GFPuv标记猕猴桃溃疡病菌的生物学特性 及其在土壤、根系中的定殖

doi:10.3864/j.issn.0578-1752.2013.02.007

Abstract

Abstract: 【Objective】The objective of this study is to obtain fluorescence-labeled Pseudomonas syringae pv. actinidiae (Psa) strain to further understand the infection and pathogenic processes.【Method】The Psa strain was tagged with GFPuv by electroporation. Fluorescence microscope and the method of plate dilution were used to detect the colonization of tagged strain in soil and root. 【Result】 The transformants showed strong green fluorescence under UV-light indicating GFPuv expression. A 700 bp fragment of the GFPuv gene was amplified from the genome DNA of the transformants. There were no differences between the engineered PSAmx7-GFPuv1 strain and wild-type strain including morphology, optimum temperature, pH and pathogenicity. The green fluorescence could be kept until 20 times of subcultures. The PSAmx7-GFPuv1 could survive in sterile soil for three months, and three weeks or so in non-sterile soil. The PSAmx7-GFPuv1 also could infect and colonise in root.【Conclusion】The GFPuv-tagged Psa was obtained successfully through electroporation in this study. The presence of GFPuv did not have signi?cant impact on the wild-type strain. The tagged strain could survive for a long period of time in sterile soil, colonise and multiply in root.

Key words: Pseudomonas syringae pv. actinidiae , pDSK-GFPuv , electroporation , soil survival , root colonization

HUANG Qi-Ling, GAO Xiao-Ning, ZHAO Zhi-Bo, QIN Hu-Qiang, HUANG Li-Li. Transformed GFPuv into Pseudomonas syringae pv. actinidiae and Its Biological Characteristics and Colonization in Soil and Roots of Kiwifruit[J].Scientia Agricultura Sinica, 2013, 46(2): 282-291.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.chinaagrisci.com/EN/10.3864/j.issn.0578-1752.2013.02.007

https://www.chinaagrisci.com/EN/Y2013/V46/I2/282

References

[1]Koh Y J, Kim G H, Jung J S, Lee Y S, Hur J S. Outbreak of bacterial canker on Hort16A (Actinidia chinensis Planchon) caused by Pseudomonas syringae pv. actinidiae in Korea. New Zealand Journal of Crop and Horticultural Science, 2010, 38(4): 275-282.

[2]Serizawa S, Ichikawa T, Takikawa Y, Tsuyumu S, Goto M. Occurrence of bacterial canker of kiwifruit in Japan: description of symptoms, isolation of the pathogen and screening of bactericides. Annals of the Phytopathological Society of Japan, 1989, 55(4): 427-436.

[3]Ferrante P, Scortichini M. Identfication of Pseudomonas syringae pv. actinidiae as causal agent of bacterial canker of yellow kiwifruit (Actinidia chinensis Planchon) in central Italy. Journal of Phytopathology, 2009, 157: 768-770.

[4]Vanneste J, Poliakoff F, Audusseau C, Cornish D, Paillard S, Rivoal C, Yu J . First report of Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker of kiwifruit in France. Plant Disease, 2011, 95(10): 1311.

[5]Abelleira A, López M M, Peñalver J, Aguín O, Mansilla J P, Picoaga A, García M J. First report of bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae in Spain. Plant Disease, 2011, 95(12): 1583.

[6]Everett K R. Pseudomonas syringae pv. actinidiae and other pathovars of Pseudomonas syringae. Australasian Plant Disease Notes, 2011, 6(1): 61-67.

[7]Renzi M, Copini P, Taddei A R, Rossetti A, Gallipoli L, Mazzaglia A, Balestra G M. Bacterial canker on kiwifruit in Italy: Anatomical changes in the wood and in the primary infection sites. Phytopathology, 2012, 102: 827-840.

[8]Shimomura O, Johnson F H, Saiga Y. Extraction, purification, and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. Journal of Cellular and Comparative Physiology, 1962, 59(3): 223-239.

[9]张雨丽, 张桂征, 苏红梅, 蒙艺英, 闭立辉. 绿色荧光蛋白在转基因研究中的应用. 生物技术通报, 2010(9): 16-20.

Zhang Y L, Zhang G Z, Su H M, Meng Y Y, Bi L H. Applications of green fluorescent protein in transgenic research. Biotechnology Bulletin, 2010(9): 16-20. (in Chinese)

[10]邓超, 黄大昉, 宋福平. 绿色荧光蛋白及其应用. 中国生物工程杂志, 2011, 31(1): 96-102.

