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苦瓜枯萎病原菌的绿色荧光蛋白基因标记

陈燕萍 刘欣 肖荣凤 朱育菁 林永胜 刘波

陈燕萍,刘欣,肖荣凤,等. 苦瓜枯萎病原菌的绿色荧光蛋白基因标记 [J]. 福建农业学报,2020,35(11):1228−1233 doi: 10.19303/j.issn.1008-0384.2020.11.008
引用本文: 陈燕萍,刘欣,肖荣凤,等. 苦瓜枯萎病原菌的绿色荧光蛋白基因标记 [J]. 福建农业学报,2020,35(11):1228−1233 doi: 10.19303/j.issn.1008-0384.2020.11.008
CHEN Y P, LIU X, XIAO R F, et al. Transformation of Green Fluorescent Protein of Fusarium oxysporum Isolated from Diseased Bitter Gourd [J]. Fujian Journal of Agricultural Sciences,2020,35(11):1228−1233 doi: 10.19303/j.issn.1008-0384.2020.11.008
Citation: CHEN Y P, LIU X, XIAO R F, et al. Transformation of Green Fluorescent Protein of Fusarium oxysporum Isolated from Diseased Bitter Gourd [J]. Fujian Journal of Agricultural Sciences,2020,35(11):1228−1233 doi: 10.19303/j.issn.1008-0384.2020.11.008

苦瓜枯萎病原菌的绿色荧光蛋白基因标记

doi: 10.19303/j.issn.1008-0384.2020.11.008
基金项目: 福建省科技计划公益类专项(2018R1017-9);福建省农业科学院科技创新团队建设项目(STIT2017-2-8)
详细信息
    作者简介:

    陈燕萍(1985−),女,硕士,助理研究员,主要从事生物技术及生物防治(E-mail:chenyanping071@sina.com

    通讯作者:

    刘波(1957−),男,博士,研究员,主要从事微生物生物技术与农业生物药物研究(E-mail:fzliubo@163.com

  • 中图分类号: S 436.8

Transformation of Green Fluorescent Protein of Fusarium oxysporum Isolated from Diseased Bitter Gourd

  • 摘要:   目的  苦瓜枯萎病是由尖孢镰刀菌苦瓜专化型(Fusarium oxysporum f. sp. momodicae)侵染引起的一种重要土传病害。为有效防控该病害,对其病原菌进行绿色荧光蛋白基因(gfp)标记,为研究病原菌在苦瓜植株体内的侵染特性提供可视化跟踪检测手段。  方法  采用农杆菌介导的遗传转化方法对苦瓜枯萎病原菌强致病性野生型菌株FJAT-3018进行绿色荧光蛋白基因(gfp)标记,通过对转化子的菌落形态观察、生长速率和致病性测定,筛选出遗传稳定的转化子。  结果  野生型菌株FJAT-3018的转化效率约为14.5个转化子/106个孢子。经10次继代培养,筛选获得的转化子在菌落形态、生长速率和致病性方面与野生型菌株FJAT-3018无明显差异,gfp基因在转化子的菌丝体和分生孢子中均能强表达;通过激光共聚焦显微镜扫描跟踪发现,转化子能够在苦瓜植株根部和茎部侵染与定殖。  结论  绿色荧光蛋白基因已成功转入到苦瓜野生型菌株FJAT-3018中,其转化子具有良好的遗传稳定性且致病力不受影响。
  • 图  1  转化子的生物学特性

    注 A:野生型菌株FJAT-3018菌落形态;B:转化子FJAT-31290菌落形态;C:转化子菌落在蓝光切胶仪下发绿色荧光状态;D-F:转化子的菌丝、产孢细胞和分生孢子在荧光显微镜下发绿色荧光状态。

    Figure  1.  Biological characteristics of transformants

    Note: A:colony morphology of FJAT-3018; B:colony morphology of transformant, FJAT-31290; C:colony morphology of transformants under blue light; D-F:gfp gene expressions in hyphae, conidiogenous cell, and conidia, respectively, of transformants under fluorescence microscope.

    图  2  绿色荧光蛋白基因标记转化子与野生型菌株的致病性测定

    注 A:清水对照;B:转化子FJAT-31284; C:转化子FJAT-31290;D:野生型菌株FJAT-3018

    Figure  2.  Pathogenicity of transformants containing gfp and wild type FJAT-3018

    Note: A: control with water; B: transformant FJAT-31284; C: transformant FJAT-31290; D: wild-type strain FJAT-3018.

