裂壳菌L-14原生质体制备与再生体系构建
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摘要
【目的】初步构建裂壳菌L-14原生质体制备和再生体系,为同源菌株原生质体制备研究提供理论依据。【方法】研究细胞壁裂解酶组合、酶解时间、酶解温度、渗透压稳定剂种类对原生质体制备产量的影响,以获得最优制备方案,同时筛选适合的原生质体再生培养基。【结果】菌株经YEPG液体培养基培养7 d后,使用0.7 mol/L的NaCl作为渗透压稳定剂配置浓度各为20 mg/mL的崩溃酶+裂解酶复合酶液,在30℃、80 r/min条件下酶解5.5 h后可获得较高的原生质体数量,利用PDS培养基的原生质体再生率可达10.65%,显著高于其它类型培养基。【结论】确定了裂壳菌L-14的高效原生质体制备与再生体系,为该菌株遗传转化体系的建立研究奠定基础。
【Objective】 To provide theoretical basis for protoplast preparation and regeneration conditions of homologous strains, the optimization of protoplast preparation and regeneration conditions of dark septate endophyte Schizothecium sp.L-14 were studied.【Method】The effects of different enzyme combination, enzymolysis time, enzymolysis temperature and osmotic stabilizer type on the protoplast formation were studied to understand the optimization method. Meanwhile, optimal culture medium have been investigated for protoplast regeneration.【Result】The highest production rate of protoplast was obtained when the L-14 mycelia was first cultured in YEPG medium for 7 days, then taking 0.7 mol/L NaCl as osmotic stabilizer, followed by an digestion in 20 mg/mL driselase and 20 mg/mL lysing enzyme mixture system for 5.5 hours at 30 ℃,80 r/min condition. The protoplast regeneration rate reached 10.65 % when cultured in PDS medium, which was significantly higher than cultured in other medium.【Conclusion】 An efficient method for protoplast preparation and regeneration conditions of Schizothecium sp.L-14 was produced, which would lay a foundation for strain transformation, distribution and biocontrol characteristics research in the future.
【Objective】 To provide theoretical basis for protoplast preparation and regeneration conditions of homologous strains, the optimization of protoplast preparation and regeneration conditions of dark septate endophyte Schizothecium sp.L-14 were studied.【Method】The effects of different enzyme combination, enzymolysis time, enzymolysis temperature and osmotic stabilizer type on the protoplast formation were studied to understand the optimization method. Meanwhile, optimal culture medium have been investigated for protoplast regeneration.【Result】The highest production rate of protoplast was obtained when the L-14 mycelia was first cultured in YEPG medium for 7 days, then taking 0.7 mol/L NaCl as osmotic stabilizer, followed by an digestion in 20 mg/mL driselase and 20 mg/mL lysing enzyme mixture system for 5.5 hours at 30 ℃,80 r/min condition. The protoplast regeneration rate reached 10.65 % when cultured in PDS medium, which was significantly higher than cultured in other medium.【Conclusion】 An efficient method for protoplast preparation and regeneration conditions of Schizothecium sp.L-14 was produced, which would lay a foundation for strain transformation, distribution and biocontrol characteristics research in the future.
引文
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[17]陈亮,伦莹莹,孙庚牛,等.苹果腐烂病原菌原生质体制备与再生条件优化[J].山东农业科学,2014,46(8):109-112.
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[19]杜婵娟,付岗,叶云峰,等.拮抗木霉gz-2菌株原生质体制备及转化条件优化[J].南方农业学报,2017,48(10):1817-1823.
[2]Kwasna H,Bateman G L,Ward E.Determining species diversity of microfungal communities in forest tree roots by pure-culture isolation and DNA sequencing[J].Applied Soil Ecology,2008,40(1):44-56.
[3]张杰.云南两个重金属矿区(暮乃、个旧)深色有隔内生真菌(DSE)的研究[D].昆明:云南大学,2010:30-31.
[4]谢玲,刘斌,覃丽萍,等.甘蔗根围深色有隔内生真菌(DSE)的分离及接种效应[J].西南农业学报,2014,27(2):635-640.
[5]解琳琳.西北荒漠花棒根系DSE真菌物种多样性和耐盐性研究[D].保定:河北大学,2017:22-23.
[6]徐全智,孙牧笛,李帆,等.宁夏枸杞内生真菌的分离及多样性分析[J].北方园艺,2017,41(10):103-109.
[7]Usuki F,Narisawa K.A mutualistic symbiosis between a dark septate endophytic fungus,Heteroconium chaetospira,and a nonmycorrhizal plant,Chinese cabbage[J].Mycorrhiza,2007,99(2):175-184.
[8]Narisawa K,Hambleton S,Currah R S.Heteroconium chaetospira,a dark septate root endophyte allied to the Herpotrichiellaceae (Chaetothyriales ) obtained from some forest soil samples in Canada using bait plant[J].Mycoscience,2007,48(5):274-281.
[9]Knapp D G,Kovács G M,Zajta E,et al.Dark septate endophytic pleosporalean genera from semiarid areas[J].Persoonia,2015(35):87-100.
[10]覃丽萍,苏琴,张雯龙,等.甘蔗优良DSE促生菌株的筛选及田间接种效果[J].热带作物学报,2015,36(10):1861-1865.
[11]农倩,张雯龙,蓝桃菊,等.一株抗香蕉枯萎病DSE菌株的筛选鉴定及抗病机理初探[J].热带作物学报,2017,38(3):559-564.
[12]Ge C Y,Duan Y B,Zhou M G,et al.A protoplast transformation system for gene deletion and complementation in Sclerotinia sclerotiorum[J].Journal of Phytopathology,2013,161(11):800-806.
[13]Feng L,Li C,Xu C,et al.Protoplast formation and regeneration of Rhodococcus sp.BX2[J].Information Technology and Agricultural Engineering,2012(134):113-120.
[14]沈慧敏,高利,李超,等.小麦光腥黑粉菌原生质体的制备与再生[J].中国植保导刊,2017,37(10):14-18.
[15]Sander C S,Hipler U C,Wollina U,et al.Inhibitory effect of terbinafine on reactive oxygen species (ROS) generation by Candida albicans[J].Mycoses,2010,45(5-6):152-155.
[16]沈慧敏,李超,高利,等.小麦矮腥黑粉菌原生质体的制备与再生[J].植物保护,2018,44(2):140-144.
[17]陈亮,伦莹莹,孙庚牛,等.苹果腐烂病原菌原生质体制备与再生条件优化[J].山东农业科学,2014,46(8):109-112.
[18]李淼,曾柏全,冯金儒.高产纤维素酶菌株原生质体制备及再生条件[J].中南林业科技大学学报,2013,33(12):174-180.
[19]杜婵娟,付岗,叶云峰,等.拮抗木霉gz-2菌株原生质体制备及转化条件优化[J].南方农业学报,2017,48(10):1817-1823.