中国中药杂志

2014, v.39(01) 52-58

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颠茄托品烷生物碱合成途径基因表达分析与生物碱积累研究
Expression pattern of genes involved in tropane alkaloids biosynthesis and tropane alkaloids accumulation in Atropa belladonna

强玮;王亚雄;张巧卓;李金弟;夏科;吴能表;廖志华;
QIANG Wei;WANG Ya-xiong;ZHANG Qiao-zhuo;LI Jin-di;XIA Ke;WU Neng-biao;LIAO Zhi-hua;Key Laboratory of Eco-environments in Three Gorges Reservoir Region of Ministry of Education,School of Life Science;Chongqing Engineering Research Center for Sweetpotato,School of Life Science,Southwest University;

摘要(Abstract):

托品烷类生物碱(tropane alkaloids,TAs)是临床上广泛使用的抗胆碱药物,颠茄是药典收录的TAs最主要的商业栽培药源植物。基于颠茄转录组测序数据构建TAs合成途径中9个结构基因(ODC,ADC,AIH,CPA,SPDS,PMT,CYP80F1,H6H,TRⅡ)UniGene序列的数字表达谱,采用qPCR对其中4个已报道基因(PMT,CYP80F1,H6H,TRⅡ)的表达水平进行验证分析,同时采用HPLC测定不同组织中TAs含量。数字表达谱分析结果表明4个TAs上游合成途径基因(ODC,ADC,AIH,CPA)和2个支路途径基因(SPDS,TRⅡ)在颠茄各器官中均有表达,但在须根中高水平表达;3个TAs合成途径特异的结构基因PMT,CYP80F1和H6H均只在须根中大量表达,主根中其次。qPCR检测PMT,CYP80F1,H6H,TRⅡ的表达结果与数字表达谱基本一致,但PMT,CYP80F1,H6H在主根中表达量很低。莨菪碱质量分数在嫩茎中最高(3.364 mg·g-1),幼叶,根,幼果和果萼中其次(分别为1.526,1.598,1.271,1.413 mg·g-1);东莨菪碱质量分数在果萼中最高(1.003 mg·g-1),嫩茎和幼叶(分别为0.600,0.601 mg·g-1)中其次;2种生物碱在老茎(莨菪碱0.283 mg·g-1,东莨菪碱0.043 mg·g-1)和老叶(莨菪碱0.313 mg·g-1,东莨菪碱0.080 mg·g-1)中质量分数均为最低。该研究结果表明须根是颠茄TAs生物合成主要器官,而地上幼嫩组织是TAs主要存贮积累器官,TAs合成后存在转运过程。PMT下游基因均只在须根中表达,筛选颠茄须根的转录组数据库就可能为解决该途径中不清晰的合成步骤和相关转录调控因子提供有力的帮助。
Atropa belladonna is a medicinal plant and main commercial source of tropane alkaloids( TAs) including scopolamine and hyoscyamine,which are anticholine drugs widely used clinically. Based on the high throughput transcriptome sequencing results,the digital expression patterns of UniGenes representing 9 structural genes( ODC,ADC,AIH,CPA,SPDS,PMT,CYP80F1,H6H, TRII) involved in TAs biosynthesis were constructed,and simultaneously expression analysis of 4 released genes in NCBI( PMT, CYP80F1,H6H,TRII) for verification was performed using qPCR,as well as the TAs contents detection in 8 different tissues. Digital expression patterns results suggested that the 4 genes including ODC,ADC,AIH and CPA involved in the upstream pathway of TAs,and the 2 branch pathway genes including SPDS and TRII were found to be expressed in all the detected tissues with high expression level in secondary root. While the 3 TAs-pathway-specific genes including PMT,CYP80F1,H6H were only expressed in secondary roots and primary roots,mainly in secondary roots. The qPCR detection results of PMT,CYP80F1 and H6H were consistent with the digital expression patterns,but their expression levels in primary root were too low to be detected. The highest content of hyoscyamine was found in tender stems( 3. 364 mg·g- 1),followed by tender leaves( 1. 526 mg·g- 1),roots( 1. 598 mg·g- 1),young fruits( 1. 271 mg· g- 1) and fruit sepals( 1. 413 mg·g- 1). The highest content of scopolamine was detected in fruit sepals( 1. 003 mg·g- 1),then followed by tender stems( 0. 600 mg·g- 1) and tender leaves( 0. 601 mg·g- 1). Both old stems and old leaves had the lowest content of hyoscyamine and scopolamine. The gene expression profile and TAs accumulation indicated that TAs in Atropa belladonna were mainly biosynthesized in secondary root,and then transported and deposited in tender aerial parts. Screening Atropa belladonna secondary root transcriptome database will facilitate unveiling the unknown enzymatic reactions and the mechanisms of transcriptional control.

关键词(KeyWords): 颠茄;托品烷生物碱;数字表达谱;qPCR
Atropa belladonna;tropane alkalonids;digital expression pattern;qPCR

Abstract:

Keywords:

基金项目(Foundation): 教育部新世纪人才支持计划项目(NCET-12-0930);; 国家高技术研究发展计划(863)项目(2011AA100605);; 国家自然科学基金项目(31370333);; 重庆市科技攻关项目(CSTC2012GGYYJS80013);; 西南大学中央高校基本科研业务费专项(XDJK2013A024)

作者(Author): 强玮;王亚雄;张巧卓;李金弟;夏科;吴能表;廖志华;
QIANG Wei;WANG Ya-xiong;ZHANG Qiao-zhuo;LI Jin-di;XIA Ke;WU Neng-biao;LIAO Zhi-hua;Key Laboratory of Eco-environments in Three Gorges Reservoir Region of Ministry of Education,School of Life Science;Chongqing Engineering Research Center for Sweetpotato,School of Life Science,Southwest University;

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