137-143Yang F,Zhu R,Lin N,Liu F H.Construction of male sterility relatedgenesatp6andatp9RNAivectorsinramie.Chinese Agricultural Science Bulletin,2013,29(21):137-143
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[19]Sirikantaramas S,Morimoto S,Shoyama Y.The gene controlling marijuanapsychoactivity.JournalofBiologicalChemistry,2004,279(38):39767-39774
[20]KojomaM,SekiH,YoshidaS,MuranakaT.DNA polymorphismsinthetetrahydrocannabinolicacid(THCA)synthase gene in"drug-type"and"fiber-type"Cannabis sativaL.Forensic Science International,2006,159(2-3):132-140
[21]Chuang C F,Meyerowitz E M.Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana.Proceedings of the National Academy of Sciences of the United States of America,2000,97(9):4985-4990
[22]Waterhouse P M,Helliwell C A.Exploring plant genomes by RNA induced gene silencing.Nature Reviews Genetics,2003,4:29-38
[23]Wesley S V,Helliwell C A,Smith N A,Wang M B,Rouse D T,Liu Q,Gooding P S,Singh S P,Abbott D,Stoutjesdijk P A,RobinsonSP,Gleave AP,Green AG,WaterhouseP M.Construct design for efficient,effective and high-throughput gene silencing in plants.Plant Journal,2001,27:581-590
[24]姜颖,曹言勇,路运才,唐保军,王利锋,李会勇.拟南芥中RING型E3泛素连接酶基因AtGW2的克隆和功能分析.植物遗传资源学报,2011,12(3):448-454Jiang Y,Cao Y Y,Lu Y C,Tang B J,Wang L F,Li H Y.Cloning andfunctionalanalysisofArabidopsisthalianaAtGW2,a RING-type E3 ubiquitin ligase protein.Journal of Plant Genetic Resources,2011,12(3):448-454
[25]Kaiser J.Plant genetics.From genome to functional genomics.Science,2000,288(5472):1715-1715
[26]Slusarkiewicz-Jarzina A,Ponitka A,Kaczmarek Z.Influence of cultivar,explant source and plant growth regulator on callus inductionandplantregenerationofCannabissativaL.Acta Biologica Cracoviensia,2005,47(2):145-151
[27]WielgusK,Luwanska A,LassocinskiW,Kaczmarek Z.EstimationofCannabissativaL.tissuecultureconditions essential for callus induction and plant regeneration.Journal of Natural Fibers,2008,5(3):199-207
[28]刘以福,唐祥发.大麻组织培养首次获得绿苗.中国麻作,1984(2):29Liu Y F,Tang X F.The first time for obtaining green seedlings byCannabissativatissueculture.PlantFiberandProducts,1984(2):29
[29]尹品训,杨明,郭鸿彦,刘正博,胡学礼,陈裕.大麻组织培养中玻璃化苗研究初报.云南农业科技,2004(4):12YinPX,YangM,GuoH Y,LiuZB,HuXL,Chen Yu.A preliminarystudyonvitrificationinCannabissativatissue culture.Yunnan AgriculturalScienceand Technology,2004(4):12
[30]夏冰,李凝,佟金凤,高南.工业用途的大麻快速繁殖方法,CN 1887043.2007Xia B,Li N,Dong J F,Gao N.Rapid Propagation of Cannabis for Industrial Use,CN 1887043.2007
[31]姜颖,夏尊民,韩承伟,李秋芝,赵越,曹洪勋.工业大麻高效再生体系的初步研究.中国麻业科学,2015(3):126-129JiangY,XiaZM,HanCW,LiQZ,ZhaoYue,CaoH X.Preliminarystudyonefficientregenerationsystemof industrial hemp(Cannabis sativa L.).Plant Fiber Sciences in China,2015(3):126-129
