Qiang Wei, Wang Ya-Xiong, Zhang Qiao-Zhuo, Li Jin-Di, Xia Ke, Wu Neng-Biao, Liao Zhi-Hua
Zhongguo Zhong Yao Za Zhi. 2014 Jan;39(1):52-8.
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 x g(-1)), followed by tender leaves (1.526 mg x g(-1)), roots (1.598 mg x g(-1)), young fruits (1.271 mg x g(-1)) and fruit sepals (1.413 mg x g(-1)). The highest content of scopolamine was detected in fruit sepals (1.003 mg x g(-1)), then followed by tender stems (0.600 mg x g(-1)) and tender leaves (0.601 mg x 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.
颠茄是一种药用植物，也是包括东莨菪碱和莨菪碱在内的托烷生物碱（TAs）的主要商业来源，这些都是临床上广泛使用的抗胆碱药物。基于高通量转录组测序结果，构建了代表参与TAs生物合成的9个结构基因（ODC、ADC、AIH、CPA、SPDS、PMT、CYP80F1、H6H、TRII）的单基因数字表达模式，同时使用qPCR对NCBI中4个已发布基因（PMT、CYP80F1、H6H、TRII）进行表达分析以进行验证，并检测了8种不同组织中的TAs含量。数字表达模式结果表明，参与TAs上游途径的4个基因包括ODC、ADC、AIH和CPA，以及包括SPDS和TRII在内的2个分支途径基因在所有检测组织中均有表达，在次生根中表达水平较高。而包括PMT、CYP80F1、H6H在内的3个TAs途径特异性基因仅在次生根和初生根中表达，主要在次生根中表达。PMT、CYP80F1和H6H的qPCR检测结果与数字表达模式一致，但它们在初生根中的表达水平过低无法检测到。莨菪碱含量最高的是嫩茎（3.364 mg x g(-1)），其次是嫩叶（1.526 mg x g(-1)）、根（1.598 mg x g(-1)）、幼果（1.271 mg x g(-1)）和果萼（1.413 mg x g(-1)）。东莨菪碱含量最高的是果萼（1.003 mg x g(-1)），其次是嫩茎（0.600 mg x g(-1)）和嫩叶（0.601 mg x g(-1)）。老茎和老叶中莨菪碱和东莨菪碱的含量最低。基因表达谱和TAs积累表明，颠茄中的TAs主要在次生根中生物合成，然后运输并沉积在地上嫩组织中。筛选颠茄次生根转录组数据库将有助于揭示未知的酶促反应和转录调控机制。
