PEG介导的猴头菌遗传转化体系的建立  

Establishment of genetic transformation system of Hericium erinaceus using PEG mediated method

作　　者：刘莉[1] 肖招燕[1] 郭丽琼[1,2] 林俊芳[1,2] 尤琳烽[1] 廖静文[1] 

机构地区：[1]华南农业大学食品学院,广东广州510640 [2]华南农业大学生物质能研究所,广东广州510640

出　　处：《菌物学报》2014年第1期121-128,共8页Mycosystema

基　　金：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。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.

关 键 词：猴头菌 原生质体制备 PEG介导转化 

分 类 号：S646[农业科学—蔬菜学]