[32]赵越,魏国江,潘冬梅,韩承伟,韩喜财,徐磊.农杆菌介导的汉麻遗传转化影响因素的研究进展.黑龙江农业科学,2015(9):157-160Zhao Y,WeiGJ,PanDM,HanCW,HanXC,XuLei.Research progress on the influencing factors of AgrobacteriummediatedgenetictransformationofCannabissativaL..Heilongjiang Agricultural Sciences,2015(9):157-160
[33]Hartsel S C,Loh W H,Robertson L W.Biotransformation of cannabidiol to cannabielsoin by suspension cultures of Cannabis sativa and Saccharum officinarum.Planta Medica,1983,48(1):17-19
[34]Loh H T,Hartsel S C,Robertson L W.Tissue culture of Cannabis sativa L. and in vitro,biotransformation of phenolics.Zeitschrift Für Pflanzenphysiologie,1983,111(5):395–400
[35]Feeney M,Punja Z K.Tissue culture and Agrobacteriummediated transformation of hemp(Cannabis sativa L.).In Vitro Cellular&Developmental Biology-Plant,2003,39(6):578-585
[2]Radu S,Robu T.Effects and efficiency of dietary hemp seed and flaxseed oils on the human metabolic function.Journal of Environmental Protection and Ecology,2014,15(1):326-331
[3]Rodriguezleyva D,Pierce G N.The cardiac and haemostatic effects of dietary hempseed.Nutrition&Metabolism,2010,7(1):32
[4]Lee M J,Park M S,Hwang S,Hong Y K,Choi G,Suh Y S,Han S Y,Kim D,Jeun J,Oh C T,Lee S J,Han S J,Kim D,KimES,JeongG,ChoKS.Dietaryhempseedmealintake increasesbodygrowthandshortensthelarvalstageviathe upregulationofcellgrowthandsterollevelsinDrosophila melanogaster.Molecules&Cells,2010,30(1):29-36
[5]胡尊红,郭鸿彦,胡学礼,陈璇,刘旭云,郭孟壁,张庆滢,许艳萍,郭丽芬,杨明.大麻品种遗传多样性的AFLP分析.植物遗传资源学报,2012,13(4):555-561HuZH,GuoH Y,HuXL,ChenX,LiuX Y,GuoMB,ZhangQ Y,Xu YP,GuoLF,YangM.Geneticdiversityof CannabissativaL.basedon AFLPanalysis.JournalofPlant Genetic Resources,2012,13(4):555-561
[6]张华,张建春,张杰.汉麻-一种高值特种生物质资源及应用.高分子通报,2011(8):1-7Zhang H,Zhang J C,ZHANG J.Hemp-a high value biological resource and its application chinese polymer bulletin,2011(8):1-7
[7]常丽,唐慧娟,李建军,黄思齐,陈安国,赵立宁,李德芳.大麻CBDA1基因的生物信息学分析.安徽农业科学,2017,45(29):144-148Chang L,Tang H J,Li J J,Huang S Q,Chen A G,Zhao L N,LiDF.BioinformaticanalysisofCBDA1geneinCannabis sativa.Journal of Anhui Agricultural Science,2017,45(29):144-148
[8]陈建华,臧巩固,赵立宁,李育君,唐慧娟.大麻化学成分研究进展与开发我国大麻资源的探讨.中国麻业科学,2003,25(6):266-271Chen J H,Zang G G,Zhao L N,Li Y J,Tang H J.The progress ofstudiesoncannabioidsandsuggestionofexploitinghemp resource in China.Plant Fiber Science in China,2003,25(6):266-271
[9]陈璇,杨明,郭鸿彦.大麻植物中大麻素成分研究进展.植物学报,2011,46(2):197-205.Chen X,Yang M,Guo H Y.Research advances in cannabinoids of Cannabis sativa.Chinese Bulletin of Botany,2011,46(2):197–205