    图  3  利用激光共聚焦观察转化子在苦瓜根部和茎部的侵染

    注:A、B:转化子FJAT-31284和FJAT-31290分别在苦瓜根部组织的侵染;C、D:转化子FJAT-31284和FJAT-31290分别在苦瓜茎部组织的侵染。

    Figure  3.  Root and stem tissues of transformant-infected bitter gourd under confocal laser scanning microscope

    Note: A and B:hyphae inside roots of bitter gourd inoculated with transformants FJAT-31284 and FJAT-31290, respectively; C and D:hyphae inside stems of bitter gourd inoculated with transformants FJAT-31284 and FJAT-31290, respectively.

    表  1  农杆菌转化所需的试剂及培养基配方

    Table  1.   Preparation of stock solutions and media for Agrobacterium-mediated transformation

    试剂名称
    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 20.00 g 1.00 mL 1.00 mL 1.00 mL
    KH2PO4 14.50 g
    M-N缓冲液 M-N buffer MgSO4.7H2O 3.00 g 2.00 mL 2.00 mL 2.00 mL
    NaCl 1.50 g
    1%氯化钙(二水)1% CaCl2·2H20 CaCl2.2H2O 1.00 g 0.10 mL 0.10 mL 0.10 mL
    微量元素 Spore elements ZnSO4.7H2O 0.01 g 1.00 mL 1.00 mL 1.00 mL
    CuSO4.5H2O 0.01 g
    H3BO3 0.01 g
    MnSO4.H2O 0.01 g
    NaMoO4.2H2O 0.01 g
    20%硝酸铵 20% NH4NO3 NH4NO3 20.00 g 0.25 mL 0.25 mL 0.25 mL
    20%葡萄糖 20% Glucose Glucose 20.00 g 1.00 mL 1.00 mL 1.00 mL
    0.01%硫酸铁 0.01% FeSO4 FeSO4 00.01 g 1.00 mL 1.00 mL 1.00 mL
    50%甘油 50% Glycerol Glycerol 50.00 mL 1.00 mL 1.00 mL
    1 mol·L−1 2-吗啉乙磺酸(pH 5.3)1 mol·L−1 MES(pH 5.3) MES 21.32 g 4.00 mL 4.00 mL
    卡那霉素 Kanamycin stock Kanamycin stock 0.15 mL 0.15 mL 0.15 mL
    乙酰丁香酮 Acetosyringone Acetosyringone 0.20 mL 0.20 mL
    琼脂 Agar Agar 1.50 g
    去离子水 Deionized H20 Deionized H2O 93.50 mL 88.30 mL 88.30 mL
    注:表中葡萄糖、FeSO4、卡那霉素和MES采用0.22 μm微孔过滤器灭菌,其他试剂于120 ℃高压灭菌20 min。FeSO4,卡那霉素,乙酰丁香酮和MES储存在−20 ℃冰箱,其他试剂均储存在4 ℃冰箱。
    Note: Glucose, FeSO4, kanamycin, and MES were separately sterilized by filtration through a 0.22 μm filter. Other reagents were autoclaved for 20 min at 120 ℃. Reagents were stored at 4 ℃, except −20 ℃ for FeSO4, kanamycin, acetosyringone, and MES.
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  • [1] SUN S K, HUANG J W. A new Fusarium wilt of bitter gourd in Taiwan [J]. Plant Disease, 1983, 67(2): 226−227. doi: 10.1094/PD-67-226
    [2] 关峰, 万新建, 张景云, 等. 苦瓜枯萎病研究进展 [J]. 中国瓜菜, 2018, 31(5):1−4. doi: 10.3969/j.issn.1673-2871.2018.05.001

    GUAN F, WAN X J, ZHANG J Y, et al. Research progress on Fusarium wilt of bitter gourd [J]. China Cucurbits and Vegetables, 2018, 31(5): 1−4.(in Chinese) doi: 10.3969/j.issn.1673-2871.2018.05.001
    [3] SARROCCO S, FALASCHI N, VERGARA M, et al. Use of Fusarium oxysporum f. sp. dianthi transformed with marker genes to follow colonization of carnation roots [J]. Journal of Plant Pathology, 2007, 89(1): 47−54.
    [4] 黄亚丽, 潘玮. 根癌农杆菌介导丝状真菌遗传转化的研究进展 [J]. 生物技术通报, 2007(3):111−114. doi: 10.3969/j.issn.1002-5464.2007.03.024

