农杆菌介导的刺芹侧耳遗传转化体系的建立-北京玛格泰克科技发展.PDFVIP

菌物学报 jwxt@ 15 July 2015, 34(4): 653-661 Http:// Mycosystema ISSN1672-6472 CN11-5180/Q © 2015 IMCAS, all rights reserved. 研究论文 Research paper DOI: 10.13346/j.mycosystema.150048 农杆菌介导的刺芹侧耳遗传转化体系的建立 盛立柱1, 2 宋冰2 戴月婷2 李丹2 付永平2* 李玉2* 1 吉林农业大学农学院 吉林 长春 130118 2 吉林农业大学食药用菌教育部工程中心 吉林 长春 130118 摘 要：以刺芹侧耳菌丝球为受体，潮霉素（Hyg ）为筛选标记，应用农杆菌介导法对刺芹侧耳菌丝进行了遗传转化研究。 潮霉素敏感性测试结果表明，刺芹侧耳Hyg 耐受浓度为50mg/L 。农杆菌介导的刺芹侧耳菌丝最佳遗传转化体系为：菌液 浓度OD600=0.6–0.7 ，侵染时间30–35min，共培养时间2d，侵染液和共培养培养基中乙酰丁香酮（AS ）浓度为1mg/mL； 经潮霉素抗性筛选、PCR 鉴定和GUS 活性的组织化学分析，表明外源基因GUS 已转入到刺芹侧耳菌丝中，并获得表达。 本实验成功地建立了稳定的农杆菌介导的刺芹侧耳遗传转化体系。 关键词：刺芹侧耳，菌丝球，农杆菌，GUS 染色，转化体系 Construction of the genetic transformation system of Pleurotus eryngii by Agrobacterium-mediated method SHENG Li-Zhu1, 2 SONG Bing2 DAI Yue-Ting2 LI Dan2 FU Yong-Ping2* LI Yu2* 1 College of Agronomy, Jilin Agricultural University, Changchun, Jilin 130118, China 2 Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi, College of Agronomy, Jilin Agricultural University, Changchun, Jilin 130118, China Abstract : The mycelial pellets of Pleurotus eryngii were used as receptor for transformation by Agrobacterium -mediated method, and the genetic transformation system of P. eryngii was established. Sensitivity test showed that the tolerance concentration for P. eryngii against hygromycin was 50mg/L. The opti mum genetic transformation system for Agrobacterium -mediated P. eryngii mycelia was OD600=0.6–0.7 of the bacterial concentration, 30–35min of infection duration, 2 days of the co-cultivation period and 1mg/mL of acetosyringone (AS) concentration. The results of PCR identification and the histochemical analysis of β-glucuronidase (GUS) activities indicated that the exogenous gene gus was transferred into the mycelia