Journal of Nuclear Agricultural Sciences ›› 2019, Vol. 33 ›› Issue (2): 278-289.DOI: 10.11869/j.issn.100-8551.2019.02.0278

• Food Irradiation·Food Science • Previous Articles     Next Articles

Optimizing Conditions for Protoplasts Preparation and Regeneration of Agaricus bisporus Portobello

WANG Jinbin1,2,**, LI Wen1,**, JIANG Wei1, BAI Lan1, LIU Hua1, LI Zhengpeng1, WU Wenhui2, TANG Xueming1,2,*   

  1. 1 Key Lab of Agricultural Genetic Breeding of Shanghai/Shanghai Academy of Agricultural Sciences, Shanghai 201106;
    2 College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306;
  • Received:2017-08-02 Online:2019-02-10 Published:2018-12-20

褐色双孢蘑菇原生质体制备、再生条件的研究

王金斌1,2,**, 李文1,**, 蒋玮1, 白蓝1, 刘华1, 李正鹏1, 吴文惠2, 唐雪明1,2,*   

  1. 1 上海市农业科学院/上海市农业遗传育种重点实验室,上海 201106;
    2 上海海洋大学食品学院,上海 201306;
  • 通讯作者: *唐雪明,男,研究员,主要从事生物技术研究。E-mail: xueming70@foxmail.com
  • 作者简介:王金斌,男,助理研究员,主要从事食品安全与检测技术研究。E-mail:wangjinbin2013@126.com;李文,女,助理研究员,主要从事功能食品技术研发和利用研究。E-mail:liwen@saas.sh.cn。**同为第一作者。
  • 基金资助:
    上海市重点攻关项目[沪农科攻字(2015)第6-1-5号],上海市农业科学院卓越团队[农科创2017(13-07)]

Abstract: In order to improve the yield and regeneration rate of Agaricus bisporus Portabello protoplast. Mushroom strain was ZONGXIU No.1 was selected, and two regression analysis with Design-Expert 8.0.6 software were used to optimize the preparation conditions of protoplast. The results showed that the yield of protoplast was arrived at the highest with 4.39×107·mL-1 in the condition of lywallzyme concentration1.35%, snailase concentration 7.01%, enzymolysis time 3.00 h, mycelium age 8.36 days and enzymolysis temperature 30.4℃. The highest protoplast regeneration rate with 15.1% was found in the condition of lywallzyme concentration 1.32%, snailase concentration 6.96%, enzymolysis time 2.97 h, mycelium age 9.00 days and enzymolysis temperature 31.0℃. The ultraviolet mutagenesis and chemical mutagenesis were used to mutate the protoplast growth characteristic, and eighteen strains with improved growth characteristics were obtained. The results could provide theoretical basis for protoplasts transformation, genome rearrangement and genome targeted editing for Agaricus bisporus Portobello.

Key words: Agaricus bisporus Portobello, protoplast, regeneration, response surface methodology, mutagenesis

摘要: 为了提高褐色双孢蘑菇原生质体产量和再生率,本研究以褐色双孢蘑菇棕秀一号为材料,利用Design-Expert 8.0.6软件进行二次回归分析对褐色双孢蘑菇制备原生质体的条件进行优化。结果表明,原生质体最佳制备条件为溶壁酶1.35%、蜗牛酶7.01%、酶解温度30.4℃、褐色双孢蘑菇菌丝体菌龄8.36 d、酶解时间3.00 h,在此条件下原生质体产量高达4.39×107 个·mL-1;当溶壁酶含量为1.32%、蜗牛酶含量为6.96%、褐色双孢蘑菇菌丝体菌龄9.00 d、酶解时间2.97 h、酶解温度31.0℃时,原生质体的再生率最高(15.1%)。利用紫外诱变和化学诱变技术对褐色双孢蘑菇原生质体进行生长特性诱变,筛选获得了18 株生长速率提高的突变菌株。本研究为褐色双孢蘑菇在遗传转化、基因组重排、基因编辑技术等后续分子遗传学研究提供了理论基础。

关键词: 褐色双孢蘑菇, 原生质体, 再生, 响应面分析法, 诱变