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建兰茎腐病原菌尖孢镰刀菌F-02的绿色荧光蛋白基因标记

姚锦爱 张鸿 黄鹏 陈峰 余德亿

姚锦爱, 张鸿, 黄鹏, 陈峰, 余德亿. 建兰茎腐病原菌尖孢镰刀菌F-02的绿色荧光蛋白基因标记[J]. 福建农业学报, 2019, 34(1): 70-75. doi: 10.19303/j.issn.1008-0384.2019.01.011
引用本文: 姚锦爱, 张鸿, 黄鹏, 陈峰, 余德亿. 建兰茎腐病原菌尖孢镰刀菌F-02的绿色荧光蛋白基因标记[J]. 福建农业学报, 2019, 34(1): 70-75. doi: 10.19303/j.issn.1008-0384.2019.01.011
YAO Jin-ai, ZHANG Hong, HUANG Peng, CHEN Feng, YU De-yi. Green Fluorescent Protein Genetic Marker of Fusarium oxysporum F-02 of Stem Rot Disease on Cymbidium ensifolium[J]. Fujian Journal of Agricultural Sciences, 2019, 34(1): 70-75. doi: 10.19303/j.issn.1008-0384.2019.01.011
Citation: YAO Jin-ai, ZHANG Hong, HUANG Peng, CHEN Feng, YU De-yi. Green Fluorescent Protein Genetic Marker of Fusarium oxysporum F-02 of Stem Rot Disease on Cymbidium ensifolium[J]. Fujian Journal of Agricultural Sciences, 2019, 34(1): 70-75. doi: 10.19303/j.issn.1008-0384.2019.01.011

建兰茎腐病原菌尖孢镰刀菌F-02的绿色荧光蛋白基因标记

doi: 10.19303/j.issn.1008-0384.2019.01.011
基金项目: 

福建省科技计划项目——省属公益类科研院所基本科研专项 2018R1024-2

福建省财政专项——福建省农业科学院科技创新团队建设项目 STIT2017-2-2

详细信息
    作者简介:

    姚锦爱(1978-), 女, 副研究员, 主要从事植病生防方面的研究(E-mail:yaoja@163.com)

    通讯作者:

    余德亿(1972-), 男, 研究员, 主要从事病虫害生物防治方面的研究(E-mail:yudy_2004@126.com)

  • 中图分类号: S436.8

Green Fluorescent Protein Genetic Marker of Fusarium oxysporum F-02 of Stem Rot Disease on Cymbidium ensifolium

  • 摘要:   目的  开展建兰茎腐病原菌尖孢镰刀菌F-02的绿色荧光蛋白标记研究,直观观察尖孢镰刀菌在建兰植株上的侵染为害,明确病原菌的致病过程,为开展建兰茎腐病原菌的相关研究提供良好技术手段。  方法  采用农杆菌介导法对建兰茎腐病原菌尖孢镰刀菌F-02进行绿色荧光蛋白(GFP)转化,并检测转化子的遗传稳定性。  结果  GFP基因可被成功导入到尖孢镰刀菌F-02中并获得表达,转化子在蓝色光源激发下,菌丝和分生孢子均能稳定散发绿色荧光;转化子10次继代培养后菌落生长良好,菌丝和分生孢子仍可稳定散发出绿色荧光,对寄主植物的致病力也未发生改变,  结论  GFP基因可在建兰茎腐病原菌尖孢镰刀菌F-02中稳定存在,并对其致病力没有影响。
  • 图  1  菌株F-02与农杆菌共培养后在混合纤维微孔滤上的生长情况

    Figure  1.  Co-culture of F-02 and agrobacterium on mixed fiber microporous membrane

    图  2  转化子在筛选培养基上生长形态

    Figure  2.  Morphology of transformants on selective medium

    图  3  尖孢镰刀菌F-02的绿色荧光表达

    注:A、B分别为荧光显微镜下观察菌丝和分生孢子。

    Figure  3.  GFP-expressing F.oxysporum F-02

    Note: A, B: Mycelia and conidia observed of fluorescence microscope.

    图  4  GFP标记的尖孢镰刀菌F-02侵染植株特征

    注:A为侵染植株假鳞茎,B、C为菌丝侵染植株表皮。

    Figure  4.  Morphologies of plant infected by GFP-expressing F.oxysporum F-02

    Note: A: infected pseudobulb of the plant, B-C: Mycelia infected epidermis of the plant.

    表  1  农杆菌转化所需储存液及培养基的配制

    Table  1.   Ingredients in stock solutions and media used for agrobacterium transformation

    制剂
    Reagent
    100 mL储存液Stock solution to 100 mL 100 mL培养基所需药品用量Amount required to make 100 mL
    药品
    Chemical
    用量
    Amount required
    MM液体培养基
    MM liquid medium
    IM液体培养基
    IM liquid medium
    CM固体培养基
    CM solid medium
    K-缓冲液K-buffer (pH 7.0) K2HPO4 20 g 1 mL 1 mL 1 mL
    KH2PO4 14.5 g
    M-N缓冲液M-N buffer MgSO4·7H2O 3 g 2 mL 2 mL 2 mL
    NaCl 1.5 g
    1%二水合氯化钙1% CaCl2·2H2O CaCl2·2H2O 1 g 0.1 mL 0.1 mL 0.1 mL
    微量元素Spore elements ZnSO4·7H2O 0.01 g 1 mL 1 mL 1 mL
    CuSO4·5H2O 0.01 g
    H3BO3 0.01 g
    MnSO4·H2O 0.01 g
    Na2MoO4·2H2O 0.01 g
    20%硝酸铵20% NH4NO3 NH4NO3 20 g 0.25 mL 0.25 mL 0.25 mL
    20%葡萄糖20% Glucose Glucose 20 g 1 mL 1 mL 1 mL
    0.01%硫酸铁0.01% FeSO4 FeSO4 0.01 g 1 mL 1 mL 1 mL
    50%甘油50% Glycerol Glycerol 50 mL - 1 mL 1 mL
    1 mol·L-1乙磺酸1 mol·L-1 MES (pH 5.3) MES 21.32 g - 4 mL 4 mL
    卡那霉素Kanamycin stock 0.15 mL 0.15 mL 0.15 mL
    乙酰丁香酮Acetosyringone - 0.2 mL 0.2 mL
    琼脂Agar 1.5g
    去离子水Sterile H2O 93.5 mL 88.3 mL 88.3 mL
    下载: 导出CSV
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出版历程
  • 收稿日期:  2018-10-19
  • 修回日期:  2018-11-22
  • 刊出日期:  2019-01-28

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