Deng C, Huang D F, Song F P. Green fluorescence protein and its application. China Biotechnology, 2011, 31(1): 96-102. (in Chinese)

[11]Wang K, Kang L, Anand A, Lazarovits G, Mysore K S. Monitoring in planta bacterial infection at both cellular and whole plant levels using the green ?uorescent protein variant GFPuv. New Phytologist, 2007, 174: 212-223.

[12]Tombolini R, Unge A, Davey M E, Bruijn F J, Jannsson J K. Flow cytometric and microscopic analysis of GFP-taagged Pseudomonas fluorescens bactreria. FEMS Microbiology Ecology, 1997, 22: 17-28.

[13]张云峰, 邵磊. 根癌农杆菌电击转化条件的研究. 淮阴师范学院学报: 自然科学版, 2009, 8(3): 243-245.

Zhang Y F, Shao L. Studies on electroporation transformation of Agrobacterium tumefaciens. Journal of Huaiyin Teachers College: Natural Science Edition, 2009, 8(3): 243-245. (in Chinese)

[14]孙磊, 孔文涛, 孔健. 乳酸乳球菌电转化条件的研究. 山东大学学报: 理学报, 2005, 40(3): 121-124.

Sun L, Kong W T, Kong J. The factors affected transformation efficiency of Lactococcus lactis by electroporation. Journal of Shandong University, 2005, 40(3): 121-124. (in Chinese)

[15]Frederick M A, Roger B, Robert E K, David D. Current Protocols in Molecular Biology. John Wiley & Sons, 2003.

[16]周莉娟. GFP在南方根结线虫 (Meloidogyne incogita)拮抗菌Alcaligenes faecalis研究中的应用[D]. 福州: 福建农林大学, 2005.

Zhou L J. Application of GFP on the study of Meloidogyne incogita antagonistic Alcaligenes faecalis[D]. Fuzhou: Fujian Agriculture and Forestry University, 2005. (in Chinese)

[17]崔明学, 张成刚, 靳素英, 李明祺. 基模势对luxAB标记的快生型大豆根瘤菌在土壤中存活的影响. 应用生态学报, 1997, 8(5): 519-526.

Cui M X, Zhang C G, Jin S Y, Li M Q. Effect of matric potential on the survival of luxAB marked Rhizobium fredii in soil. Chinese Journal of Applied Ecology, 1997, 8(5): 519-526. (in Chinese)

[18]Sarathchandra S U, DI Menna M E, Burch G, Brown J A, Watson R N, Bell N L, Cox N R. Eeffcts of plant-parasitic nematodes and rhizosphere microorganisms on the growth of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.). Soil Biology & Biochemistry, 1995, 27(1): 9-16.

[19]倪培华, 温惠萍, 王丹艺, 周同, 王鸿利. 绿色荧光蛋白及其应用. 中国实验诊断学, 2001, 5(5): 280-282.

Ni P H, Wen H P, Wang D Y, Zhou T, Wang H L. Application of green fluorescent protein. Chinese Journal of Laboratory Diagnosis, 2001, 5(5): 280-282. (in Chinese)

[20]安千里, 杨学健, 董越梅, 冯丽洁, 匡柏建, 李久蒂. 用共聚焦激光扫描显微镜观测GFP标记的内生固氮菌Klebsiella oxytoca SAZ侵染水稻根. 植物学报, 2001, 43(6): 558-564.

An Q L, Yang X J, Dong Y M, Feng L J, Kuang B J, Li J D. Using confocal laser scanning microscope to visualize the infection of rice roots by GFP_labelled Klebsiella oxytoca SA2, an endophytic diazotroph. Acta Botanica Sinica, 2001, 43(6): 558-564. (in Chinese)

[21]王国芬. 桑细菌性枯萎病病原学及其致病机理研究[D]. 杭州: 浙江大学, 2009.

Wang G F. Etiology and pathogenesis of the Enterobacter cloacae complex causing mulberry wilt disease[D]. Hangzhou: Zhejiang University, 2009. (in Chinese)

[22]李法峰, 平淑珍, 苏宝林, 林敏. 粪产碱菌的Tn5转座诱变及吲哚乙酸生物合成特性的研究. 微生物学报, 2000, 40(5): 551-555.

