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

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

姚锦爱^{1, 2,},
张鸿³,
黄鹏¹,
陈峰¹,
余德亿^{1, 2, ,}

1.
福建省农业科学院植物保护研究所, 福建福州 350013
2.
福建省作物有害生物监测与治理重点实验室, 福建福州 350003
3.
福建省农业科学院作物研究所, 福建福州 350013

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
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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

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

YAO Jin-ai^{1, 2
,},
ZHANG Hong³,
HUANG Peng¹,
CHEN Feng¹,
YU De-yi^{1, 2
, ,}

1.
Institute of Plant Protection, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350013, China
2.
Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, Fuzhou, Fujian 350003, China
3.
Institute of Crop Sciences, Fujian Academy of Agricultural Science, Fuzhou, Fujian 350013, China

摘要

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。
- 建兰 /
- 茎腐病 /
- 尖孢镰刀菌 /
- 绿色荧光蛋白 /
- 基因标记
Abstract: Objective Green fluorescent protein(GFP) genetic marker of Fusarium oxysporum F-02 of stem rot disease on Cymbidium ensifolium were conducted in this study in order to observe the inflection of F. oxysporum on C. ensifolium plants, confirm the process of the pathogen, and provide powerful technic for further studies on F. oxysporum. Method GFP was transformed into F.oxysporum strain F-02 mediated by Agrobacterium, and the genetic stability of GFP in transformants was investigated. Result GFP gene can be successfully introduced into F.oxysporum and stable green fluorescence can be produced in the hypha and spores under the excitation of blue light source.After 10 successive transfers, the progenies grew well with stable green fluorescence and similar pathogenicity as parental transfrormant. Conclusion GFP gene was inherited stablely in C.ensifolinm and no effect on pathogenicity in F-02 strain.
- Cymbidium ensifolium /
- stem rot /
- Fusarium oxysporum /
- green fluorescent protein /
- genetic marker

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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

下载: 全尺寸图片幻灯片

图 2 转化子在筛选培养基上生长形态

Figure 2. Morphology of transformants on selective medium

下载: 全尺寸图片幻灯片

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.

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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.

下载: 全尺寸图片幻灯片

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
制剂 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 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

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参考文献(20)

