颠茄pmt、trI、h6h双基因策略对颠茄毛状根托品烷类生物碱含量的影响
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摘要
颠茄是我国药典规定托品烷类生物碱来源的唯一药用植物,也是莨菪碱、东莨菪碱最主要的商业药源(国家药典委员会2011版)。与莨菪碱相比,东莨菪碱副作用小、药理作用强,所以需求量十分巨大。野生颠茄中东莨菪含量非常低,而在商用栽培颠茄中,东莨菪碱含量也仅为干重的0.01-0.08%。近年来,随着托品烷类生物碱生物合成途径分子生物学研究的深入,使得在分子水平实现包括莨菪碱和东莨菪碱在内的TAs生物合成的代谢调控取得了重要进展。特别是目前东莨菪碱代谢途径上的一些功能基因的确定,为利用托品烷类代谢工程技术提高颠茄中东莨菪碱含量打下了基础。
在托品烷类生物碱代谢途径中,pmt、trI和h6h是重要的基因。其中,pmt为上游重要基因,trI为中间分支点基因,h6h为下游重要基因。研究表明,在颠茄毛状根和颠茄植株中,外源pmt、trI、h6h的高度表达对莨菪碱和东莨菪碱含量的变化都是有所相关的：将烟草pmt基因转入白曼陀罗,莨菪碱的含量提高2倍多,东莨菪碱的含量提高3倍多；在颠茄中高度表达曼陀罗trI,莨菪碱的含量提高了3倍多,东莨菪碱的含量提高了5倍多；将莨菪的h6h导入到埃及莨菪中,最好的克隆产生了17mg/L的东莨菪碱,是对照克隆的100倍以上。而内源基因在其中的作用尚不清楚。针对这点,在之前研究基础上,本文通过内源pmt、trI、h6h的双基因策略研究对颠茄毛状根莨菪碱和东莨菪碱含量的影响。
分别构建携带目的基因的植物高效双元表达载体p1304+-pmt-trI、p1304+-pmt-h6h和p1304+-trI-h6h,转化“卸甲”的根癌农杆菌C58C1获得工程菌C58C1-p1304+-pmt-trI、C58C1-p1304+-pmt-h6h和C58C1-p1304+-trI-h6h,遗传转化颠茄叶片。在C58C1-p1304+-pmt-trI组合中,经检测rolC、hygr和pmt、trI基因后成功筛选出6个转基因发根系,分别进行液体培养基扩大培养然后检测托品烷类生物碱(TAs)含量和基因表达量。其中有4个发根系的总TAs含量得到了提高,依次为PT11>PT17>PT13>T3,PT11的总TAs含量约为空菌发根对照(NC)的2.5倍；有2个发根系的含量维持不变或明显降低,但东莨菪碱的含量有所提高。莨菪碱和东莨菪碱含量提高最多的PT11相比空白对照分别提高了2倍和5.7倍。基因表达量上,除了PT2的pmt基因表达量较空白发根低,其他转基因发根系的pmt和h6h基因的表达量都比空白对照高。在C58C1-p1304+-pmt-h6h组合中,经检测rolC、hygr和pmt、h6h基因后成功筛选出5个转基因发根系,分别进行液体培养基扩大培养然后检测TAs含量和基因表达量。其中有2个发根系的总TAs含量得到了提高,为PH2>PH14,PH2的总TAs含量约为空菌发根对照(NC)的2.7倍；有3个发根系的含量维持不变或明显降低,但东莨菪碱的含量有所提高。在这5个发根系中,莨菪碱和东莨菪碱含量提高最多的PH2比空白对照分别提高了1.8倍和8.2倍。除了PH32的pmt基因表达量低于空白对照,其余转基因发根系的pmt和h6h基因表达量都有所提高或持平。在C58C1-p1304+-trI-h6h组合中,经检测rolC、hygr和trI、h6h基因后成功筛选出4个转基因发根系,分别进行液体培养基扩大培养然后检测TAs含量和基因表达量。其中有3个发根系的总TAs含量得到了提高,为HT2>HTA1>HTA3>HT16,HT2的总TAs含量约为空菌发根的2.6倍；有1个发根系的含量降低,但东莨菪碱的含量有所提高。莨菪碱和东莨菪碱含量提高最多的HT2比NC分别提高了2.1倍和5.6倍。HT2的h6h表达量为最高,其东莨菪碱含量也为最高,HTA1、HTA3、HT16的h6h基因表达量与NC接近,但trI基因的表达量有很大的提高。上述基因的具体表达量与含量水平之间并不完全一致,这可能是由于双转的基因在表达上产生了共抑制,使转基因发根在基因的高表达下反而含量保持不变或有所降低。但总体上,双基因策略在托品烷类生物碱合成途径中可能存在协同作用。转基因发根个体间托品烷类生物碱合成能力有个体差异,但是并没有改变两个基因过量表达能够提高东莨菪碱含量的趋势,说明双基因的遗传转化能够打破颠茄托品烷类生物碱合成途径中的限速反应步骤,从而推动代谢流更多的向托品烷类生物碱合成方向流动,提高了莨菪碱和东莨菪碱合成能力,最终表现为两者含量的提高。
pmt是东莨菪碱生物合成途径中的第一个重要酶基因,其过量表达能够为下游的东莨菪碱合成提供更为充足的前体物质；trI是东莨菪碱生物合成途径中的一个分支点,使代谢流更多的流向东莨菪碱；h6h是该途径中的最后一个重要酶基因,其超量表达能够高效转化来至于途径上游提供的代谢流,合成东莨菪碱。本研究的实验结果证明了颠茄内源pmt、trI和h6h在颠茄托品烷类生物碱合成途径中具有提高总TAs作用,为进一步开展颠茄的TAs代谢工程提供了理论支持
Atorpa belladonna is the only medicinal plant species and the main commercial source of hyoscyamine and scopolamine in China(authorized by pharmacopoeia of The People's Republic of China2011). Compared to hyoscyamine, scopolamine is the most potent reagent with relatively low side effects, so its demand is large. The content of scopolamine is very low in wild A. belladonna, and about the commercial cultivation of A. belladonna, content of scopolamine is only0.01-0.08%of dry weight. In recent years, with the development of molecular biology research of tropine alkaloids (TAs) biosynthetic pathway, great progress of metabolic regulation of TAs biosynthesis including hyoscyamin and scopolamine, has been made at molecular level. Several important functional genes involved in the biosynthetic pathway of scopolamine have been identified that can be used to genetically modify the TAs pathway.
