农杆菌介导的猪苓遗传转化体系的建立及优化  

Establishment and optimization of Agrobacterium tumefaciens-mediated genetic transformation system for Polyporus umbellatus

作　　者：池丽 韩鹏杰 焦红红 刘天睿 周骏辉[4] 袁媛 CHI Li;HAN Peng-jie;JIAO Hong-hong;LIU Tian-rui;ZHOU Jun-hui;YUAN Yuan(School of Pharmacy,Shaanxi University of Chinese Medicine,Xianyang 712046,China;Shandong Analysis and Test Center,Qilu University of Technology,Ji'nan 250014,China;Institute of Traditional Chinese Medicine Health Industry,China Academy of Chinese Medical Sciences,Nanchang 330115,China;National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Experimental Research Center,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]陕西中医药大学药学院,陕西咸阳712046 [2]齐鲁工业大学山东省分析测试中心,山东济南250014 [3]中国中医科学院中医药健康产业研究所,江西南昌330115 [4]中国中医科学院中药资源中心,北京100700 [5]中国中医科学院医学实验中心,北京100700

出　　处：《中国中药杂志》2024年第15期4015-4021,共7页China Journal of Chinese Materia Medica

基　　金：中国中医科学院科技创新工程项目(CI2023D001,CI2023E002-04);国家杰出青年科学基金项目(82325049);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(ZZ16-ND-12)。

摘　　要：猪苓菌核栽培中种苓质量不稳定是影响猪苓菌核品质和产量的关键问题,该文拟对根癌农杆菌介导的猪苓遗传转化体系进行研究,为通过分子育种从而获得优质种苓提供技术保障。采用根癌农杆菌介导法探究抗生素浓度、菌株类型、菌液浓度、受体材料、侵染时间、共培养时间和筛选条件对猪苓遗传转化效率的影响,利用潮霉素抗性标记基因、特异引物PCR以及荧光检测方法筛选并检测转化子。结果表明,根癌农杆菌GV3101菌株可对猪苓菌丝进行遗传转化,菌液浓度A_(600 nm)=0.6,受体材料猪苓菌丝,侵染30 min,共培养3 d为最佳侵染条件;9μg·mL^(-1)潮霉素初筛和13μg·mL^(-1)潮霉素复筛两步筛选法为最优筛选条件。经潮霉素抗性筛选、特异引物PCR检测和荧光检测分析,结果表明外源基因eGFP已转入猪苓菌丝内整合到基因组中并成功表达,在最优条件下,转化率可达到2.3%,遗传转化周期从大于90 d缩短到小于60 d。该研究建立并优化了根癌农杆菌介导的猪苓菌丝遗传转化体系,为解析猪苓生长发育的分子机制及分子育种奠定基础。The unstable quality of Polyporus umbellatus sclerotia during cultivation is the key factor affecting the quality and yield of P.umbellatus sclerotia.In order to provide technical support for obtaining superior P.umbellatus by molecular breeding,the genetic transformation system mediated by Agrobacterium tumefaciens was studied in this paper.A.tumefaciens-mediated method was used to investigate the effects of antibiotic concentration,strain type,A.tumefaciens concentration,receptor material,infection time,co-culture time,and screening conditions on the genetic transformation efficiency of P.umbellatus.The transformants were screened and detected by hygromycin resistance marker genes,polymerase chain reaction(PCR)of specific primers,and fluorescence detection methods.The results showed that the A.tumefaciens GV3101 strain could genetically transfer P.umbellatus mycelium cells,and the optimal conditions for infection were as follows:the A.tumefaciens concentration A_(600 nm)=0.6,P.umbellatus mycelium cells as receptor material,infection time of 30 min,and co-culture time of 3 days.The two-step screening method involving hygromycin of 9 and 13μg·mL^(-1)was the best screening condition.The results of hygromycin resistance screening,PCR detection of specific primers,and fluorescence detection showed that the exogenous gene eGFP had been transferred into the P.umbellatus mycelium cells,integrated into the genome,and successfully expressed.Under optimal conditions,the conversion efficiency could be increased to 2.3%,and the genetic transformation period was shortened from more than 90 days to less than 60 days.This study established and optimized the genetic transformation system of P.umbellatus mycelium cells mediated by A.tumefaciens,laying a foundation for the analysis of the molecular mechanism of P.umbellatus during growth and molecular breeding.

关 键 词：猪苓 遗传转化体系 根癌农杆菌 条件优化 分子育种 

分 类 号：S567.3[农业科学—中草药栽培]