[10]姜颖,潘冬梅,李秋芝,王晓楠,韩承伟,曹焜,韩喜财.大麻THCA合成酶基因的克隆及生物信息学分析.山西农业大学学报:自然科学版,2017,37(5):326-329Jiang Y,PanDM,LiQZ,WangXN,HanC W,CanK,HanXC.CloningangbioinformaticsanalysisofTHCA synthase gene in hemp(Cannabis sativa L.).Journal of Shanxi Agricultural University:Natural Science Edition,2017,37(5):326-329
[11]姜颖,孙宇峰,李秋芝,潘冬梅,韩喜财.大麻中THCA合成酶基因的表达分析.中国麻业科学,2017,39(6):278-282Jiang Y,Sun YF,LiQZ,PanDM,HanXC.Expression pattern assay of THCA synthase gene in hemp(Cannabis sativa L.).Plant Fiber Sciences in China,2017,39(6):278-282
[12]Taura F,Morimoto S,Shoyama Y,Mechoulam R.First direct evidence for the mechanism ofΔ1-tetrahydrocannabinolic acid biosynthesis.Journal of the American Chemical Society,1995,117(38):9766-9767
[13]Sirikantaramas S,Morimoto S,Shoyama Y,Ishikawa Y,Wada Y,Shoyama Y,Taura F.The gene controlling marijuana psychoactivity.JournalofBiologicalChemistry,2004,279(38):39767-39774
[14]陈建华,赵立宁,藏巩固.去除大麻中四氢大麻醇(THC)的方法:四氢大麻酸合成酶基因突变法.中国麻业,2002,24(4):50-52ChenJH,ZhaoLN,ZangGG.EliminatingtheTHC,tetrahydrocannabinolpathwayinCannabissativaby mutagenesis of tetrahydrocannabinolic acid synthase.Plant Fiber and Products,2002,24(4):50-52
[15]杨飞,朱睿,林娜,刘飞虎.苎麻雄性不育相关基因atp6和atp9 RNA干扰载体的构建.中国农学通报,2013,29(21):137-143Yang F,Zhu R,Lin N,Liu F H.Construction of male sterility relatedgenesatp6andatp9RNAivectorsinramie.Chinese Agricultural Science Bulletin,2013,29(21):137-143
[16]黄志鹏,赵艳红,明庭会,余明丽,冉珊敏,钱景华,周瑞阳,陈鹏.红麻6-磷酸葡萄糖酸脱氢酶(6PDGH)基因克隆及RNAi载体构建.南方农业学报,2014,45(12):2110-2116HuangZD,Zhao YH,Ming TH,YuML,RanSM,Qian JH,ZhouR Y,ChenP.6-phosphogluconatedehydrogenase(6PDGH)gene cloning of kenaf(Hibiscus cannabinus L.)and itsRNAinterferencevectorconstruction.JournalofSouthern Agriculture,2014,45(12):2110-2116
[17]张旭,孙宇峰,田媛,石雨,崔宝玉,高宝昌.SPE-HPLC法同时测定汉麻提取物中3种大麻酚含量.化学试剂,2018,40(6):547-550,554ZhangX,SunYF,TianY,ShiY,CuiBY,GaoBC.Determinationofthreecannabinoidsinhempextractionby SPE-HPLC.Journal of Chemical Reagents,2018,40(6):547-550,554
[18]Taura F,Morimoto S Y.Cannabinerolic acid,a cannabinoid from Cannabis sativa.Phytochemistry,1995,39(2):457-458
[19]Sirikantaramas S,Morimoto S,Shoyama Y.The gene controlling marijuanapsychoactivity.JournalofBiologicalChemistry,2004,279(38):39767-39774
[20]KojomaM,SekiH,YoshidaS,MuranakaT.DNA polymorphismsinthetetrahydrocannabinolicacid(THCA)synthase gene in"drug-type"and"fiber-type"Cannabis sativaL.Forensic Science International,2006,159(2-3):132-140
[21]Chuang C F,Meyerowitz E M.Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana.Proceedings of the National Academy of Sciences of the United States of America,2000,97(9):4985-4990
[22]Waterhouse P M,Helliwell C A.Exploring plant genomes by RNA induced gene silencing.Nature Reviews Genetics,2003,4:29-38
