北冬虫夏草原生质体的制备及诱变育种
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摘要
通过对北冬虫夏草(Cordyceps militaris Link)菌种液体发酵培养,离心分离获得菌丝体菌液,分别从渗透压稳定剂、裂解酶、裂解时间及菌龄4个方面优化原生质体制备的条件.结果表明:原生质体制备的最优渗透压稳定剂为0.6 mol/L MgSO_4,最优酶解液为1%纤维素酶,最佳菌龄为5 d,最优裂解时间为2 h.L_(20)(5~4)的正交试验表明,优化条件下制备原生质体为1.46×10~7个/m L.紫外诱变实验结果表明:北冬虫夏草半致死率时的照射剂量约为40 s,诱变距离与致死率成反比,在诱变总时间相同的情况下,诱变受诱变次数影响较小,分次处理和一次处理效果基本相同.以多糖含量为指标,筛选诱变菌株,诱变株多糖含量高于普通菌株,其中17~#菌株多糖含量达6.01%,筛选菌株的稳定性实验表明:继代培养3次,筛选的菌株产多糖能力下降较少,表明筛选菌株具有较好的稳定性.
After enlarged culture of Cordyceps militaris Link and liquid fermentation,centrifugal separation was carried out to obtain mycelial liquid.The conditions of protoplast preparation were optimized from four aspects:osmotic pressure stabilizer,lysozyme,lysis time and bacterial age.The results showed that the optimal osmotic pressure stabilizer for protoplast preparation was 0.6 mol/L sulfatase,and the optimal osmotic pressure stabilizer was 0.6 mol/L sulfatase.While the optimum enzyme solution was 1%cellulase,the optimum bacterial age was 5 days,and the optimum cracking time was 2 hours.The orthogonal experiment of L_(20)(5~4)showed that 1.46×10~7protoplasts could be prepared under the optimum conditions.The results of ultraviolet mutagenesis experiment showed that the irradiation dose was about 40 seconds,and the mutagenesis distance was inversely proportional to the lethality.When the total time of mutagenesis was the same,the number of mutation was less affected,and the effects of multiple and single treatment were similar.The polysaccharide content was then used as an indicator to screen the mutagenized strains,and we successfully got the mutants that showed higher content of the polysaccharide than that of common strains.The polysaccharide content of 17~#was 6.01%.The stability test of the screened strains showed that the ability of polysaccharide production of screened strains decreased less after three subcultures and the screened strains had better stability.
After enlarged culture of Cordyceps militaris Link and liquid fermentation,centrifugal separation was carried out to obtain mycelial liquid.The conditions of protoplast preparation were optimized from four aspects:osmotic pressure stabilizer,lysozyme,lysis time and bacterial age.The results showed that the optimal osmotic pressure stabilizer for protoplast preparation was 0.6 mol/L sulfatase,and the optimal osmotic pressure stabilizer was 0.6 mol/L sulfatase.While the optimum enzyme solution was 1%cellulase,the optimum bacterial age was 5 days,and the optimum cracking time was 2 hours.The orthogonal experiment of L_(20)(5~4)showed that 1.46×10~7protoplasts could be prepared under the optimum conditions.The results of ultraviolet mutagenesis experiment showed that the irradiation dose was about 40 seconds,and the mutagenesis distance was inversely proportional to the lethality.When the total time of mutagenesis was the same,the number of mutation was less affected,and the effects of multiple and single treatment were similar.The polysaccharide content was then used as an indicator to screen the mutagenized strains,and we successfully got the mutants that showed higher content of the polysaccharide than that of common strains.The polysaccharide content of 17~#was 6.01%.The stability test of the screened strains showed that the ability of polysaccharide production of screened strains decreased less after three subcultures and the screened strains had better stability.
引文
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[3]袁建国,程显好,侯永勤,等.功能因子冬虫夏草多糖的研究开发[J].中国食品添加剂,1999,18(2):18-22.YUAN Jianguo,CHENG Xianhao,HOU Yongqin,et al.Study on polysaccharide from cordyceps as functional factor[J]. China Food Additives,1999,18(2):18-22.
[4]车振明,王燕,周黎黎,等.原生质体紫外诱变选育蛹虫草新菌种的研究[J].食品与发酵工业,2004,30(8):35-38.CHE Zhenming,WANG Yan,ZHOU Lili,et al.Study on the breeding of a new variety of Cordyceps militaris by mutated with ultraviolet radiation[J].Food and Fermentation Industries,2004,30(8):35-38.
[5]潘淼,孙立洁,陈祥松,等.高山被孢霉高产ARA的原生质体融合子筛选[J].河南师范大学学报(自然科学版),2017,45(1):39-46.PAN Miao,SUN Lijie,CHEN Xiangsong,et al.Screening of protoplast fusion of Mortierella alpina with highyield[J].Journal of Henan Normal University(Natural Science Edition),2017,45(1):39-46.
[6]郑锦荣.金针菇与巨大口蘑原生质体融合育种研究[D].广州:华南农业大学,2016.ZHENG Jinrong.Studies on Flammulina velutipes and Tricholoma giganterm massee protoplast fusion breeding[D].Guangzhou:South China Agricultural University,2016.
[7]王海磊,张永梅,刘磊,等.新西兰青霉(Penicillium novae-zeelandiae)原生质体的制备与再生研究[J].河南师范大学学报(自然科学版),2015,43(6):112-117.WANG Hailei,ZHANG Yongmei,LIU Lei,et al. Formation and regeneration of Penicillium novae-zeelandiae HSD04B protoplasts and influence factors[J].Journal of Henan Normal University(Natural Science Edition),2015,43(6):112-117.
[8]朱蕴兰,陈安徽,王陶,等.冬虫夏草原生质体诱变育种研究[J].食品科学,2010,31(5):256-260.ZHU Yunlan,CHEN Anhui,WANG Tao,et al. Ultraviolet-induced protoplast mutagenesis for breeding high-performance strains of Cordyceps sinensis[J].Food Science,2010,31(5):256-260.