Among the TAs biosynthetic genes, pmt、trI and h6h are important ones in TAs metabolic pathways, separately encoding putrescine N-methyltransferase (PMT; EC2.1.1.53), tropinone reductase-I (TRI; EC1.1.1.206) and hyoscyamine6β-hyroxylase (H6H; EC1.14.11.11), and located in the upstream, middle branch point and downstream of the pathway respectively. Many researches have shown that in A. belladona hair root and plant, the high expression levels of pmt, trI and h6h are related to changes of hyoscyamine and scopolamine contents. Overexpression of tobacoo pmt in Datura metel increased hyoscyamine content2times more than control, and3times for scopolamine content. Strong expression of D.stramonium trI in A.belladona is accompanied with3-fold more hyoscyamine and5-fold more scopolamine compared with control roots. Introduce h6h of Hyoscyamus niger into Hyoscyamus muticus hair root. The best clone produces17mg/L DW of scopolamine, which is over100times more than the control clones. But the effects of endogenous genes are not clear. Based on research before, this paper study the influence of two-gene strategy of A. belladonna (pmt、trl、h6h)for the tropine alkaloids content of A. belladonna hairy root. Constructing two-gene efficient expression vectors of p1304+-pmt-trl, p1304+-pmt-h6h and p]304+-trI-h6h, respectively. Transform the constructed vectors into "disarmed" C58C1to obtain C58C1-p1304+-pmt-trI, C58C1-p1304+-pmt-h6h and C58C1-pl304+-trI-fh6h, for genetic transformation of A. belladonna.
In transgenic C58C1-pl304+-pmt-trI hair root, PCR studies confirmed their presence in6hairy root clones. Continue training in liquid medium, then detect content of TAs and genes expression level. TAs contents in4hair root clones have been improved, PT11> PT17>PT13>PT3in turn. Total TAs content in PT11is2.5times than control hair root. There are2hair root transgenic lines contents remain unchanged or reduce significantly, but scopolamine content has been improved. The best line(PT11) increases2times in hyoscyamine content and5.7times in scopolamine content. Except PT2pmt gene, the pmt and trI genes expression levels of the other hair root are higher than NC. In transgenic C58C1-pl304+-pmt-h6h hair root, obtain5transgenic hair root clones. Total TAs content in2hair root clones enhanced, PH2>PH14. PH2TAs content increased2.7times than NC. There are3hair root transgenic clones hyoscyamine contents remain unchanged or reduce significantly, but scopolamine content have been improved. The best line(PH2) increases1.8times of hyoscyamine content and8.2times of scopolamine content. Except PH32pmt gene, the pmt and h6h genes expression levels of the other hair root are higher than NC. In transgenic C58Cl-p1304+-trI-h6h hair root, In transgenic C58C1-p1304+trI-h6h hair root, it has succeed to get4hairy root clones. TAs content in3hair root clones have been improved, HT2>HTA1>HTA3in turn. Total TAs content in HT2is2.6times than NC. TAs contents of One transgenic line reduced, but scopolamine content enhanced. The best line(HT2) increases2.1times of hyoscyamine content and5.6times of scopolamine content. Expression of HT2h6h genes are far higher than NC. The expression of HTA1、HTA3and HT16h6h gene are closed to NC, but trI enhanced significantly. The genes expression is not completely consistent with the level of TAs content. This may be an expression suppression due to the two transgenic genes. Under high expression of genes, the transgenic hair root content remain the same or reduced. Above all, two genes in the tropine alkaloids biosynthetic pathway may exist synergies. Despite of existing difference among the individual in transgenic hair root, the trend of improving scopolamine content doesn't change. It proves that two-gene strategy can break the rate-limited step in A.belladonna TAs biosynthetic pathway, and produce more TAs content including hyoscyamine and scopolamine.