    HUANG Y L, PAN W. Transformation in Filamentous fungi mediated by Agrobacterium tumefaciens [J]. Biotechnology Bulletin, 2007(3): 111−114.(in Chinese) doi: 10.3969/j.issn.1002-5464.2007.03.024
    [5] 徐进, 莫明和, 张克勤. 绿色荧光蛋白(GFP)在真菌研究中的应用 [J]. 生物技术, 2004, 14(6):74−77. doi: 10.3969/j.issn.1004-311X.2004.06.037

    XU J, MO M H, ZHANG K Q. The application of gene fluorescent protein (GFP) in Fungi [J]. Biotechnology, 2004, 14(6): 74−77.(in Chinese) doi: 10.3969/j.issn.1004-311X.2004.06.037
    [6] 肖荣凤, 朱育菁, 李燕丹, 等. 西瓜尖孢镰刀菌FOV-135的绿色荧光蛋白基因转化 [J]. 福建农业学报, 2009, 24(6):521−524. doi: 10.3969/j.issn.1008-0384.2009.06.007

    XIAO R F, ZHU Y J, LI Y D, et al. Green fluorescent protein gene transformation on Fusarium oxysporum f. sp. niveum strain, FOV-135 [J]. Fujian Journal of Agricultural Sciences, 2009, 24(6): 521−524.(in Chinese) doi: 10.3969/j.issn.1008-0384.2009.06.007
    [7] 张鸿, 林志坚, 林赵淼, 等. T-DNA随机插入法获得甘薯蔓割病菌非致病生防菌株 [J]. 中国生物防治学报, 2016, 32(5):610−618.

    ZHANG H, LIN Z J, LIN Z M, et al. Obtaining nonpathogenic biological control strains against sweetpotato Fusarium wilt by Agrobacterium-mediated transformation [J]. Chinese Journal of Biological Control, 2016, 32(5): 610−618.(in Chinese)
    [8] 孙科. 基因枪法介导的抗赤霉病防卫基因转化小麦的研究[D]. 武汉: 华中农业大学, 2013.

    SUN K. Studies on the transformation of wheat with defense genes resistant to FHB by microprojectile bombardment[D]. Wuhan: Hua zhong Agricultural University, 2013. (in Chinese)
    [9] 张俊华, 刘烨, 韩雨桐, 等. 农杆菌介导稻瘟病菌绿色荧光蛋白(GFP)遗传转化研究 [J]. 东北农业大学学报, 2014, 45(11):1−7. doi: 10.3969/j.issn.1005-9369.2014.11.001

    ZHANG J H, LIU Y, HAN Y T, et al. GFP genetic transformation of Magnaporthe grisea mediated by Agrobacterium tumefaciens [J]. Journal of Northeast Agricultural University, 2014, 45(11): 1−7.(in Chinese) doi: 10.3969/j.issn.1005-9369.2014.11.001
    [10] NAHALKOVA J, FATEHI J. Red fluorescent protein (DsRed2) as a novel reporter in Fusarium oxysporumf. sp. lycopersici [J]. FEMS Microbiology Letters, 2003, 225(2): 305−309. doi: 10.1016/S0378-1097(03)00534-2
    [11] VISSER M., GORDON T R. WINGFIELD B D, et al Transformation of Fusarium oxysporum f. sp. cubense causal agent of Fuasrium wilt of banana, with the green fluorescent protein (GFP) gene [J]. Australasian Plant Pathology, 2004, 33(1): 69−75. doi: 10.1071/AP03084
    [12] 张欣. 香蕉枯萎病菌遗传多态性及绿色荧光蛋白基因转化的研究[D]. 儋州: 华南热带农业大学, 2007.
    [13] NONOMURA T, TAJIMA H, KITAGAWA Y, et al. Distinguishable staining with neutral red for GFP-marked and GFP-nonmarked Fusarium oxysporum strains simultaneously colonizing root surfaces [J]. Journal of General Plant Pathology, 2003, 69(1): 45−48. doi: 10.1007/s10327-002-0018-7
    [14] WU L, CONNER R L, WANG X M, et al. Variation in growth, colonization of maize, and metabolic parameters of GFP- and DsRed-Labeled Fusarium verticillioides strains [J]. Phytopathology, 2016, 106(8): 890−899.
    [15] KHANG C H, PARK S Y, RHO H S, et al. Filamentous fungi (Magnaporthe grisea and Fusarium oxysporum)[M]//Agrobacterium Protocols Volume 2. Totowa, NJ: Humana Press, 2006: 403-420.
    [16] 张旭, Theo van de Lee, 陆维忠, 等. 小麦赤霉菌绿色荧光蛋白标记突变体的侵染研究 [J]. 中国农业科学, 2008, 41(10):3077−3082. doi: 10.3864/j.issn.0578-1752.2008.10.021

