生物技术通报 ›› 2018, Vol. 34 ›› Issue (7): 166-173.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0160

• 研究报告 • 上一篇    下一篇

大丽轮枝菌原生质体的制备及再生

赵小强, 陈志荣, 何芳, 沈楠, 高峰, 黄家风   

  1. 石河子大学农学院 农业部西北荒漠绿洲作物有害生物综合治理重点实验室,石河子 832003
  • 收稿日期:2018-02-24 出版日期:2018-07-26 发布日期:2018-08-01
  • 作者简介:赵小强,男,硕士研究生,研究方向:分子植物病理学;E-mail:1963458511@qq.com
  • 基金资助:
    国家自然科学基金项目(31760497,31560494),农业部西北荒漠绿洲作物有害生物综合治理重点实验室开放课题(KFJJ20170104)

Preparation and Regeneration of Protoplast from Verticillium dahliae

ZHAO Xiao-qiang, CHEN Zhi-rong, HE Fang, SHEN Nan, GAO Feng, HUANG Jia-feng   

  1. College of Agriculture,Shihezi University/ Key Laboratory of Integrated Pest Management on Crop in Northwestern Oasis,Ministry of Agriculture,Shihezi 832003
  • Received:2018-02-24 Published:2018-07-26 Online:2018-08-01

摘要: 为建立大丽轮枝菌(Verticillium dahliae)原生质体的制备和再生体系,采用单因素分析法分析了大丽轮枝菌摇培时间、裂解酶浓度、酶解时间和温度、渗透压稳定剂的种类和浓度及pH对原生质体释放的影响;从再生培养基、培养基Agar浓度和酶解时间对原生质体的再生条件进行了优化;并对优化条件下获得的原生质体进行了GFP瞬时表达。结果表明,将分生孢子培养18 h收集菌丝体,以1.2 mol/L的KCl作为渗透压稳定剂,在pH 6.0的条件下,加入10 mg/mL的裂解酶,30℃酶解4 h时,获得的原生质体产量最高,达到3.3×107个/mL;在Agar浓度为0.5%的TB3再生培养基中进行原生质体再生,再生率最高,可达22.45%;GFP瞬时表达结果表明,优化条件下获得的原生质体可用于遗传转化材料。

关键词: 大丽轮枝菌, 原生质体制备, 再生条件, 转化

Abstract: In order to establish optimal conditions on preparation and regeneration of protoplast from Verticillium dahlia,the single-factor experiment was used to analyze the effects of culture time,concentration of lyase,enzymatic digesting time and temperature,type and concentration of osmotic stabilizer,and pH on the release of protoplasts. The conditions of regeneration medium,agar concentration,and enzymatic lysing time on protoplast regeneration were optimized. In order to observe protoplast visually,the recombinant plasmid containing GFP was transformed by PEG-CaCl2-mediated method. The results showed that the yield of protoplast was the highest with 3.3×107cells/mL under the optimal conditions:collecting the mycelia when conidia cultured for 18 h,using 1.2 mol/L KCl as osmotic stabilizer,then adding 10 mg/mL lyase at pH 6.0,and lysing for 4 h at 30℃. Protoplast regeneration was achieved in TB3 medium with 0.5% agar,and the regeneration rate was up to 22.45%. The result from GFP transient-expression indicated that the protoplasts under optimal conditions may be used for genetic transformation.

Key words: Verticillium dahliae, protoplast preparation, protoplast regeneration, transformation