pmt is the first important enzyme gene in TAs biosynthetic pathway. Its overexpression can provide more precursor. trI gene is a branch point of TAs biosynthetic pathway. It makes more flux to scopolamine. The last important enzyme gene is h6h. The overexpression of h6h can deliver more scopolamine. This study proves that A. belladonna endogenous genes Abpmt, Abtrl and Abh6h, can enhance TAs content.
在托品烷类生物碱代谢途径中,pmt、trI和h6h是重要的基因。其中,pmt为上游重要基因,trI为中间分支点基因,h6h为下游重要基因。研究表明,在颠茄毛状根和颠茄植株中,外源pmt、trI、h6h的高度表达对莨菪碱和东莨菪碱含量的变化都是有所相关的：将烟草pmt基因转入白曼陀罗,莨菪碱的含量提高2倍多,东莨菪碱的含量提高3倍多；在颠茄中高度表达曼陀罗trI,莨菪碱的含量提高了3倍多,东莨菪碱的含量提高了5倍多；将莨菪的h6h导入到埃及莨菪中,最好的克隆产生了17mg/L的东莨菪碱,是对照克隆的100倍以上。而内源基因在其中的作用尚不清楚。针对这点,在之前研究基础上,本文通过内源pmt、trI、h6h的双基因策略研究对颠茄毛状根莨菪碱和东莨菪碱含量的影响。
分别构建携带目的基因的植物高效双元表达载体p1304+-pmt-trI、p1304+-pmt-h6h和p1304+-trI-h6h,转化“卸甲”的根癌农杆菌C58C1获得工程菌C58C1-p1304+-pmt-trI、C58C1-p1304+-pmt-h6h和C58C1-p1304+-trI-h6h,遗传转化颠茄叶片。在C58C1-p1304+-pmt-trI组合中,经检测rolC、hygr和pmt、trI基因后成功筛选出6个转基因发根系,分别进行液体培养基扩大培养然后检测托品烷类生物碱(TAs)含量和基因表达量。其中有4个发根系的总TAs含量得到了提高,依次为PT11>PT17>PT13>T3,PT11的总TAs含量约为空菌发根对照(NC)的2.5倍；有2个发根系的含量维持不变或明显降低,但东莨菪碱的含量有所提高。莨菪碱和东莨菪碱含量提高最多的PT11相比空白对照分别提高了2倍和5.7倍。基因表达量上,除了PT2的pmt基因表达量较空白发根低,其他转基因发根系的pmt和h6h基因的表达量都比空白对照高。在C58C1-p1304+-pmt-h6h组合中,经检测rolC、hygr和pmt、h6h基因后成功筛选出5个转基因发根系,分别进行液体培养基扩大培养然后检测TAs含量和基因表达量。其中有2个发根系的总TAs含量得到了提高,为PH2>PH14,PH2的总TAs含量约为空菌发根对照(NC)的2.7倍；有3个发根系的含量维持不变或明显降低,但东莨菪碱的含量有所提高。在这5个发根系中,莨菪碱和东莨菪碱含量提高最多的PH2比空白对照分别提高了1.8倍和8.2倍。除了PH32的pmt基因表达量低于空白对照,其余转基因发根系的pmt和h6h基因表达量都有所提高或持平。在C58C1-p1304+-trI-h6h组合中,经检测rolC、hygr和trI、h6h基因后成功筛选出4个转基因发根系,分别进行液体培养基扩大培养然后检测TAs含量和基因表达量。其中有3个发根系的总TAs含量得到了提高,为HT2>HTA1>HTA3>HT16,HT2的总TAs含量约为空菌发根的2.6倍；有1个发根系的含量降低,但东莨菪碱的含量有所提高。莨菪碱和东莨菪碱含量提高最多的HT2比NC分别提高了2.1倍和5.6倍。HT2的h6h表达量为最高,其东莨菪碱含量也为最高,HTA1、HTA3、HT16的h6h基因表达量与NC接近,但trI基因的表达量有很大的提高。上述基因的具体表达量与含量水平之间并不完全一致,这可能是由于双转的基因在表达上产生了共抑制,使转基因发根在基因的高表达下反而含量保持不变或有所降低。但总体上,双基因策略在托品烷类生物碱合成途径中可能存在协同作用。转基因发根个体间托品烷类生物碱合成能力有个体差异,但是并没有改变两个基因过量表达能够提高东莨菪碱含量的趋势,说明双基因的遗传转化能够打破颠茄托品烷类生物碱合成途径中的限速反应步骤,从而推动代谢流更多的向托品烷类生物碱合成方向流动,提高了莨菪碱和东莨菪碱合成能力,最终表现为两者含量的提高。
pmt是东莨菪碱生物合成途径中的第一个重要酶基因,其过量表达能够为下游的东莨菪碱合成提供更为充足的前体物质；trI是东莨菪碱生物合成途径中的一个分支点,使代谢流更多的流向东莨菪碱；h6h是该途径中的最后一个重要酶基因,其超量表达能够高效转化来至于途径上游提供的代谢流,合成东莨菪碱。本研究的实验结果证明了颠茄内源pmt、trI和h6h在颠茄托品烷类生物碱合成途径中具有提高总TAs作用,为进一步开展颠茄的TAs代谢工程提供了理论支持
Atorpa belladonna is the only medicinal plant species and the main commercial source of hyoscyamine and scopolamine in China(authorized by pharmacopoeia of The People's Republic of China2011). Compared to hyoscyamine, scopolamine is the most potent reagent with relatively low side effects, so its demand is large. The content of scopolamine is very low in wild A. belladonna, and about the commercial cultivation of A. belladonna, content of scopolamine is only0.01-0.08%of dry weight. In recent years, with the development of molecular biology research of tropine alkaloids (TAs) biosynthetic pathway, great progress of metabolic regulation of TAs biosynthesis including hyoscyamin and scopolamine, has been made at molecular level. Several important functional genes involved in the biosynthetic pathway of scopolamine have been identified that can be used to genetically modify the TAs pathway.
