不同发育时期大麻素合成相关酶基因表达特征与大麻素含量的相关分析
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摘要
本研究采用高效液相色谱法和实时定量PCR方法,分别测定了大麻植株不同发育时期主要大麻素含量变化以及大麻素合成途径16个相关酶基因的表达丰度,分析了大麻素含量与各基因表达量之间的相关性。结果表明,大麻素含量在幼苗期和快速生长期(简称快生期)接近零,生殖阶段(盛蕾期,盛花期和始果期)快速增加,以始果期苞片中含量最高。大麻素合成过程中4个合成途径内部存在基因协同表达特征,4个合成途径各自酶基因相对表达量变化规律相似。此外,发育过程合成相关酶基因表达量与大麻素含量相关性分析表明,CMK、MDS、HDS、HDR和GPP(lsu)等5个基因表达量和大麻素含量呈现显著正相关(相关系数>0.9)。结果说明大麻素合成相关酶基因可能通过协同作用来调控大麻素合成与积累,MEP和GPP途径后端的酶基因在大麻素合成过程中起着重要作用,而Cannabinoid途径中OLS、PT和THCAS三个大麻中特有的酶基因主要在始果期苞片腺毛中对大麻素合成积累起着关键作用。这些结果为今后利用基因工程手段改良大麻素含量提供了研究基础。
In this study,the HPLC and real-time PCR were used to determine the cannabinoids content and the expression levels of 16 related enzymes in cannabinoids biosynthesis at different developmental stages of Cannabis sativa L.,aiming to analyze the correlation between cannabinoids content and gene expression.The results showed that the cannabinoids content was close to zero at seedling stage and rapid-growing stage,but increased rapidly at the reproductive stage(budding,flowering and initial-fruiting),with the highest content in the bract at initial-fruiting stage.In the process of cannabinoids synthesis,the 4 biosynthetic pathways were characterized by gene co-expression,and the relative expression of each enzyme gene in the 4 biosynthetic pathways was similar.Moreover,the correlation analysis between expression level of related enzymes and cannabinoids content showed that the expression levels of 5 enzyme genes(CMK,MDS,HDS,HDR and GPP(lsu)) had significant positive correlation with cannabinoids content(R>0.9).It was revealed that the related enzyme genes in cannabinoids biosynthesis might regulate the synthesis and accumulation of cannabinoids through coordinated expression.It alsodemonstrated that the back-end enzyme genes in MEP and GPP pathway played important roles in cannabinoids biosynthesis,while the three specific enzyme genes in C.sativa(OLS,PT and THCAS) played key roles in the biosynthesis and accumulation of cannabinoids in the glandular hairs of bract at initial-fruiting stage.These results would provide a basis for modifying cannabinoids content through genetic engineering in the future.
In this study,the HPLC and real-time PCR were used to determine the cannabinoids content and the expression levels of 16 related enzymes in cannabinoids biosynthesis at different developmental stages of Cannabis sativa L.,aiming to analyze the correlation between cannabinoids content and gene expression.The results showed that the cannabinoids content was close to zero at seedling stage and rapid-growing stage,but increased rapidly at the reproductive stage(budding,flowering and initial-fruiting),with the highest content in the bract at initial-fruiting stage.In the process of cannabinoids synthesis,the 4 biosynthetic pathways were characterized by gene co-expression,and the relative expression of each enzyme gene in the 4 biosynthetic pathways was similar.Moreover,the correlation analysis between expression level of related enzymes and cannabinoids content showed that the expression levels of 5 enzyme genes(CMK,MDS,HDS,HDR and GPP(lsu)) had significant positive correlation with cannabinoids content(R>0.9).It was revealed that the related enzyme genes in cannabinoids biosynthesis might regulate the synthesis and accumulation of cannabinoids through coordinated expression.It alsodemonstrated that the back-end enzyme genes in MEP and GPP pathway played important roles in cannabinoids biosynthesis,while the three specific enzyme genes in C.sativa(OLS,PT and THCAS) played key roles in the biosynthesis and accumulation of cannabinoids in the glandular hairs of bract at initial-fruiting stage.These results would provide a basis for modifying cannabinoids content through genetic engineering in the future.
