首页 | 本学科首页   官方微博 | 高级检索  
     

PEG介导的猴头菌遗传转化体系的建立
引用本文:刘莉,肖招燕,郭丽琼,林俊芳,尤琳烽,廖静文. PEG介导的猴头菌遗传转化体系的建立[J]. 菌物学报, 2014, 33(1): 121-128
作者姓名:刘莉  肖招燕  郭丽琼  林俊芳  尤琳烽  廖静文
作者单位:华南农业大学食品学院 广东 广州 510640;华南农业大学食品学院 广东 广州 510640;华南农业大学食品学院 广东 广州 510640 华南农业大学生物质能研究所 广东 广州 510640;华南农业大学食品学院 广东 广州 510640 华南农业大学生物质能研究所 广东 广州 510640;华南农业大学食品学院 广东 广州 510640;华南农业大学食品学院 广东 广州 510640
基金项目:国家自然科学基金(No. 31071837,No. 31272217)“食用真菌组合表达紫杉醇及其中间产物的基础研究”和“食用真菌组合表达白藜芦醇的研究”
摘    要:对猴头菌Hericium erinaceus原生质体制备的各种因素进行比较研究,结果表明,猴头菌原生质体制备的最佳体系为:液体培养5d的猴头菌丝,以0.6mol/L KCl作为稳渗剂,加入含1.0%纤维素酶+1.0%蜗牛酶+1.0%溶壁酶的复合酶,在30℃酶解猴头菌丝3h时,原生质体得率达到3.0×106个/mL。潮霉素敏感性测试表明,猴头菌在PDSA固体培养基上的潮霉素最低筛选浓度为60μg/mL。采用PEG介导的原生质体法,将质粒pBgGI-hph(含有灵芝gpd1-Gl启动子和潮霉素抗性基因hph)转化猴头菌原生质体,经潮霉素初步筛选以及PCR鉴定,表明有4株猴头菌拟转化子的基因组扩增出hph基因;转化子经过多次转接后进行Southern杂交验证,结果表明4个转化子的基因组中均稳定整合了hph抗性基因。

关 键 词:猴头菌  原生质体制备  PEG介导转化

Establishment of genetic transformation system of Hericium erinaceus using PEG mediated method
LIU Li,XIAO Zhao-Yan,GUO Li-Qiong,LIN Jun-Fang,YOU Lin-Feng and LIAO Jing-Wen. Establishment of genetic transformation system of Hericium erinaceus using PEG mediated method[J]. Mycosystema, 2014, 33(1): 121-128
Authors:LIU Li  XIAO Zhao-Yan  GUO Li-Qiong  LIN Jun-Fang  YOU Lin-Feng  LIAO Jing-Wen
Affiliation:College of Food Science, South China Agriculture University, Guangzhou, Guangdong 510640, China;College of Food Science, South China Agriculture University, Guangzhou, Guangdong 510640, China;College of Food Science, South China Agriculture University, Guangzhou, Guangdong 510640, China Institute of Biomass Energy, Guangzhou, Guangdong 510640, China;College of Food Science, South China Agriculture University, Guangzhou, Guangdong 510640, China Institute of Biomass Energy, Guangzhou, Guangdong 510640, China;College of Food Science, South China Agriculture University, Guangzhou, Guangdong 510640, China;College of Food Science, South China Agriculture University, Guangzhou, Guangdong 510640, China
Abstract:The method for protoplast preparation of Hericium erinaceus was studied. The 5-day-old mycelia treated with the mixture of 1.0% lywallzyme, 1.0% cellulase and 1.0% snailase in 0.6mol/L KCl at 30℃ for 3h could produce an efficient yield of protoplasts to 3.0×106/mL. Drug resistance test of H. erinaceus against hygromycin B showed that the lowest selection concentration were 60μg/mL. By PEG-mediated protoplast transformation, the expression plasmid pBgGI-hph (containing promoter gpd1-Gl derived from Ganoderma lucidum and selectable marker gene hph conferring resistance to hygromycin B) was successfully transformed into the protoplast of H. erinaceus. Identifications of the putative transformants were performed by hygromycin B blotting and PCR analysis with hph gene. The results showed that there are four positive transgenic H. erinaceus with hph gene. After several generations of subculture, southern blotting analysis showed that the marker hph gene was integrated into the genomic DNA of transformants.
Keywords:Hericium erinaceus   preparation of protoplast   PEG-mediated protoplast transformation
本文献已被 CNKI 等数据库收录!
点击此处可从《菌物学报》浏览原始摘要信息
点击此处可从《菌物学报》下载全文
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号