GFP-Marking and Colonization on Cucumber of Bacillus subtilis BSD-2

doi:10.7668/hbnxb.2016.04.018

Abstract

Abstract: For the purpose of exploring the colonization on the cucumber,the Bacillus subtilis BSD-2 had remarkable control effect on the Botrytis cinerea. In this study,the plasmid of pGFP4412 that contains green fluorescent protein gene(GFP) was transformed into the Bacillus subtilis BSD-2 by modified electroporation.The plasmid stability,growth curve and the inhibition activity of GFP-labeled strains were measured.The results showed that the GFP-tagged strain could emit green fluorescence successfully.The tagged strain nearly had the same trend with the wild type strain in growth.The stability of GFP-marked in engineering B.subtilis BSD-2 strain was 86% after transference of culture 56 hours continuously without selective pressure.Inhibition activity showed that the GFP-marked strain exhibited the comparable ability as the wild type strain to inhibit Fusarium oxysporum and Botrytis cinerea.Observation by fluorescence microscope indicated that the GFP-tagged BSD-2 could colonize on the root tissue after inoculated 24 hours.It could be seen on the leaf veins after five days.It still could be observed on the leaf veins after 50 days.All of these data indicated that the GFP-marked strain could colonize on the cucumber so properly that stopped pathogens to invade the plant.

Key words: Bacillus subtilis, GFP, Fusarium oxysporum, Botrytis cinerea, Inhibitory activity

CLC Number:

Q78
S436.421

HAO Huijuan, LIU Hongwei, YIN Shuli, LIU Qianqian, ZHANG Liping, SONG Shuishan. GFP-Marking and Colonization on Cucumber of Bacillus subtilis BSD-2[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2016, 31(4): 106-111. doi: 10.7668/hbnxb.2016.04.018.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.2016.04.018