Among the TAs biosynthetic genes, pmt、trI and h6h are important ones in TAs metabolic pathways, separately encoding putrescine N-methyltransferase (PMT; EC2.1.1.53), tropinone reductase-I (TRI; EC1.1.1.206) and hyoscyamine6β-hyroxylase (H6H; EC1.14.11.11), and located in the upstream, middle branch point and downstream of the pathway respectively. Many researches have shown that in A. belladona hair root and plant, the high expression levels of pmt, trI and h6h are related to changes of hyoscyamine and scopolamine contents. Overexpression of tobacoo pmt in Datura metel increased hyoscyamine content2times more than control, and3times for scopolamine content. Strong expression of D.stramonium trI in A.belladona is accompanied with3-fold more hyoscyamine and5-fold more scopolamine compared with control roots. Introduce h6h of Hyoscyamus niger into Hyoscyamus muticus hair root. The best clone produces17mg/L DW of scopolamine, which is over100times more than the control clones. But the effects of endogenous genes are not clear. Based on research before, this paper study the influence of two-gene strategy of A. belladonna (pmt、trl、h6h)for the tropine alkaloids content of A. belladonna hairy root. Constructing two-gene efficient expression vectors of p1304+-pmt-trl, p1304+-pmt-h6h and p]304+-trI-h6h, respectively. Transform the constructed vectors into "disarmed" C58C1to obtain C58C1-p1304+-pmt-trI, C58C1-p1304+-pmt-h6h and C58C1-pl304+-trI-fh6h, for genetic transformation of A. belladonna.
In transgenic C58C1-pl304+-pmt-trI hair root, PCR studies confirmed their presence in6hairy root clones. Continue training in liquid medium, then detect content of TAs and genes expression level. TAs contents in4hair root clones have been improved, PT11> PT17>PT13>PT3in turn. Total TAs content in PT11is2.5times than control hair root. There are2hair root transgenic lines contents remain unchanged or reduce significantly, but scopolamine content has been improved. The best line(PT11) increases2times in hyoscyamine content and5.7times in scopolamine content. Except PT2pmt gene, the pmt and trI genes expression levels of the other hair root are higher than NC. In transgenic C58C1-pl304+-pmt-h6h hair root, obtain5transgenic hair root clones. Total TAs content in2hair root clones enhanced, PH2>PH14. PH2TAs content increased2.7times than NC. There are3hair root transgenic clones hyoscyamine contents remain unchanged or reduce significantly, but scopolamine content have been improved. The best line(PH2) increases1.8times of hyoscyamine content and8.2times of scopolamine content. Except PH32pmt gene, the pmt and h6h genes expression levels of the other hair root are higher than NC. In transgenic C58Cl-p1304+-trI-h6h hair root, In transgenic C58C1-p1304+trI-h6h hair root, it has succeed to get4hairy root clones. TAs content in3hair root clones have been improved, HT2>HTA1>HTA3in turn. Total TAs content in HT2is2.6times than NC. TAs contents of One transgenic line reduced, but scopolamine content enhanced. The best line(HT2) increases2.1times of hyoscyamine content and5.6times of scopolamine content. Expression of HT2h6h genes are far higher than NC. The expression of HTA1、HTA3and HT16h6h gene are closed to NC, but trI enhanced significantly. The genes expression is not completely consistent with the level of TAs content. This may be an expression suppression due to the two transgenic genes. Under high expression of genes, the transgenic hair root content remain the same or reduced. Above all, two genes in the tropine alkaloids biosynthetic pathway may exist synergies. Despite of existing difference among the individual in transgenic hair root, the trend of improving scopolamine content doesn't change. It proves that two-gene strategy can break the rate-limited step in A.belladonna TAs biosynthetic pathway, and produce more TAs content including hyoscyamine and scopolamine.