引文
Alexander S.P.,2016,Therapeutic potential of cannabis-related drugs,Prog.Neuropsychopharmacol Biol.Psychiatry,64:157-166
Cao S.X.,Zhang C.,Tang Y.F.,and Qi H.Y.,2014,Protein characteristic of the plant lipoxygenase and the function on fruit ripening and senescence and adversity stress,Zhiwu Shengli Xuebao(Plant Physiology Communications),50(8):1096-1108(曹嵩晓,张冲,汤雨凡,齐红岩,2014,植物脂氧合酶蛋白特性及其在果实成熟衰老和逆境胁迫中的作用,植物生理学报,50(8):1096-1108)
Chen X.,Guo M.B.,Guo H.Y.,Xu Y.P.,Zhang Q.Y.,Guo R.,and Yang M.,2016,Effects of main environment factors on THC accumulation in different development stages of Cannabis sativa,Xibu Linye Kexue(Journal of West China Forestry Science),45(3):44-50(陈璇,郭孟璧,郭鸿彦,许艳萍,张庆滢,郭蓉,杨明,2016,主要环境因子对大麻不同发育期四氢大麻酚积累的影响,西部林业科学,45(3):44-50)
Chen X.,Guo M.B.,Zhang Q.Y.,Xu Y.P.,Guo H.Y.,Yang M.,and Yang Q.H.,2013,Chemotype and genotype of cannabinoids in hemp landrace from southern Yunnan,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),33(9):1817-1822(陈璇,郭孟璧,张庆滢,许艳萍,郭鸿彦,杨明,杨清辉,2013,滇南农家大麻品种中大麻素化学型及基因型研究,西北植物学报,33(9):1817-1822)
Chen X.,Yang M.,and Guo H.Y.,2011,Research advances in cannabinoids of Cannabis sativa,Zhiwu Xuebao(Chinese Bulletin of Botany),46(2):197-205(陈璇,杨明,郭鸿彦,2011,大麻植物中大麻素成分研究进展,植物学报,46(2):197-205)
De Meijer E.P.,Bagatta M.,Carboni A.,Crucitti P.,Moliterni V.M.Ranalli P.,and Mandolino G.,2003,The inheritance of chemical phenotype in Cannabis sativa L.,Genetics,163:335-346
Flores-Sanchez I.J.,and Verpoorte R.,2008,PKS activities and biosynthesis of cannabinoids and flavonoids in Cannabis sativa L.plants,Plant Cell Physiol.,49(12):1767-1782
He S.L.,Zheng J.G.,Wang X.F.,Wang Y.H.,Xu M.,Li B.L.,and Lin M.,2002,Plant secondary metabolism:function,regulation and gene engineering,Yingyong Yu Huanjing Shengwu Xuebao(Chinese Journal of Application and Environmenta Biology),8(5):558-563(何水林,郑金贵,王晓峰,王燕华许明,李斌莲,林明,2002,植物次生代谢:功能,调控及其基因工程,应用与环境生物学报,8(5):558-563)
Liu H.,Zhang J.T.,Chen H.Y.,Yi Y.J.,Liu J.Y.,and Zhang H.X.2013,Fatty acid desaturation and plant responses to biotic and abiotic stresses,Dongbei Nongye Daxue Xuebao(Journal of Northeast Agricultural University),44(1):154-160(刘华,张建涛,陈海燕,衣艳君,刘家尧,张洪霞,2013,脂肪酸去饱和酶参与植物对胁迫的响应,东北农业大学学报44(1):154-160)
Livak K.J.,and Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative PCR and the2-△△CT method,Methods,25(4):402-408
Mahlberg P.G.,and Kim E.S.,2004,Accumulation of cannabinoids in glandular trichomes of Cannabis(Cannabaceae)Journal of Industrial Hemp,9(1):15-36
Rajan T.S.,Giacoppo S.,Iori R.,De Nicola G.R.,Grassi G.,Pollastro F.,Bramanti P.,and Mazzon E.,2016,Anti-inflammatory and antioxidant effects of a combination of cannabidiol and moringin in LPS-stimulated macrophages,Fitoterapia112:104-115
Sirikantaramas S.,Taura F.,Tanaka Y.,Ishikawa Y.,Morimoto S.,and Shoyama Y.,2005,Tetrahydrocannabinolic acid synthase,the enzyme controlling marijuana psychoactivity is secreted into the storage cavity of the glandular trichomes Plant Cell Physiol.,46(9):1578-1582
Su Y.,Li R.,and Chen J.M.,2015,Expression of gfp gene in transgenic gfp barley(Hordeum vulgare L.)by semi-quantitative RT-PCR analysis,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(9):1934-1938(苏琰,李融,陈建民,2015,转gfp基因大麦中gfp基因表达的半定量RT-PCR方法分析,基因组学与应用生物学,35(9):1934-1938)
