李慧;钱润;田娜;李仰华;蒋超;袁媛;黄璐琦;
LI Hui;QIAN Run;TIAN Na;LI Yang-hua;JIANG Chao;YUAN Yuan;HUANG Lu-qi;Aademician Workstation, Jiangxi University of Traditional Chinese Medicine;School of Pharmacy, Anhui University of Chinese Medicine;School of Traditional Chinese Medicine, Guangdong Pharmaceutical University;State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;

• 1:江西中医药大学院士工作站
• 2:安徽中医药大学药学院
• 3:广东药科大学中药学院
• 4:中国中医科学院中药资源中心道地药材国家重点实验室培育基地

摘要(Abstract)：

天麻作为我国名贵中药材,具有良好的药用价值。《中国植物志》中将天麻分为红天麻(原变型)Gastrodia elata f.elata、乌天麻G.elata f.glauca、绿天麻G.elata f.viridis、黄天麻G.elata f.flavid和松天麻G.elataf.alba等5个变型,其中红天麻与乌天麻的天麻素和多糖含量均较高,为栽培的优良品系。目前市场上流通的天麻商品多为红天麻、乌天麻或杂交天麻,但其传统性状鉴别特征并不明显,无法快速、准确区分。该研究旨在建立一种能够高效、准确鉴别红天麻、乌天麻及其杂交天麻的聚合酶链式反应(polymerase chain reaction,PCR)方法。根据天麻重测序结果,筛选获得特异性单核苷酸多态性(single nucleotide polymorphism, SNP)变异位点,并利用变异位点设计2对特异性引物W291-F/W291-R和H255-F/H255-R。分别采集红天麻、乌天麻及其杂交天麻样品,建立并优化PCR鉴别方法,并对其耐受性和适用性进行考察与验证。使用特异性引物H255-F/H255-R、退火温度为48℃、循环数为33时,红天麻与红乌杂交天麻在255 bp处出现单一明亮条带;使用特异性引物W291-F/W291-R、退火温度为51℃,循环数为31时,乌天麻与红乌杂交天麻在291 bp处出现单一明亮条带。建立的特异性PCR方法可准确鉴定红天麻、乌天麻及其杂交天麻。
Gastrodia elata is a kind of traditional Chinese medicinal materials and has good medicinal value. G. elata is divided into five varieties, which includes G. elata f. elata(proto variant), G. elata f. glauca, G. elata f. viridis, G. elata f. flavid and G. elata f. alba. Among them, G. elata f. elata and G. elata f. glauca have excellent characteristics and higher contents of gastrodin and polysaccharides. The hybrid of G. elata f. elata and G. elata f. glauca is present in markets, but the characteristics between hybrid and parent are not obvious and distinguished quickly and accurately. The aim of this study is to establish a PCR specific PCR identification method, which can identify G. elata f. elata, G. elata f. glauca and their hybrid. Based on the re-sequencing results of G. elata, we screened for the single nucleotide polymorphism(SNP) variation sites, and designed two pairs of specific primers(W291-F/W291-R and H255-F/H255-R). We further collected G. elata f. elata, G. elata f. glauca and their hybrid samples from different regions, established and optimized PCR method, and investigated and verified their tolerance and applicability. The results showed that when the annealing temperature was 48 ℃ and the number of cycles was 33, 255 bp specific band were obtained from G. elata f. glauca and hybrid by using specific primers W291-F/W291-R. When the annealing temperature was 51 ℃ and the number of cycles was 33, 291 bp specific band were obtained from G. elata f. elata and hybrid by using specific primers H255-F/H255-R. Our method could be used as a promising method to identify G. elata f. elata, G. elata f. glauca and their hybrid.

关键词(KeyWords)： 天麻;杂交;聚合酶链式反应;分子鉴定
Gastrodia elata;hybrid;polymerase chain reaction;molecular identification

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金重大项目(81891013);; 中央本级重大增减支项目(2060302);; 中央级公益性科研院所基本科研业务费专项(ZZ10-008);; 广西科技重大专项(桂科AA18242040)

作者(Author): 李慧;钱润;田娜;李仰华;蒋超;袁媛;黄璐琦;
LI Hui;QIAN Run;TIAN Na;LI Yang-hua;JIANG Chao;YUAN Yuan;HUANG Lu-qi;Aademician Workstation, Jiangxi University of Traditional Chinese Medicine;School of Pharmacy, Anhui University of Chinese Medicine;School of Traditional Chinese Medicine, Guangdong Pharmaceutical University;State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;

Email:

DOI: 10.19540/j.cnki.cjcmm.20200527.102

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