pmt is the first important enzyme gene in TAs biosynthetic pathway. Its overexpression can provide more precursor. trI gene is a branch point of TAs biosynthetic pathway. It makes more flux to scopolamine. The last important enzyme gene is h6h. The overexpression of h6h can deliver more scopolamine. This study proves that A. belladonna endogenous genes Abpmt, Abtrl and Abh6h, can enhance TAs content.
引文
[1]中国科学院中国植物志编辑委员会.中国植物志[M].北京：科学出版社出版,1978：18-19.
[2]肖培根.新编中药志[M].北京：化学工业出版社,2002.
[3] Chi J. Motion sickness patch making a comeback [J]. Drug Topics,2004,161:21.
[4]唐克轩.中草药生物技术[M].复旦大学出版社,2005.
[5] Patterson S, O'Hagan D. Biosynthetic studies on the tropane alkaloid hyoscyamine in Datura stramonium; hyoscyamine is stable to in vivo oxidation and is not derived from littorine via a vicinal interchange process [J]. Phytochemistry,2002, 61 (3):323-329.
[6] Matsuda J, Okabe S, Hashimoto T, et al. Molecular cloning of hyoscyamine 6β-hydroxylase, a 2-oxoglutaratedependent dioxygenase, from cultured roots of Hyoscyamus niger [J]. J Biol Chem,1991 (266):9460-9464.
[7] Nasomjai P, Reed D W, Tozer D J, et al. Mechanistic Insights into the Cytochrome P450-Mediated Oxidation and Rearrangement of Littorine in Tropane Alkaloid Biosynthesis [J]. ChemBioChem,2009,10 (14):2382-2393.
[8] Yun D Y, Hashimoto T, Yamada Y. Metabolic engineering of medicinal plants: Transgenic Atropa belladonna with an improved alkaloid composition [J]. PNAS, 1992(89):11799-11803.
[9] Suzuki K, Yamada Y, Hashimoto T, Expression of Atropa belladonna Putrescine N-Methyltransferase Gene in Root Pericycle [J]. Plant Cell Physiol,1999,40: 289-297.
[10] Teuber M, Azemi M, Namjoyan F, et al. Putrescine N-methyltransferases-a structure-function analysis [J]. Plant Mol Biol,2007,63 (6):787-801.
[11] Liu T, Zhu P, Cheng K, et al. Molecular cloning and expression of putrescine N-methyltransferase from the hairy roots of Anisodus tanguticus [J]. Planta Med, 2005,71 (10):987-989.
[12]Kai G, Zhang Y, Chen J, et al. Molecular characterization and expression analysis of two distinct putrescine N-methyltransferases from roots of Anisodus acutangulus [J]. Physiol Plant,2009,135 (2):121-129.
[13]Nakajima K, Hashimoto T, Yamada Y. Two tropinone reductases with different stereospecificities are short-chain dehydrogenases evolved from a common ancestor [J]. PNAS,1993,90 (20):9591-9595.
[14]Nakajima K, Hashimoto T, Yamada Y. cDNA encoding tropinone reductase-II from Hyoscyamus niger [J]. Plant Physiol,1993,103 (4):1465-1466.
[15] Kaiser H, Richter U, keiner R, et al. Immunolocalisation of two tropinone reductases in potato(Solanum tuberosum L.) root, stolon, and tuber sprouts [J]. Planta,2006,225:127-137.
[16]Kai G, Li L, Jiang Y, et al. Molecular cloning and characterization of two tropinone reductases in Anisodus acutangulus and enhancement of tropane alkaloid production in AaTRI-transformed hairy roots [J]. Biotechnol Appl Biochem,2009, 54 (3):177-186.
[17] Li R, Reed D W, Liu E, et al, Functional Genomic Analysis of Alkaloid Biosynthesis in Hyoscyamus niger Reveals a Cytochrome P450 Involved in Littorine Rearrangement [J]. Chem & Biol,2006,13:513-520.
[18] Suzuki K, Yun D Y, Chen X Y, et al, An Atropa belladonna hyoscyamine 6(3-hydroxylase gene is differentially expressed in the root pericycle and anthers [J]. Plant Mol Biol,1999,40(1):141-152.
