基于农杆菌介导的茄子枯萎病菌遗传转化体系建立及GFP标记菌株的构建

doi:10.3969/j.issn.1674-7968.2023.09.018

摘要
图/表
参考文献
相关文章 (10)

全文: PDF (3209 KB) HTML (1 KB)
输出: BibTeX | EndNote (RIS)

摘要由尖孢镰刀菌茄子专化型(Fusarium oxysporum f. sp. melongenae)引起的茄子(Solanum melongena)枯萎病严重制约茄子的生产,其致病机制尚不明确。本研究以茄子枯萎病菌的分生孢子为转化受体,利用携带GFP表达载体pCAMsgfp的根癌农杆菌(Agrobacterium tumefaciens)介导转化茄子枯萎病菌,建立并优化了农杆菌介导的茄子枯萎病菌遗传转化体系。结果表明,当农杆菌光密度(optical density)值为0.5时,在200 μmol/L乙酰丁香酮诱导条件下,与浓度为1×10⁶个/mL的病原菌分生孢子悬浮液等体积混合后,在共培养温度为25 ℃~28 ℃条件下共培养48 h,茄子枯萎病菌的转化效率最高可达319±10.21个转化子/10⁶个孢子。GFP的特异性引物PCR鉴定结果以及荧光观察表明GFP基因能够正常表达。本研究建立并优化了根癌农杆菌介导的茄子枯萎病菌的遗传转化体系,并获得了遗传稳定的GFP标记菌株,为病原菌与茄子互作的机制研究提供技术支撑。

	服务

	把本文推荐给朋友
	加入我的书架
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	严亚琴
	胡天华
	王五宏
	胡海娇
	魏庆镇
	汪精磊
	包崇来

关键词 ：茄子枯萎病, 尖孢镰刀菌茄子专化型, 农杆菌介导的遗传转化, GFP

Abstract：Fusarium wilt caused by Fusarium oxysporum f. sp. melongenae seriously restricts the production of eggplant (Solanum melongena). However, its pathogenic mechanism is still unclear. In this study, a transformation system of F. oxysporum f. sp. melongenae was established and optimized, in which GFP expression vector pCAMsgfp was transformed into conidia using , ATMT (Agrobacterium tumefaciens-mediated transformation) method. The results showed that a satisfactory transformation with an efficiency of 319±10.21 transformants per 1×10⁶ spores could be achieved in a system of 1×10⁶ spores per milliliter of F. oxysporum f. sp. melongenae spore suspension which were co-cultured with Agrobacterium tumefaciens suspension at a concentration of OD₆₀₀ 0.5 under the co-culture medium containing 200 μmol/mL acetosyringone at 25~28 ℃ for 48 h. PCR identification of the GFP gene and fluorescence observation showed that GFP gene could be expressed normally. In this study, the genetic transformation system of F. oxysporum f.sp. melongenae mediated by ATMT was established and optimized, and the GFP-labeled strain was successfully obtained, which could provide powerful technic for further explore the infection process and the pathogenic mechanism of F. oxysporum f. sp. melongenae.

Key words： Fusarium wilt Fusarium oxysporum f. sp. melongenae Agrobacterium tumefaciens-mediated transformation GFP

收稿日期: 2022-05-17

ZTFLH:

S182

基金资助:浙江省自然科学基金(LQ22C150007); 浙江省“十四五”农业新品种选育重大科技专项(2021C02065-1-3)

通讯作者: * baocl@mail.zaas.ac.cn

引用本文:

严亚琴, 胡天华, 王五宏, 胡海娇, 魏庆镇, 汪精磊, 包崇来. 基于农杆菌介导的茄子枯萎病菌遗传转化体系建立及GFP标记菌株的构建[J]. 农业生物技术学报, 2023, 31(9): 1980-1988.
YAN Yan-Qin, HU Tian-Hua, WANG Wu-Hong, HU Hai-Jiao, WEI Qing-Zhen, WANG Jing-Lei, BAO Chong-Lai. Establishing of the Transformation System of Fusarium oxysporum f. sp. melongenae by Agrobacterium tumefaciens-mediated Transformation (ATMT) and Constructing of GFP Labeled Strain. 农业生物技术学报, 2023, 31(9): 1980-1988.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2023.09.018 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2023/V31/I9/1980