Sun M.Y.,Hua W.,Liu J.,Huang S.M.,Wang X.F.,Liu G.H.,and Wang H.Z.,2012,Design of new genome-and gene-sourced primers and identification of QTL for seed oil content in a specially high-oil Brassica napus cultivar,PLo SOne,7(10):e47037
United Nations Office on Drugs and Crime,2009,Recommended methods for the identification and analysis of Cannabis and Cannabis products,United Nations Publication,New York,USA,pp.14
van Bakel H.,Stout J.M.,Cote A.G.,Tallon C.M.,Sharpe A.G.,Hughes T.R.,and Page J.E.,2011,The draft genome and transcriptome of Cannabis sativa,Genome Biology,12:102
Wang L.,Zhang Y.X.,Shi L.L.,and Liu Y.J.,2007,Application of functional genomic and metabolomic techniques to the studies on biosynthesis and regulation of plant secondary metabolites,Beijing Linye Daxue Xuebao(Journal of Beijing Forestry University),29(5):153-159(王莉,张艳霞,史玲玲,刘玉军,2007,功能基因组学和代谢组学技术在植物次生代谢物合成及调控研究中的应用,北京林业大学学报,29(5):153-159)
Xiang L.E.,Yan Z.H.,Wang G.J.,Liu W.H.,Tang K.X.,and Liao Z.H.,2012,Relative expression of genes involved in artemisinin biosynthesis and artemisinin accumulation in different tissues of Artemisia annua.,Zhongguo Zhongyao Zazhi(China Journal of Chinese Materia Medica),37(9):1169-1173(向礼恩,严铮辉,王贵君,刘万宏,唐克轩,廖志华,2012,青蒿素生物合成途径基因组织表达分析与青蒿素积累研究,中国中药杂志,37(9):1169-1173)
Xie X.X.,Wu W.D.,Lin B.Y.,Lin Z.P.,Gao S.,and Qian K.,2016,Genetic transformation of hydroperoxide lyase gene in lettuce,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),37(5):856-861(谢鑫鑫,吴卫东,林碧英,林忠平,高山,钱昆,2016,脂氢过氧化物裂解酶基因对生菜遗传转化的研究,热带作物学报,37(5):856-861)
Zhang S.T.,Chen H.L.,Cui H.,Yang H.J.,and Liu G.S.,2012,Multiple regulation mechanisms of MEP pathway in plant,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),32(7):1500-1504(张松涛,陈红丽,崔红,杨惠娟,刘国顺,2012,植物MEP途径的代谢调控机制,西北植物学报,32(7):1500-1504)
Cao S.X.,Zhang C.,Tang Y.F.,and Qi H.Y.,2014,Protein characteristic of the plant lipoxygenase and the function on fruit ripening and senescence and adversity stress,Zhiwu Shengli Xuebao(Plant Physiology Communications),50(8):1096-1108(曹嵩晓,张冲,汤雨凡,齐红岩,2014,植物脂氧合酶蛋白特性及其在果实成熟衰老和逆境胁迫中的作用,植物生理学报,50(8):1096-1108)
Chen X.,Guo M.B.,Guo H.Y.,Xu Y.P.,Zhang Q.Y.,Guo R.,and Yang M.,2016,Effects of main environment factors on THC accumulation in different development stages of Cannabis sativa,Xibu Linye Kexue(Journal of West China Forestry Science),45(3):44-50(陈璇,郭孟璧,郭鸿彦,许艳萍,张庆滢,郭蓉,杨明,2016,主要环境因子对大麻不同发育期四氢大麻酚积累的影响,西部林业科学,45(3):44-50)
Chen X.,Guo M.B.,Zhang Q.Y.,Xu Y.P.,Guo H.Y.,Yang M.,and Yang Q.H.,2013,Chemotype and genotype of cannabinoids in hemp landrace from southern Yunnan,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),33(9):1817-1822(陈璇,郭孟璧,张庆滢,许艳萍,郭鸿彦,杨明,杨清辉,2013,滇南农家大麻品种中大麻素化学型及基因型研究,西北植物学报,33(9):1817-1822)
Chen X.,Yang M.,and Guo H.Y.,2011,Research advances in cannabinoids of Cannabis sativa,Zhiwu Xuebao(Chinese Bulletin of Botany),46(2):197-205(陈璇,杨明,郭鸿彦,2011,大麻植物中大麻素成分研究进展,植物学报,46(2):197-205)
De Meijer E.P.,Bagatta M.,Carboni A.,Crucitti P.,Moliterni V.M.Ranalli P.,and Mandolino G.,2003,The inheritance of chemical phenotype in Cannabis sativa L.,Genetics,163:335-346
Flores-Sanchez I.J.,and Verpoorte R.,2008,PKS activities and biosynthesis of cannabinoids and flavonoids in Cannabis sativa L.plants,Plant Cell Physiol.,49(12):1767-1782
