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

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

福建省农业科学院农业生物资源研究所，福建　福州　350003

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
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出版历程
- 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

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

Institute of Agricultural Biological Resource Research, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350003, China

摘要

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。
- 苦瓜 /
- 枯萎病 /
- 绿色荧光蛋白 /
- 基因标记
Abstract: Objective As a genetic marker, the green fluorescent protein gene (gfp) of Fusarium oxysporum f. sp. momodicae (Fom), one of the most serious fungal pathogens that caused the fusarium wilt on bitter gourd, was identified and transformed into bacterium to facilitate the study on the infection process and control of the disease. Method A highly pathogenic wild-type strain, FJAT-3018, isolated from diseased bitter gourds was transformed with the gene encoding gfp using an Agrobacterium-mediated method. The genetically stable transformants, FJAT-31290 and FJAT-31284, were verified by their colony morphology, growth rate, and pathogenicity. Result The efficiency of transforming FJAT-3018 was approximately 14.5 transformants per 10⁶ spores. After 10 generations of subculture, the transformants did not significantly differ from the wild-type strain with respect to the colony morphology, growth rate, and pathogenicity. Strong constitutive expression of gfp could be seen in the fungal hyphae and conidia of the transformants. In addition, under a confocal laser scanning microscope, the transformant-infected bitter gourd plants showed growth of the fungal hyphae inside the roots and stem xylem. Conclusion The gfp gene had been successfully transformed into FJAT-31290 and FJAT-31284 with genetic stability and without apparent pathogenicity deviation from the highly pathogenic wild-type strain FJAT-3018.
- Bitter gourd /
- Fusarium oxysporum /
- green fluorescent protein /
- genetic marker

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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.

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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.

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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.

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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
试剂名称 Reagent	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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