[1] 侯甲男, 李武, 杨晓杰, 等. 2019. 棉花尖孢镰刀菌绿色荧光蛋白的转化[J].分子植物育种, 17(10): 3247-3252.
(Hou J N, Li W, Yang X J, et al.2019. Transformation of green fluorescent protein in Fusarium oxysporum of cotton[J]. Molecular Plant Breeding, 17(10): 3247-3252.)
[2] 胡海娇, 魏庆镇, 王五宏等. 2018. 茄子枯萎病研究进展[J]. 分子植物育种, 16(08): 2630-2637.
(Hu H J, Wei Q Z, Wang W H, et al.2018. Research progress on resistance mechanism of eggplant on Fusarium wilt[J]. Molecular Plant Breeding, 2018, 16(08): 2630-2637.)
[3] 胡懋, 曾杨璇, 苗华彪, 等. 2021. 根癌农杆菌介导真菌遗传转化的研究及应用[J].微生物学通报, 48(11): 4344-4363.
(Hu M, Zeng Y X, Miao H B, et al.2021. Research and application of Agrobacterium tumefaciens-mediated fungal genetic transformation[J]. Microbiology China, 48(11): 4344-4363.)
[4] 贾娜娜. 2015. 梨腐烂病菌GFP标记及其在梨组织中侵染的显微观察病原菌侵染方式[D]. 硕士学位论文, 华中农业大学, 导师: 王国平, pp. 10-13.
(Jia L L.2015. Microscopic observation of Valsa mali var. pyri marked with GFP in different pear tissues[D]. Thesis for M.S., Huazhong Agriculture University, Supervisor: Wang G P, pp. 10-13.)
[5] 梁慎, 常高正, 赵卫星, 等. 2012. 农杆菌介导尖孢镰刀菌西瓜专化型转化体系的建立及突变体筛选[J].河南农业科学, 444(01): 82-86.
(Liang S, Chang G Z, Zhao W X, et al.2012. Agrobacterium tumefaciens-mediated transformation of Fusarium oxysporum f. sp. niveumis and screening of mutants with special traits[J]. Journal of Henan Agricultural Science, 444(01): 82-86.)
[6] 刘丽媛, 朱立华, 刘力伟, 等. 2017. 层出镰刀菌T-DNA插入突变体库的构建与分析[J].河北农业大学学报, 191(01):71-75.
(Liu L Y, Zhu L H, Liu L W, et al.2017. Construction and analysis of the T-DNA insertional mutation library for Fusarium proliferatum[J]. Journal of Hebei Agricultural University, 191(01): 71-75.)
[7] 刘叶, 阿依保他•托合达白, 郭楠楠, 等. 2021. 农杆菌介导的棉花枯萎病菌转化体系的优化[J].微生物学通报, 48(09): 2991-3001.
(Liu Y, Ayibaota•T H D B, Guo N N, et al.2021. Optimization of Agrobacterium tumefaciens-mediated transformation system for Fusarium oxysporum[J]. Microbiology China, 48(09): 2991-3001.)
[8] 彭成彬, 陈美霞, 魏日凤, 等. 2021. 茶树炭疽菌分离鉴定与遗传转化体系建立[J].西南农业学报, 34(10): 2167-2173.
(Peng C B, Cheng M X, Wei R F, et al.2021. Isolation and identification of Anthrax from tea plant and establishment of genetic transformation system[J]. Southwest China Journal of Agricultural Sciences, 34(10): 2167-2173.)
[9] 宋培玲, 皇甫海燕, 史志丹, 等. 2021. 农杆菌介导油菜黑胫病菌遗传转化[J]. 中国油料作物学报, 186(02): 293-299.
(Song P L, Huangfu H Y,Shi Z D, et al.2021. Agrobacterium tumefaciens-mediated transformation of Leptosphaeria biglobosa[J]. Chinese Journal of Oil Crop Sciences, 186(02): 293-299.)
[10] 张磊, 郭燕, 王云, 等. 2018. 香蕉枯萎病菌TR4原生质体转化和基因敲除体系构建[J]. 植物病理学报, 48(01): 137-140.
(Construction of PEG-mediated genetic transformation and gene knockout system in Fusarium oxysporum f. sp. cubense TR4[J]. Acta Phytopathologica Sinica, 48(1): 137-140.)
[11] 朱磊. 2011. 茄科尖孢镰刀菌绿色荧光蛋白基因转化体系的优化及其定殖特性的研究[D]. 硕士学位论文, 福建农林大学, 导师: 刘波. pp. 15-19.
(Zhu L.2011. The research on the optimized transformation system of the green fluorescent protein gene to Fusarium oxysporum from Solanaceae crop and its' colonization characteristics[D]. Thesis for M.S., Fujian Agriculture and Forestry University, Supervisor: Liu B, pp. 15-19.)
[12] Andargie M, Li L, Feng A, et al.2015. Colonization of rice roots by a green fluorescent protein-tagged isolate of Ustilaginoidea virens[J]. American Journal of Plant Sciences, 6(14): 2272-2279.
[13] Aragona M, Campos-Soriano L, Piombo E, et al.2021. Imaging the invasion of rice roots by the bakanae agent Fusarium fujikuroi using a GFP-tagged isolate[J]. European Journal of Plant Pathology, 161(1): 25-36.
[14] Armesto C, Maia F G M, Abreu M S D, et al.2012. Genetic transformation with the gfp gene of Colletotrichum gloeosporioides isolates from coffee with blister spot[J]. Brazilian Journal of Microbiology, 43(3): 1222-1229.
[15] Boyaci H F, Unlu A, Abak K.2011. Genetic analysis of resistance to wilt caused by Fusarium (Fusarium oxysporum melongenae) in eggplant (Solanum melongena)[J]. Indian Journal of Agricultural Sciences, 81(9): 812-15.
[16] Campos-Soriano L, Valè G, Lupotto E, et al.2013. Investigation of rice blast development in susceptible and resistant rice cultivars using a gfp-expressing Magnaporthe oryzae isolate[J]. Plant Pathology, 62(5): 1030-1037.