[1]	YAO J A, HUANG P, LAN C Z, et al.Stem rot on Cymbidium ensifolium (Orchidaceae) caused by Fusarium oxysporum in China[J]. Canadian Journal of Plant Pathology, 2018, 40 (1): 105-108. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=10.1080/07060661.2017.1411976
[2]	姚锦爱, 黄鹏, 陈峰, 等.建兰茎腐病原菌尖孢镰刀菌的生物学特性研究[J].福建农业学报, 2018, 33(2): 190-194. doi: 10.19303/j.issn.1008-0384.2018.02.015 YAO J A, HUANG P, CHEN F, et al.Biological Characteristics of Fusarium oxysporum, a Pathogen of Stem Rot on Cymbidium ensifolium[J]. Fujian Journal of Agricultural Sciences, 2018, 33(2): 190-194.(in Chinese) doi: 10.19303/j.issn.1008-0384.2018.02.015
[3]	肖荣凤, 朱育菁, 李燕丹, 等.西瓜尖孢镰刀茵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 Q, 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
[4]	BOTTIN A, LARCHE L, VILLALBA F, et al.Green fluorescent protein (gfp) as gene expression reporter and vital marker for studying development and microbe-plant interaction in the tobacco pathogen Phythophthora parasitica var.nicotianae[J]. FEMS Microbiology Letters, 1999, 176: 51-56. doi: 10.1111/fml.1999.176.issue-1
[5]	WEST P V, REID B, CAMPBELL T A, et al.Green fluorescent protein (gfp) as a reporter gene for the plant pathogenic oomycete Phytophthora palmivora[J]. FEMS Microbiology Letters, 1999, 178: 71-80. doi: 10.1111/fml.1999.178.issue-1
[6]	NAHALKOVA J, FATEHI J.Red fluorescent protein (DsRed2) as a novel reporter in Fusarium oxysporum f.sp. 1ycopersici[J]. FEMS Microbiology Letters.2003, 225: 305-309. doi: 10.1016/S0378-1097(03)00534-2
[7]	VISSER M, GORDON T R, WINGFIELD B D, et al.Transformation of Fusarium oxysporum f.sp cubense, causal agent of Fusarium wilt of banana, with the green fluorescent protein (GFP) gene[J]. Australasian Plant Pathology, 2004, 33: 69-75. doi: 10.1071/AP03084
[8]	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[M].广州:华南热带农业大学出版社, 2007. ZHANG X.Studies on Genetic Diversity and Transformation of the Green Fluorescent Protein Gene in Fusarium Oxysporum f.sp. Cubense, Causal Agent of Banana Fusarium Wilt[M]. Guangzhou: South China Agicultural University Press, 2007.(in Chinese)
[9]	NONOMURA T, TAJIMA H, KITAGAWA Y, et al.Distinguishable staining with neutral red for GFP-marked and GFP-nonmarked Fusarium oxysporum strains sim-ultaneously colonizingroot surfaces[J]. Journal of General Plant Pathology, 2003, 69(1): 45-48. doi: 10.1007/s10327-002-0018-7
[10]	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. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=1cee1a23e666d3f298771bb7c88e57c1
[11]	KHANG C H, PARK S Y, RHO H S, et al.Filamentous fungi (Magnaporthe grisea and Fusarium oxysporum)//Agrobacterium Protocols[M]. Totowa: Humana Press, 2007:403-420.
[12]	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
[13]	许志刚.普通植物病理学(第4版)[M].北京:中国农业出版社, 2003:23-25. XU Z G, General Plant Pathology(4th Edition)[M]. Beijing: China Agriculture Press, 2003:23-25.(in Chinese)
[14]	许有嫔, 廖海澄, 陈金华, 等.利用绿色荧光蛋白GFP研究稻瘟病菌与水稻的互作[J].植物保护, 2017, 3(6): 53-61. doi: 10.3969/j.issn.0529-1542.2017.06.008 XU Y B, LIAO H C, CHEN J H, et al.Fluorescent microscopic analysis of the plant infection process of Magnaporthe oryzae using green fluorescent protein[J].Plant Protection, 2017, 3(6): 53-61.(in Chinese) doi: 10.3969/j.issn.0529-1542.2017.06.008
[15]	CAI X D, FU J, GUO W W.Mitochondrial Genome of Callus Protoplast Has a Role in Mes ophyll Protoplast Regeneration in Citrus: Evidence From Transgenic GFP Somatic Homo-Fusion[J]. Horticultural Plant Journal, 2017, 3 (5): 177-182. doi: 10.1016/j.hpj.2017.10.001
[16]	李二峰, 王殿东, 杨宇红, 等.根癌农杆菌介导甘蓝枯萎病菌的遗传转化[J].中国蔬菜, 2011, 14: 28-34. http://d.old.wanfangdata.com.cn/Periodical/zgsc201114005 LI E F, WANG D D, YANG Y H, et al.Agrobacterium Tumefaciens Mediated Transformation of Cabbage Fusarium Wilt Pathogen[J]. China Vegetables, 2011, 14: 28-34.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/zgsc201114005
[17]	李敏慧, 张荣, 姜大刚, 等.根癌农杆菌介导的香蕉枯萎病菌4号生理小种的转化[J].植物病理学报, 2009, 39(4): 405-412. http://d.old.wanfangdata.com.cn/Periodical/zwblxb200904010 LI M H, ZHANG R, JIANG D G, et al.Agrobacterium tumefaciens Mediated Transformation of Fusarium oxysporum f.sp. cubense race 4[J]. Acta Phytopathologica Sinica, 2009, 39(4): 405-412.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/zwblxb200904010
[18]	任海英, 方丽, 李岗, 等.农杆菌介导的甜瓜蔓枯病菌遗传转化体系的建立[J].生物工程学报, 2010, 26(6): 802-808. http://d.old.wanfangdata.com.cn/Periodical/swgcxb201006014 REN H Y, FANG L, LI G, et al.Transformation of Didymella bryoniae mediated by Agrobacterium tumefaciens[J]. Chinese Journal of Biotechnology, 2010, 26(6): 802-808.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/swgcxb201006014
[19]	段学清, 郑莉茹, 刘文波, 等.GFP绿色荧光法辅助筛选玉米转基因植株[J].玉米科学, 2017, 25(2): 31-38. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=ymkx201702006 DUAN X Q, ZHENG L R, LIU W B, et al.Production of Transgenic Maize Plants via the GFP Green Fluorescence-assisted Selection[J]. Journal of Maize Sciences, 2017, 25(2): 31-38.(in Chinese) http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=ymkx201702006
[20]	陈春艳, 马晖玲, 董文科, 等.CPB-LAR-GFP表达载体的构建及其在紫花苜蓿愈伤组织中的表达[J].草原与草坪, 2017, 37(2): 25-30. http://d.old.wanfangdata.com.cn/Periodical/cyycp201701006 CHEN C Y, MA H L, DONG W K, et al.Construction of CPB-LAR-GFP expression vector and its transformation in callus of alfalfa[J]. Grassland and Turf, 2017, 37(2):25-30.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/cyycp201701006