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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 results showed that the optimal system for preparation of Hericium erinaceus protoplasts was: Hericium hyphae cultured in liquid for 5 days, with 0.6mol / L KCl as the stabilizer Agent was added with 1.0% cellulase + 1.0% snail enzyme + 1.0% lysozyme complex enzyme, Enzymatic hydrolysis of Hericium at 30 ℃ 3h, the protoplast yield reached 3.0 × 106 个 / mL. Hygromycin sensitivity test showed that the minimum screening concentration of hygromycin on Herba PDSA solid medium was 60μg / mL. The plasmid pBgGI-hph (containing the gpd1-Gl promoter of Ganoderma lucidum and hygromycin resistance gene hph) was transformed into protoplast of Hericium erinaceus by PEG-mediated protoplast method. The results of preliminary screening and PCR identification of hygromycin showed that there was The hph gene was amplified from the genomes of four Hericium erinaceus transformants. Southern blotting confirmed that the hph gene was integrated into the genomes of four transformants.