原生质体紫外诱变法选育高产γ-氨基丁酸秀珍菇菌株
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摘要
采用原生质体紫外诱变育种方法,进行高产γ-氨基丁酸的秀珍菇菌株选育工作,对原生质体制备条件、再生条件和诱变株γ-氨基丁酸含量进行了筛选分析。结果表明:MM缓冲液中,20 mg/mL的溶壁酶能够有效去除秀珍菇细胞壁,获得2×10~6个/mL的原生质体;在含有0.5 mol/L甘露醇的YMG培养基上,原生质体再生率最高,可达到0.65%±0.13%。35 W紫外灯照射1～2 min时,致死率可达到76%～86%;菌丝体和子实体中,3株突变株γ-氨基丁酸含量较出发菌株分别提高了20.7%、196.3%、126.8%和21.3%、237.8%、97.3%。诱变菌株的获得,可为高值化富含γ-氨基丁酸秀珍菇生产菌株的培育提供优异的育种材料。
An ultraviolet ray-mediated protoplast mutagenesis method was applied to breed γ-aminobutyric acid(GABA)-rich Pleurotus geesteranus strains, screening conditions for protoplast preparation, protoplast regeneration and GABA concentrations in mutants. The results show that, concentration of protoplast of Pleurotus geesterans could reach 2×10~6 cloes/mL when 20 mg/mL(wt) lywallzyme being dissolved in MM buffer. The protoplast regeneration rate could top 0.65%±0.13% on a YMG plus 0.5 mol/L mannitol medium. The lethal ratio could range from 76% to 86% when protoplasts being irradiated under a 35 W ultraviolet lamp for 1～2 minutes. GABA concentration in mycelia and fruiting bodies from 3 mutants has increased by 20.7% and 196.3%, 126.8% and 21.3%, 237.8% and 97.3% respectively, compared to those from the original strains. The acquisition of GABA-rich mutant strains can provide excellent breeding materials for cultivation of high-value GABA-rich Pleurotus geesteranus products.
An ultraviolet ray-mediated protoplast mutagenesis method was applied to breed γ-aminobutyric acid(GABA)-rich Pleurotus geesteranus strains, screening conditions for protoplast preparation, protoplast regeneration and GABA concentrations in mutants. The results show that, concentration of protoplast of Pleurotus geesterans could reach 2×10~6 cloes/mL when 20 mg/mL(wt) lywallzyme being dissolved in MM buffer. The protoplast regeneration rate could top 0.65%±0.13% on a YMG plus 0.5 mol/L mannitol medium. The lethal ratio could range from 76% to 86% when protoplasts being irradiated under a 35 W ultraviolet lamp for 1～2 minutes. GABA concentration in mycelia and fruiting bodies from 3 mutants has increased by 20.7% and 196.3%, 126.8% and 21.3%, 237.8% and 97.3% respectively, compared to those from the original strains. The acquisition of GABA-rich mutant strains can provide excellent breeding materials for cultivation of high-value GABA-rich Pleurotus geesteranus products.
引文
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[2] 冯志勇,王志强,郭力刚,等.秀珍菇生物学特性研究[J].食用菌学报,2003,10(3):11.
[3] 韩建东,宫志远,姚强,等.金针菇菌渣栽培秀珍菇的营养成分分析[J].中国食用菌,2013,32(6):30.
[4] SHEN H S,LIU H Z,CHEN J C,et a1.Tetramethylpyrazine from Pleurotus geesteranus[J].Natural Product Communications,2015,10(9):1553.
[5] HE P X,GENG L J,MAO D B,et a1.Production,preliminary characterization,and bioactives of exopolysaccharides from Pleurotus geesteranus 5#[J].Preparative Biochemistry Biotechnology,2013,43(1):108.
[6] MAO D B,MA Y P,GENG L J,et a1.Fermentation characteristics in stirred-tank reactor of exopolysaccharides with hypolipidemic activity produced by Pleurotus geesteranus 5(#)[J].Biological Sciences,2013,85(4):1473.
[7] RANOGAJEC A,BELUHAN S,SMIT Z.Analysis of nucleosides and monophosphate nucleotides from mushrooms with reversedphase HPLC[J].J Separation Science,2010,33(8):1024.
[8] WANG Q,LI H,CHEN T T,et a1.Yield,polysaccharides content and antioxidant propeties of Pleurotus abalonus and Pleurotus geesteranus produced on asparagus straw as substrate[J].Scientia Horticulturae,2012,134(2):2229.
[9] LV G Y,ZHANG Z F,PAN H J,et a1.Antioxidant properties of different solvents extracts from three edible mushrooms[C]//3rd International Conference on Bioinformatics and Biomedical Engineering.Beijing:ICBBE,2009.
[10] 陈发川,柯斌榕,吴小平.秀珍菇菌种复壮方法比较[J].热带作物报,2011,32(11):2037.
[11] 许谦.药用真菌桑黄原生质体的制备和诱变[J].中国食用菌,2016,35(4):67.
[12] 秦婷婷,冯昆达,张涛,等.原生质体诱变筛选高产纽莫康定B0的菌株[J].中国抗生素杂志,2016,41(3):212.
[13] 李守勉,李明,邢蕾,等.食用菌原生质体技术应用的研究[J].安徽农业科学,2007,35(25):7770.
[14] 王丽宁,赵妍,张宝粉,等.利用原生质体紫外诱变技术选育耐高温香菇菌株[J].微生物学通报,2014,41(7):1350.
[15] 李红梅.原生质体紫外诱变技术选育烟酰胺高产菌株[J].应用化工,2013,42(1):72.
[16] 于长青,李丽娜.深黄被孢霉高产花生四烯酸菌株的紫外诱变原生质体育种[J].微生物学报,2009,49(1):44.
[17] 何小慧.杏鲍菇原生质体再生变异菌株的筛选[D].福州:福建师范大学,2014.
[18] 梁臣,陈忠.γ-氨基丁酸及其受体功能的研究与应用现状[J].动物医学进展,2015(4):108.
[19] 赵宏飞,宋伟,裴家伟,等.食品中三种γ-氨基丁酸检测方法比较[J].中国乳品工业,2008,36(11):51.