高卢蜜环菌PCR-RFLP快速鉴别方法研究  

Molecular identification of Armillaria gallica by PCR-RFLP analysis

作　　者：梁宇庭 蒋超[1] 周骏辉[1] 胡启跳 袁媛[1] LIANG Yu-ting;JIANG Chao;ZHOU Jun-hui;HU Qi-tiao;YUAN Yuan(State Key Laboratory of Dao-di Herbs Breeding Base,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]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

出　　处：《中国中药杂志》2019年第17期3622-3626,共5页China Journal of Chinese Materia Medica

基　　金：国家科技基础性工作专项(2015FY111500);国家自然科学基金重大项目(81891013,81891010);中央级公益性科研院所基本科研业务费专项资金项目(ZZ10-008,ZZ11-116);中央本级重大增支减项目(2060302)

摘　　要：蜜环菌是名贵中药天麻、猪苓栽培过程中必备的共生菌,蜜环菌的菌索侵入天麻块茎或猪苓菌核后被后者消化利用,成为它们重要的营养源。不同性质的蜜环菌影响天麻、猪苓的生长。作者收集了13个地区的市售蜜环菌,经过IGS鉴定均为高卢蜜环菌。在基因水平和代谢水平上筛选出了蔗糖含量具有显著差异的2种高卢蜜环菌,命名为A型和B型,并建立了简单、准确的高卢蜜环菌DNA分子标记鉴定方法。通过比较高卢蜜环菌A型和B型蔗糖酶基因序列差异,根据差异片段设计聚合酶链反应-限制性内切酶酶切长度多态(PCR-RFLP)鉴别方法,即引物ZTM.F/ZTM.R可扩增高卢蜜环菌A型和B型,产生约304 bp长度条带,限制性内切酶EcoR V可以识别高卢蜜环菌A型和B型的差异序列,仅高卢蜜环菌B型可以被酶切形成2个片段,从而特异性鉴别高卢蜜环菌为B型。该实验建立的PCR-RFLP可以作为高卢蜜环菌A型和B型的DNA鉴别方法。在天麻和猪苓的栽培过程中,可根据品种、生长阶段、生态环境等不同需要,选择合适的菌株,因地制宜提升它们的产量和品质。Armillaria gallica is a symbiotic fungus in the cultivation process of Gastrodia elata and Polyporus.The rhizomorph of A. gallica invades the stalk of the G. elata or the Sclerotium of the Polyporus,and is digested and utilized by the latter,becoming their important source of nutrition. Different nature of A. gallica affects the growth of G. elata and Polyporus. The authors collected A. gallica from 13 commercially available regions and screened two A. gallica,A and B,at the genetic and metabolic levels,in order to distinguish between the two A. gallica market. We have established convenient and effective DNA molecular identification method.By comparing the sequence differences between the A. gallica type A and type B invertase genes,PCR-RFLP primers were designed based on differential fragment. Primer ZTM.F/ZTM.R can amplified A. gallica type A and B,producing a band of about 304 bp in length. The restriction endonuclease EcoR V could recognize the difference sequence of A and B types of A. gallica. The type B was digested to form two fragments,thereby specifically identifying the A. gallica as type B. The established methods of PCR-RFLP is an accurate identification method for A. gallica. Therefore,in the cultivation process of G. elata and Polyporus,suitable strains can be selected according to different needs of variety,growth stage and ecological environment,and the yield and quality can be improved according to local conditions.

关 键 词：高卢蜜环菌 聚合酶链反应-限制性内切酶酶切长度多态性 分子鉴定 

分 类 号：R28[医药卫生—中药学]