[19] Kai G, Chen J F, Li L, et al. Molecular Cloning and Characterization of a New cDNA Encoding Hyoscyamine 6(3-hydroxylase from Roots of Anisodus acutangulus [J]. J BioChem Mol Biol,2007 (40):715-722.
[20] Hashimoto T, Nakajima K, Ongena G, et al. Two tropinone reductases with distinct stereo specifities from cultured roots of Hyoscyamus niger [J]. Plant Physiol,1992, 100:836-845.
[21] Portsteffen A, Drager B, Nahrsted A. Two tropinone reducing enzymes from Datura stramonium transformed root cultrues. Phytochemistry.1992,31:1135-1138.
[22] Drager B. Tropinone reductases, enzymes at the branch point of tropane alkaloid metabolism. Phytochemistry.2006,67:327-337.
[23] Nakajima K, Oshita Y, Kaya M, et al. Structures and expression patterns of two tropinone reductase genes from Hyoscyamus niger [J]. Biosci Biotech Bioch.1999, 63 (10):1756-1764.
[24] Evans W C, Ghani A, Woolley V A. Distribution of littorine and other alkaloids in the roots of Datura species [J]. Phytochemistry,1972,11 (8):2527-2529.
[25] Robins R J, Bachmann P, Woolley J G. Biosynthesis of hyoscyamine involves an intramolecular rearrangement of littorine [J]. J Chem Soc, Perkin Trans 1,1994,1: 615-619.
[26] Bouwmeester H J, Matusova R, Zhongkui S, et al. Secondary metabolite signalling in host-parasitic plant interactions [J]. Current Opinion in Plant Biology,2003.6(4): 358-364.
[27] Harbome J B. Role of secondary metabolites in chemical defence mechanisms in plants[J]. Ciba Found Symp,1990,154:126-134.
[28] Yazaki K, Natural Products and Metabolites. Handbook of Plant Biotechnology, 2004.
[29] Rocha L G, Almeida J R G S, Macedo R O, et al. A review of natural products with antileishmanial activity [J]. Phytomedicine,2005,12(6-7):514-535.
[30] Flores H E, Vivanco J M, and Loyola-Vargas V M,'Radicle'biochemistry:the biology of root-specific metabolism[J]. Trends in Plant Science,1999,4:220-226.
[31] Wink M. (2010) Introduction, in Annual Plant Reviews Volume 39:Functions and Biotechnology of Plant Secondary Metabolites, Second edition (ed M. Wink), Wiley-Blackwell, Oxford, UK.
[32] Yazaki K, Transporters of secondary metabolites. Current Opinion in Plant Biology, 2005,8(3):301-307.
[33] Morita M, et al, Vacuolar transport of nicotine is mediated by a multidrug and toxic compound extrusion (MATE) transporter in Nicotiana tabacum[J]. PNAS,2009, 106(7):2447-2452.
[34] De Luca V and St Pierre B, The cell and developmental biology of alkaloid
biosynthesis[J]. Trends in Plant Science,2000,5:1360-1385.
[35] Moyano E, Jouhikainen K, and Tammela P. Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus [J]. J Exp Bot,2003,54 (381):203-211.
[36] Sato F, Hashimoto T, Hachiya A, et al. Metabolic engineering of plant alkaloid biosynthesis [J]. PNAS,2001,98 (1):367-372.
[37] Richter U, Rothe G, Fabian A K, et al. Overexpression of tropinone reductases alters alkaloid composition in Atropa belladonna root cultures [J]. J Exp Bot,2005,412: 645-652.
[38] Hashimoto T, Yun D, Yamada Y. Production of tropane alkaloids in genetically engineered root cultures [J]. Phytochemistry,1993,32 (3):713-718.
[39] Jouhikainen K, Lindgren L, Jokelainen T, et al. Enhancement of scopolamine production in Hyoscyamus muticus L. hairy root cultures by genetic engineering [J]. Planta,1999 (208):545-551.
[40] Zhang L, Ding R, Chai Y, et al. Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures [J]. PNAS,2004,101 (17):6786-6791.
[41] Liu X Q, Yang C X, Chen M, et al. Promoting scopolamine accumulation in transgenic plants of Atropa belladonna generated from hairy roots with over expression of pmt and h6h gene [J]. J Med Plants Res,2010,4 (17):1708-1713.
[42] Yang C X, Chen M, Zeng L J, et al. Improvement of tropane alkaloids production in hairy root cultures of Atropa belladonna by overexpressing pmt and h6h genes [J]. Plant Omics Journal,2011,4 (1):29-33.
[43] Kang S M. Effects of methyl jasmonate and salicylic acid on the production of ropane alkaloids and the expression of PMT and H6H inadventitious root cultures of Scopolia parviflora[J]. Plant Science.2004(166):745-751.