    ZHANG X, THEO V D L, LU W Z, et al. Infection of Fusarium graminearum on wheat spikes with green fluorescence protein-tagged revertants [J]. Scientia Agricultura Sinica, 2008, 41(10): 3077−3082.(in Chinese) doi: 10.3864/j.issn.0578-1752.2008.10.021
    [17] 姚锦爱, 张鸿, 黄鹏, 等. 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.

    YAO J A, ZHANG H, HUANG P, et al. 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.(in Chinese)
    [18] 张俊华, 牟明, 常浩, 等. 根癌农杆菌介导gfp基因转化水稻纹枯病菌及其对病原菌稳定性和致病力的影响 [J]. 东北农业科学, 2016, 41(6):67−74.

    ZHANG J H, MU M, CHANG H, et al. Agrobacterium Tumefaciens-mediated transformation of gfp gene and its effects on stability and pathogenicity for Rhizoctonia solani [J]. Journal of Northeast Agricultural Sciences, 2016, 41(6): 67−74.(in Chinese)
    [19] 任俊杰, 王丽霞, 高洪波, 等. 农杆菌介导的西瓜枯萎病菌遗传转化 [J]. 植物保护, 2015, 41(1):93−97. doi: 10.3969/j.issn.0529-1542.2015.01.018

    REN J J, WANG L X, GAO H B, et al. Agrobacterium tumefaciens-mediated transformation of Fusarium oxysporum f. sp. niveum [J]. Plant Protection, 2015, 41(1): 93−97.(in Chinese) doi: 10.3969/j.issn.0529-1542.2015.01.018
    [20] 刘朋娟, 王政逸, 王秋华, 等. 农杆菌介导的稻瘟病菌转化及致病缺陷突变体筛选 [J]. 中国水稻科学, 2006, 20(3):231−237. doi: 10.3321/j.issn:1001-7216.2006.03.001

    LIU P J, WANG Z Y, WANG Q H, et al. Agrobacterium tume f aciens-mediated transformation of Magnaporthe grisea and identification of pathogenicity defective mutant [J]. Chinese Journal of Rice Science, 2006, 20(3): 231−237.(in Chinese) doi: 10.3321/j.issn:1001-7216.2006.03.001
    [21] 郭强, 王鑫, 徐世强, 等. 农杆菌介导甘蔗梢腐病病原菌YN41的遗传转化 [J]. 基因组学与应用生物学, 2016, 35(5):1189−1194.

    GUO Q, WANG X, XU S Q, et al. Agrobacterium tumefaciens-mediated transformation of sugarcane pokkah boeng pathogen YN41 [J]. Genomics and Applied Biology, 2016, 35(5): 1189−1194.(in Chinese)
    [22] ZVIRIN T, HERMAN R, BROTMAN Y, et al. Differential colonization and defence responses of resistant and susceptible melon lines infected by Fusarium oxysporum race 1·2 [J]. Plant pathology, 2010(59): 576−585.
    [23] 李春强, 梁慧施, 夏亦荠, 等. GFP标记的尖孢镰刀菌西瓜专化型侵染西瓜过程观察 [J]. 热带作物学报, 2011, 32(10):1935−1939. doi: 10.3969/j.issn.1000-2561.2011.10.029

    LI C Q, LIANG H S H, XIAY J, et al. Observation of the infection process of watermelon by Fusarium oxysporum f. sp. niveum using the GFP marker [J]. Chinese Journal of Tropical Crops, 2011, 32(10): 1935−1939.(in Chinese) doi: 10.3969/j.issn.1000-2561.2011.10.029
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  • 收稿日期:  2020-09-14
  • 修回日期:  2020-10-14
  • 网络出版日期:  2020-11-13
  • 刊出日期:  2020-11-30

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