http://www.hbnxb.net/EN/Y2016/V31/I4/106

References

[1] Kidd B N,Kadoo N Y,Dombrecht B A,et al.Auxin signaling and transport promote susceptibility to the root-infecting fungal pathogen Fusarium oxysporum inArabidopsis [J].Molecular Plant-Microbe Interactions,2011,24(6):733-748.
[2] Jiao X L,Lu X H,Chen A J,et al.Effects of Fusarium solani and F.oxysporum infection on the metabolism of ginsenosides in American ginseng roots[J].Molecules,2015,20(6):10535-10552.
[3] Sperschneider J,Gardiner D M,Thatcher L F,et al.Genome-wide analysis in three Fusarium pathogens identifies rapidly evolving chromosomes and genes associated with pathogenicity[J].Genome Biology and Evolution,2015,7(6):1613-1627.
[4] Wilman K,Stępień L,Fabiańska I,et al.Plant-pathogenic fungi in seeds of different pea cultivars in Poland[J].Arhiv Za Higijenu Rada I Toksikologiju,2014,65(3):329-338.
[5] Jin W,Wu F.Characterization of miRNAs associated withBotrytis cinerea infection of tomato leaves[J].BMC Plant Biology,2015,15(1):1-14.
[6] Munafo J,Gianfagna T J.Antifungal activity and fungal metabolism of steroidal glycosides of Easter lily (Lilium longiflorum thunb.) by the plant pathogenic fungus,Botrytis cinerea [J].Journal of Agricultural and Food Chemistry,2011,59(11):5945-5954.
[7] Lyons R,Rusu A,Stiller J,et al.Investigating the association between flowering time and defense in the Arabidopsis thaliana-Fusarium oxysporum interaction[J].PLoS One,2015,10(6):1-24.
[8] Kfoury M,Sahraoui A L,Bourdon N,et al.Solubility,photostability and antifungal activity of phenylpropanoids encapsulated in cyclodextrins[J].Food Chemistry,2016,19(6):518-525.
[9] Antonio I,Franco N.Impact of preharvest application of biological control agents on postharvest diseases of fresh fruits and vegetables[J].Crop Protection,2000,19(8):715-723.
[10] Fisher P J,Petrini O.Location of fungal endophytes in tissues of Suaeda fruticosa:a preliminary study[J].Transactions of the British Mycological Society,1987,89(2):246-249.
[11] Kefi A,Ben Slimene I,Karkouch I,et al.Characterization of endophytic Bacillus strains from tomato plants (Lycopersicon esculentum) displaying antifungal activity against Botrytis cinerea Pers[J].World Journal of Microbiology & Biotechnology,2015,31(12):1967-1976.
[12] Katz E,Demain A L.Peptide antibiotics of Bacillus:chemistry,biogenesis,and possible functions[J].Bacteriological Reviews,1977,41(2):449-474.
[13] Evidente A,Punzo B,Andolfi A,et al.Alternethanoxins a and B,polycyclic ethanones produced by Alternaria sonchi,potential mycoherbicides for Sonchus arvensis biocontrol[J].Journal of Agricultural and Food Chemistry,2009,57(15):6656-6660.
[14] Oslizlo A,Stefanic P,Vatovec S,et al.Exploring ComQXPA quorum-sensing diversity and biocontrol potential of Bacillus spp.isolates from tomato rhizoplane[J].Microbial Biotechnology,2015,8(3):527-540.
[15] 张亮,王改兰,段建南,等.广谱生防菌对番茄枯萎病的防病效果及其机理[J].中国生物防治学报,2015,31(6):897-906.
[16] 张淑梅,李晶,姜威,等.内生细菌TF28诱导番茄抗病分子机制研究[J].中国生物防治学报,2015,31(6):913-920.
[17] 张铎,谢莉,张蕾,等.棉花黄萎病拮抗内生菌的筛选鉴定及抗菌物质研究[J].河北师范大学学报:自然科学版,2008,32(5):673-678.
[18] 胡瑞萍,张铎,张丽萍,等.枯草芽孢杆菌BSD-2一种抗菌肽的分离纯化与鉴定[J].华北农学报,2011,26(6):201-206.
[19] 周竞,程辉彩,仇燕肖,等.枯草芽孢杆菌BSD-2抗菌物质超滤浓缩工艺优化[J].农药,2015,54(11):805-808.
[20] 张根伟,张丽萍,李书生,等.复合土壤微生态制剂在连作花生上的应用效果[J].河南农业科学,2012,41(9):47-49.
[21] 王培培,郭庆港,李社增,等.枯草芽孢杆菌NCD-2菌株的高效电击转化[J].中国生物防治学报,2011,27(3):368-372.
[22] 田涛.蜡样芽孢杆菌绿色荧光蛋白标记及其在小麦上定殖的初探[D].北京:中国农业大学,2004.
[23] 武志江,李业燕,王亚军,等.百合枯萎病拮抗细菌的筛选、鉴定及其抑菌物质研究[J].微生物学通报,2015,42(7):1307-1320.
[24] 范晓静,邱思鑫,吴小平,等.绿色荧光蛋白基因标记内生枯草芽孢杆菌[J].应用与环境生物学报,2007,13(4):530-534.
[25] Mayer K,Iolyeva M E,Meyer-Grahle U,et al.Intestine-specific expression of green fluorescent protein-tagged cathepsin B:proof-of-principle experiments[J].Biological Chemistry,2008,389(8):1085-1096.
[26] 崔长征,沈萍,张甲耀,等.利用绿色荧光蛋白标记革兰氏阴性细菌的研究[J].环境科学学报,2011,31(2):276-282.
[27] Davidson M W,Campbell R E.Engineered fluorescent proteins:innovations and applications[J].Nature Methods,2009,6(10):713-717.
[28] Zhang F,Moniz H A,Walcott B,et al.Probing the impact of GFP tagging on Robo1-heparin interaction[J].Glycoconjugate Journal,2014,31(4):299-307.
[29] Kremers G J,Gilbert S G,Cranfill P J,et al.Fluorescent proteins at a glance[J].Journal of Cell Science,2011,124(124):157-160.
[30] Dower W J,Miller J F,Ragsdale C W.High efficiency transformation ofE.coli by high voltage electroporation[J].Nucleic Acids Research,1988,16(13):6127-6145.
[31] Sebti Y,Rabbani M,Sadeghi H M,et al.Effect of mutations in putative hormone binding sites on V2 vasopressin receptor function[J].Research in Pharmaceutical Sciences,2015,10(3):259-267.
[32] 刘邮洲,梁雪杰,乔俊卿,等.枯草芽胞杆菌PTS-394的GFP标记及其定殖能力[J].植物保护学报,2014,41(4):416-422.
[33] 田兆丰,刘伟成,董丹,等.生防枯草芽孢杆菌Kct99的GFP标记及其在甘蓝根部的定殖示踪[J].华北农学报,2012,27(6):53-57.
[34] 任嘉红,刘辉,姜楠,等.GFP标记溶磷草木樨中华根瘤菌CHW10B及其定殖[J].林业科学,2015,51(1):74-79.
[35] 黄其玲,高小宁,赵志博,等.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[J].中国农业科学,2013,46(2):282-291.
[36] 蒋晓玲,何鹏飞,王娅玲,等.玉米内生细菌Y19的荧光标记及其在玉米体内的定殖应用效果[J].玉米科学,2015,23(3):50-56.
[37] 魏春燕,邢永秀,莫遥,等.绿色荧光蛋白基因标记的固氮菌DX120E在甘蔗植株内的定殖[J].作物学报,2014,40(6):1132-1139.
[38] 卢靖乐,余新燕,侯毅平,等.枯草芽孢杆菌NJ-18的质粒消解及其在小麦根部的定殖[J].中国农业科学,2013,46(18):3776-3783.
[39] 杜芳,何鹏飞,吴毅歆,等.GFP标记内生枯草芽孢杆菌Y10及其在白菜体内的定殖[J].生态学杂志,2015,34(7):2064-2070.

Please choose a citation manager

Content to export