[17] Correll J C.1991. The relationship between formae speciales, races, and vegetative compatibility groups in Fusarium oxysporum[J]. Phytopathology, 81(9): 1061-1064.
[18] Czislowski E, Fraser‐Smith S, Zander M, et al.2018. Investigation of the diversity of effector genes in the banana pathogen, Fusarium oxysporum f. sp. cubense, reveals evidence of horizontal gene transfer[J]. Molecular plant pathology, 19(5): 1155-1171.
[19] Fan J, Liu J, Gong Z Y, et al.2020. The false smut pathogen Ustilaginoidea virens requires rice stamens for false smut ball formation[J]. Environmental Microbiology, 22(2): 646-659.
[20] Hu M, Luo L, Wang S, et al.2014. Infection processes of Ustilaginoidea virens during artificial inoculation of rice panicles[J]. European Journal of Plant Pathology, 139(1): 67-77.
[21] Lagopodi A L, Ram A F, Lamers G E, et al.2002. Novel aspects of tomato root colonization and infection by Fusarium oxysporum f. sp. radicis-lycopersici revealed by confocal laser scanning microscopic analysis using the green fluorescent protein as a marker[J]. Molecular Plant-Microbe Interactions, 15(2): 172-179.
[22] Li B, Gao Y, Mao H Y, et al.2019. The SNARE protein FolVam7 mediates intracellular trafficking to regulate conidiogenesis and pathogenicity in Fusarium oxysporum f. sp. lycopersici[J]. Environmental Microbiology, 21(8): 2696-2706.
[23] Li X Z, Zhou T, Hai Y.2007. Transformation of Botrytis cinerea with a green fluorescent protein (GFP) gene for the study of host-pathogen interactions[J]. Plant Pathology Journal, 6(2): 368-373.
[24] Lü G, Guo S, Zhang H, et al.2014. Colonization of Fusarium wilt-resistant and susceptible watermelon roots by a green-fluorescent-protein-tagged isolate of Fusarium oxysporum f. sp. niveum[J]. Journal of Phytopathology, 162(4): 228-237.
[25] Lu Y, Xu W, Kang A, et al.2007. Prokaryotic expression and allergenicity assessment of hygromycin B phosphotransferase protein derived from genetically modified plants[J]. Journal of Food Science, 72(7):228-232.
[26] Ma L J, Geiser D M, Proctor R H, et al.2003. Fusarium pathogenomics[J]. Annual Review of Microbiology, 67: 399-416.
[27] Mora-Lugo R, Zimmermann J, Rizk A M, et al.2014. Development of a transformation system for Aspergillus sojae based on the Agrobacterium tumefaciens-mediated approach[J]. BMC Microbiology, 14(1): 1-8.
[28] Shao C, Yin Y, Qi Z, et al.2015. Agrobacterium tumefaciens mediated transformation of the entomopathogenic fungus Nomuraea rileyi[J]. Fungal Genetics & Biology, 83: 19-25.
[29] Sørensen L Q, Lysøe E, Larsen J E, et al.2014. Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER- Brick vector construction system[J]. BMC Molecular Biology, 15(1): 241-244.
[30] Spellig T, Bottin A, Kahmann R.1996. Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungus Ustilago maydis[J]. Molecular Genetics and Genomics, 252(5): 503-509.
[31] Visser M, Gordon T R, Wingfield B D, et al.2004. 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, 33(1): 69-75.
[32] Wang S, Chen H, Tang X, et al.2017. Molecular tools for gene manipulation in filamentous fungi[J]. Applied MiCrobiology and Biotechnology, 101(22): 8063-8075.
[33] Yang Y Q, Mei Y, Li M H, et al.2011. Establishment of Agrobacterium tumefaciens-mediated transformation system for rice sheath blight pathogen Rhizoctonia solani[J]. Rice Science, 18(4): 297-303.
[34] Ye S W, Cai C Y, Ren H B, et al.2017. An efficient plant regeneration and transformation system of Ma bamboo (Dendrocalamus latiflorus Munro) started from young shoot as explant[J]. Frontiers in Plant Science, 8: 1298.
[35] Zhang P, Xu B, Wang Y, et al.2008. Agrobacterium tumefaciens- mediated transformation as a tool for insertional mutagenesis in the fungus Penicillium marneffei[J]. Mycological Research, 112(8): 943-949.
[36] Zhang Y, Zhang J, Gao J, et al.2018. The colonization process of sunflower by a green fluorescent protein-tagged isolate of Verticillium dahliae and its seed transmission[J]. Plant Disease, 102(9): 1772-1778.
[37] Zhao M, Wang C, Zhou E, et al.2019. The latest research progress on genetic transformation system of filamentous fungi[J]. Genomics and Applied Biology, 38(3): 1138-1143.