Li F F, Ping S Z, Su B L, Lin M. Tn5 mutagenesis and the characteristics of indole-3-acetic acid biosynthesis in Alcaligenes faecalis A1501. Acta Microbiologica Sinica, 2000, 40(5): 551-555. (in Chinese)

[23]Leff L G, Leff A A. Use of green fluorescent protein to monitor survival of genetically engineered bacteria in aquatic environments. Applied and Environmental Microbiology, 1996, 62(9): 3486-3488.

[24]梁英梅, 张星耀, 田呈明, 高爱琴, 王培新. 陕西省猕猴桃枝干溃疡病病原菌鉴定. 西北林学院学报, 2000, 15(1): 37-39.

Liang Y M, Zhang X Y, Tian C M, Gao A Q, Wang P X. Pathogenic indentification of kiwifruit bacterial canker in Shaanxi. Journal of Northwest Forestry University, 2000, 15(1): 37-39. (in Chinese)

[25]车建美, 蓝江林, 刘波. 转绿色荧光蛋白基因的青枯雷尔氏菌生物学特性. 中国农业科学, 2008, 41(11): 3626-3635.

Che J M, Lan J L, Liu B. GFP tagging Ralstonia solanacearum with gfp/luxAB mini-Tn5. Scientia Agricultura Sinica, 2008, 41(11): 3626-3635. (in Chinese)

[26]冯永君, 宋未. 水稻内生优势成团泛菌GFP 标记菌株的性质与标记丢失动力学. 中国生物化学与分子生物学报, 2002, 18(1): 85-91.

Feng Y J, Song W. Characteristics and label loss dynamics of Pantoea aggiomerans, predominant entophytic bacteria isolated from rice plant. Chinese Journal of Biochemistry and Molecular Biology, 2002, 18(1): 85-91. (in Chinese)

[27]Ogawa H, Inouye S, Tsuji F, Yasuda K, Umeson K. Localication, trafficking and temperture dependence of the Aequorea green fluorescent in cultured vertebrate cells. Proceedings of the National Academy of Sciences of the United States of America, 1995, 92: 11899-11903.

[28]陈燕飞. pH对微生物的影响. 太原师范学院学报: 自然科学版, 2009, 8(3): 121-124.

Chen Y F. pH to uygur biology influence. Journal of Taiyuan Normal University: Natural Science Edition, 2009, 8(3): 121-124. (in Chinese)

[29]蔺继尚, 崔明学, 靳素英, 李明祺, 赵巍. 以转座子Tn5作弗氏中华根瘤菌的可识别生态学标记的研究- Tn5 的水平转移及其对R. fredii Tn5 突变株运动的影响. 应用生态学报, 1994, 5(3): 292-298.

Lin J S, Cui M X, Jin S Y, Li M Q, Zhao W. Transposon Tn5 used as an identifiable ecological marker of R. fredii stains: Horizontal transfer frequency of transponson Tn5 and its influence on the movement of R. fredii Tn5 mutant. Chinese Journal of Applied Ecology, 1994, 5(3): 292-298. (in Chinese)

[30]Vandenhove H, Merckx R, Wilmots H, Vlassak K. Survival of Pseudomonas flurescens inocula of different physiological stages in soil. Soil Biology & Biochemistry, 1991, 23(12): 1133-1142.

[31]Serizawa S, Ichikawa T, Suzuki H. Epidemiology of bacterial canker of kiwifruit. 5. Effect of infection in fall to early winter on the disease development in branches and trunk after winter. Annals of the Phytopathological Society of Japan, 1994, 60(2): 237-244.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

Transformed GFPuv into Pseudomonas syringae pv. actinidiae and Its Biological Characteristics and Colonization in Soil and Roots of Kiwifruit

PDF

Cited

Abstract

Cite this article

share this article

References

Related Articles 2

Metrics

Comments

Recommended 0

[1]	ZHONG CuiLi,LI GuoLing,WANG HaoQiang,MO JianXin,QUAN Rong,ZHANG XianWei,LI ZiCong,WU ZhenFang,GU Ting,CAI GengYuan. Optimizing the Electroporation Condition of Porcine Fetal Fibroblasts for Large Plasmid [J]. Scientia Agricultura Sinica, 2019, 52(3): 530-538.
[2]	ZHANG Xiao-Hong, ZHAO Xue-Jing, LI Bo, LI Fei-Fei, LIU Pei-Xuan, MIN Dong-Hong. Factors Optimization of Pollen Electroporation Transformation and Identification of Transgenic Wheat [J]. Scientia Agricultura Sinica, 2013, 46(12): 2403-2411.