[44] Jaber-Vazdekis N, GonzaLez C, Ravelo A G, et al. Cloning, characterization and analysis of expression profiles of a cDNA encoding a hyoscyamine 6β-hydroxylase (H6H) from Atropa baetica Willk[J]. Plant physiol. Biochem.2009(47):20-25.
[45] Edwards K, Johmstone C, and Thompson C. A simple and rapid method for the preparation of plant genomic DNA for PCR analyses[J]. Nucleic Acids Research. 1991,6:1349.
[46] Mozo T, Hooykaas P J J. Electroporation of megaplasmids into Agrobacterium [J]. Plant molecular biology.1991,16:917-918.
[2]肖培根.新编中药志[M].北京：化学工业出版社,2002.
[3] Chi J. Motion sickness patch making a comeback [J]. Drug Topics,2004,161:21.
[4]唐克轩.中草药生物技术[M].复旦大学出版社,2005.
[5] Patterson S, O'Hagan D. Biosynthetic studies on the tropane alkaloid hyoscyamine in Datura stramonium; hyoscyamine is stable to in vivo oxidation and is not derived from littorine via a vicinal interchange process [J]. Phytochemistry,2002, 61 (3):323-329.
[6] Matsuda J, Okabe S, Hashimoto T, et al. Molecular cloning of hyoscyamine 6β-hydroxylase, a 2-oxoglutaratedependent dioxygenase, from cultured roots of Hyoscyamus niger [J]. J Biol Chem,1991 (266):9460-9464.
[7] Nasomjai P, Reed D W, Tozer D J, et al. Mechanistic Insights into the Cytochrome P450-Mediated Oxidation and Rearrangement of Littorine in Tropane Alkaloid Biosynthesis [J]. ChemBioChem,2009,10 (14):2382-2393.
[8] Yun D Y, Hashimoto T, Yamada Y. Metabolic engineering of medicinal plants: Transgenic Atropa belladonna with an improved alkaloid composition [J]. PNAS, 1992(89):11799-11803.
[9] Suzuki K, Yamada Y, Hashimoto T, Expression of Atropa belladonna Putrescine N-Methyltransferase Gene in Root Pericycle [J]. Plant Cell Physiol,1999,40: 289-297.
[10] Teuber M, Azemi M, Namjoyan F, et al. Putrescine N-methyltransferases-a structure-function analysis [J]. Plant Mol Biol,2007,63 (6):787-801.
[11] Liu T, Zhu P, Cheng K, et al. Molecular cloning and expression of putrescine N-methyltransferase from the hairy roots of Anisodus tanguticus [J]. Planta Med, 2005,71 (10):987-989.
[12]Kai G, Zhang Y, Chen J, et al. Molecular characterization and expression analysis of two distinct putrescine N-methyltransferases from roots of Anisodus acutangulus [J]. Physiol Plant,2009,135 (2):121-129.
[13]Nakajima K, Hashimoto T, Yamada Y. Two tropinone reductases with different stereospecificities are short-chain dehydrogenases evolved from a common ancestor [J]. PNAS,1993,90 (20):9591-9595.
[14]Nakajima K, Hashimoto T, Yamada Y. cDNA encoding tropinone reductase-II from Hyoscyamus niger [J]. Plant Physiol,1993,103 (4):1465-1466.
[15] Kaiser H, Richter U, keiner R, et al. Immunolocalisation of two tropinone reductases in potato(Solanum tuberosum L.) root, stolon, and tuber sprouts [J]. Planta,2006,225:127-137.
[16]Kai G, Li L, Jiang Y, et al. Molecular cloning and characterization of two tropinone reductases in Anisodus acutangulus and enhancement of tropane alkaloid production in AaTRI-transformed hairy roots [J]. Biotechnol Appl Biochem,2009, 54 (3):177-186.
[17] Li R, Reed D W, Liu E, et al, Functional Genomic Analysis of Alkaloid Biosynthesis in Hyoscyamus niger Reveals a Cytochrome P450 Involved in Littorine Rearrangement [J]. Chem & Biol,2006,13:513-520.
[18] Suzuki K, Yun D Y, Chen X Y, et al, An Atropa belladonna hyoscyamine 6(3-hydroxylase gene is differentially expressed in the root pericycle and anthers [J]. Plant Mol Biol,1999,40(1):141-152.
[19] Kai G, Chen J F, Li L, et al. Molecular Cloning and Characterization of a New cDNA Encoding Hyoscyamine 6(3-hydroxylase from Roots of Anisodus acutangulus [J]. J BioChem Mol Biol,2007 (40):715-722.
[20] Hashimoto T, Nakajima K, Ongena G, et al. Two tropinone reductases with distinct stereo specifities from cultured roots of Hyoscyamus niger [J]. Plant Physiol,1992, 100:836-845.
[21] Portsteffen A, Drager B, Nahrsted A. Two tropinone reducing enzymes from Datura stramonium transformed root cultrues. Phytochemistry.1992,31:1135-1138.