[23]Wesley S V,Helliwell C A,Smith N A,Wang M B,Rouse D T,Liu Q,Gooding P S,Singh S P,Abbott D,Stoutjesdijk P A,RobinsonSP,Gleave AP,Green AG,WaterhouseP M.Construct design for efficient,effective and high-throughput gene silencing in plants.Plant Journal,2001,27:581-590
[24]姜颖,曹言勇,路运才,唐保军,王利锋,李会勇.拟南芥中RING型E3泛素连接酶基因AtGW2的克隆和功能分析.植物遗传资源学报,2011,12(3):448-454Jiang Y,Cao Y Y,Lu Y C,Tang B J,Wang L F,Li H Y.Cloning andfunctionalanalysisofArabidopsisthalianaAtGW2,a RING-type E3 ubiquitin ligase protein.Journal of Plant Genetic Resources,2011,12(3):448-454
[25]Kaiser J.Plant genetics.From genome to functional genomics.Science,2000,288(5472):1715-1715
[26]Slusarkiewicz-Jarzina A,Ponitka A,Kaczmarek Z.Influence of cultivar,explant source and plant growth regulator on callus inductionandplantregenerationofCannabissativaL.Acta Biologica Cracoviensia,2005,47(2):145-151
[27]WielgusK,Luwanska A,LassocinskiW,Kaczmarek Z.EstimationofCannabissativaL.tissuecultureconditions essential for callus induction and plant regeneration.Journal of Natural Fibers,2008,5(3):199-207
[28]刘以福,唐祥发.大麻组织培养首次获得绿苗.中国麻作,1984(2):29Liu Y F,Tang X F.The first time for obtaining green seedlings byCannabissativatissueculture.PlantFiberandProducts,1984(2):29
[29]尹品训,杨明,郭鸿彦,刘正博,胡学礼,陈裕.大麻组织培养中玻璃化苗研究初报.云南农业科技,2004(4):12YinPX,YangM,GuoH Y,LiuZB,HuXL,Chen Yu.A preliminarystudyonvitrificationinCannabissativatissue culture.Yunnan AgriculturalScienceand Technology,2004(4):12
[30]夏冰,李凝,佟金凤,高南.工业用途的大麻快速繁殖方法,CN 1887043.2007Xia B,Li N,Dong J F,Gao N.Rapid Propagation of Cannabis for Industrial Use,CN 1887043.2007
[31]姜颖,夏尊民,韩承伟,李秋芝,赵越,曹洪勋.工业大麻高效再生体系的初步研究.中国麻业科学,2015(3):126-129JiangY,XiaZM,HanCW,LiQZ,ZhaoYue,CaoH X.Preliminarystudyonefficientregenerationsystemof industrial hemp(Cannabis sativa L.).Plant Fiber Sciences in China,2015(3):126-129
[32]赵越,魏国江,潘冬梅,韩承伟,韩喜财,徐磊.农杆菌介导的汉麻遗传转化影响因素的研究进展.黑龙江农业科学,2015(9):157-160Zhao Y,WeiGJ,PanDM,HanCW,HanXC,XuLei.Research progress on the influencing factors of AgrobacteriummediatedgenetictransformationofCannabissativaL..Heilongjiang Agricultural Sciences,2015(9):157-160
[33]Hartsel S C,Loh W H,Robertson L W.Biotransformation of cannabidiol to cannabielsoin by suspension cultures of Cannabis sativa and Saccharum officinarum.Planta Medica,1983,48(1):17-19
[34]Loh H T,Hartsel S C,Robertson L W.Tissue culture of Cannabis sativa L. and in vitro,biotransformation of phenolics.Zeitschrift Für Pflanzenphysiologie,1983,111(5):395–400
[35]Feeney M,Punja Z K.Tissue culture and Agrobacteriummediated transformation of hemp(Cannabis sativa L.).In Vitro Cellular&Developmental Biology-Plant,2003,39(6):578-585