He S.L.,Zheng J.G.,Wang X.F.,Wang Y.H.,Xu M.,Li B.L.,and Lin M.,2002,Plant secondary metabolism:function,regulation and gene engineering,Yingyong Yu Huanjing Shengwu Xuebao(Chinese Journal of Application and Environmenta Biology),8(5):558-563(何水林,郑金贵,王晓峰,王燕华许明,李斌莲,林明,2002,植物次生代谢:功能,调控及其基因工程,应用与环境生物学报,8(5):558-563)
Liu H.,Zhang J.T.,Chen H.Y.,Yi Y.J.,Liu J.Y.,and Zhang H.X.2013,Fatty acid desaturation and plant responses to biotic and abiotic stresses,Dongbei Nongye Daxue Xuebao(Journal of Northeast Agricultural University),44(1):154-160(刘华,张建涛,陈海燕,衣艳君,刘家尧,张洪霞,2013,脂肪酸去饱和酶参与植物对胁迫的响应,东北农业大学学报44(1):154-160)
Livak K.J.,and Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative PCR and the2-△△CT method,Methods,25(4):402-408
Mahlberg P.G.,and Kim E.S.,2004,Accumulation of cannabinoids in glandular trichomes of Cannabis(Cannabaceae)Journal of Industrial Hemp,9(1):15-36
Rajan T.S.,Giacoppo S.,Iori R.,De Nicola G.R.,Grassi G.,Pollastro F.,Bramanti P.,and Mazzon E.,2016,Anti-inflammatory and antioxidant effects of a combination of cannabidiol and moringin in LPS-stimulated macrophages,Fitoterapia112:104-115
Sirikantaramas S.,Taura F.,Tanaka Y.,Ishikawa Y.,Morimoto S.,and Shoyama Y.,2005,Tetrahydrocannabinolic acid synthase,the enzyme controlling marijuana psychoactivity is secreted into the storage cavity of the glandular trichomes Plant Cell Physiol.,46(9):1578-1582
Su Y.,Li R.,and Chen J.M.,2015,Expression of gfp gene in transgenic gfp barley(Hordeum vulgare L.)by semi-quantitative RT-PCR analysis,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(9):1934-1938(苏琰,李融,陈建民,2015,转gfp基因大麦中gfp基因表达的半定量RT-PCR方法分析,基因组学与应用生物学,35(9):1934-1938)
Sun M.Y.,Hua W.,Liu J.,Huang S.M.,Wang X.F.,Liu G.H.,and Wang H.Z.,2012,Design of new genome-and gene-sourced primers and identification of QTL for seed oil content in a specially high-oil Brassica napus cultivar,PLo SOne,7(10):e47037
United Nations Office on Drugs and Crime,2009,Recommended methods for the identification and analysis of Cannabis and Cannabis products,United Nations Publication,New York,USA,pp.14
van Bakel H.,Stout J.M.,Cote A.G.,Tallon C.M.,Sharpe A.G.,Hughes T.R.,and Page J.E.,2011,The draft genome and transcriptome of Cannabis sativa,Genome Biology,12:102
Wang L.,Zhang Y.X.,Shi L.L.,and Liu Y.J.,2007,Application of functional genomic and metabolomic techniques to the studies on biosynthesis and regulation of plant secondary metabolites,Beijing Linye Daxue Xuebao(Journal of Beijing Forestry University),29(5):153-159(王莉,张艳霞,史玲玲,刘玉军,2007,功能基因组学和代谢组学技术在植物次生代谢物合成及调控研究中的应用,北京林业大学学报,29(5):153-159)
Xiang L.E.,Yan Z.H.,Wang G.J.,Liu W.H.,Tang K.X.,and Liao Z.H.,2012,Relative expression of genes involved in artemisinin biosynthesis and artemisinin accumulation in different tissues of Artemisia annua.,Zhongguo Zhongyao Zazhi(China Journal of Chinese Materia Medica),37(9):1169-1173(向礼恩,严铮辉,王贵君,刘万宏,唐克轩,廖志华,2012,青蒿素生物合成途径基因组织表达分析与青蒿素积累研究,中国中药杂志,37(9):1169-1173)
Xie X.X.,Wu W.D.,Lin B.Y.,Lin Z.P.,Gao S.,and Qian K.,2016,Genetic transformation of hydroperoxide lyase gene in lettuce,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),37(5):856-861(谢鑫鑫,吴卫东,林碧英,林忠平,高山,钱昆,2016,脂氢过氧化物裂解酶基因对生菜遗传转化的研究,热带作物学报,37(5):856-861)
Zhang S.T.,Chen H.L.,Cui H.,Yang H.J.,and Liu G.S.,2012,Multiple regulation mechanisms of MEP pathway in plant,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),32(7):1500-1504(张松涛,陈红丽,崔红,杨惠娟,刘国顺,2012,植物MEP途径的代谢调控机制,西北植物学报,32(7):1500-1504)