[22] Drager B. Tropinone reductases, enzymes at the branch point of tropane alkaloid metabolism. Phytochemistry.2006,67:327-337.
[23] Nakajima K, Oshita Y, Kaya M, et al. Structures and expression patterns of two tropinone reductase genes from Hyoscyamus niger [J]. Biosci Biotech Bioch.1999, 63 (10):1756-1764.
[24] Evans W C, Ghani A, Woolley V A. Distribution of littorine and other alkaloids in the roots of Datura species [J]. Phytochemistry,1972,11 (8):2527-2529.
[25] Robins R J, Bachmann P, Woolley J G. Biosynthesis of hyoscyamine involves an intramolecular rearrangement of littorine [J]. J Chem Soc, Perkin Trans 1,1994,1: 615-619.
[26] Bouwmeester H J, Matusova R, Zhongkui S, et al. Secondary metabolite signalling in host-parasitic plant interactions [J]. Current Opinion in Plant Biology,2003.6(4): 358-364.
[27] Harbome J B. Role of secondary metabolites in chemical defence mechanisms in plants[J]. Ciba Found Symp,1990,154:126-134.
[28] Yazaki K, Natural Products and Metabolites. Handbook of Plant Biotechnology, 2004.
[29] Rocha L G, Almeida J R G S, Macedo R O, et al. A review of natural products with antileishmanial activity [J]. Phytomedicine,2005,12(6-7):514-535.
[30] Flores H E, Vivanco J M, and Loyola-Vargas V M,'Radicle'biochemistry:the biology of root-specific metabolism[J]. Trends in Plant Science,1999,4:220-226.
[31] Wink M. (2010) Introduction, in Annual Plant Reviews Volume 39:Functions and Biotechnology of Plant Secondary Metabolites, Second edition (ed M. Wink), Wiley-Blackwell, Oxford, UK.
[32] Yazaki K, Transporters of secondary metabolites. Current Opinion in Plant Biology, 2005,8(3):301-307.
[33] Morita M, et al, Vacuolar transport of nicotine is mediated by a multidrug and toxic compound extrusion (MATE) transporter in Nicotiana tabacum[J]. PNAS,2009, 106(7):2447-2452.
[34] De Luca V and St Pierre B, The cell and developmental biology of alkaloid
biosynthesis[J]. Trends in Plant Science,2000,5:1360-1385.
[35] Moyano E, Jouhikainen K, and Tammela P. Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus [J]. J Exp Bot,2003,54 (381):203-211.
[36] Sato F, Hashimoto T, Hachiya A, et al. Metabolic engineering of plant alkaloid biosynthesis [J]. PNAS,2001,98 (1):367-372.
[37] Richter U, Rothe G, Fabian A K, et al. Overexpression of tropinone reductases alters alkaloid composition in Atropa belladonna root cultures [J]. J Exp Bot,2005,412: 645-652.
[38] Hashimoto T, Yun D, Yamada Y. Production of tropane alkaloids in genetically engineered root cultures [J]. Phytochemistry,1993,32 (3):713-718.
[39] Jouhikainen K, Lindgren L, Jokelainen T, et al. Enhancement of scopolamine production in Hyoscyamus muticus L. hairy root cultures by genetic engineering [J]. Planta,1999 (208):545-551.
[40] Zhang L, Ding R, Chai Y, et al. Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures [J]. PNAS,2004,101 (17):6786-6791.
[41] Liu X Q, Yang C X, Chen M, et al. Promoting scopolamine accumulation in transgenic plants of Atropa belladonna generated from hairy roots with over expression of pmt and h6h gene [J]. J Med Plants Res,2010,4 (17):1708-1713.
[42] Yang C X, Chen M, Zeng L J, et al. Improvement of tropane alkaloids production in hairy root cultures of Atropa belladonna by overexpressing pmt and h6h genes [J]. Plant Omics Journal,2011,4 (1):29-33.
[43] Kang S M. Effects of methyl jasmonate and salicylic acid on the production of ropane alkaloids and the expression of PMT and H6H inadventitious root cultures of Scopolia parviflora[J]. Plant Science.2004(166):745-751.
[44] Jaber-Vazdekis N, GonzaLez C, Ravelo A G, et al. Cloning, characterization and analysis of expression profiles of a cDNA encoding a hyoscyamine 6β-hydroxylase (H6H) from Atropa baetica Willk[J]. Plant physiol. Biochem.2009(47):20-25.
[45] Edwards K, Johmstone C, and Thompson C. A simple and rapid method for the preparation of plant genomic DNA for PCR analyses[J]. Nucleic Acids Research. 1991,6:1349.
[46] Mozo T, Hooykaas P J J. Electroporation of megaplasmids into Agrobacterium [J]. Plant molecular biology.1991,16:917-918.