固氮细菌DX120E的gfp标记及对不同甘蔗品种的接种效应
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摘要
【目的】研究固氮菌株DX120E在甘蔗幼苗中的侵染定殖及其对不同甘蔗品种生长生理的影响,为进一步分析该固氮菌与甘蔗的相互作用机制提供参考依据。【方法】以3个甘蔗品种ROC22、B8和GT21为试验材料,将DX120E用gfp基因标记后,以浸根法接种甘蔗,观察其在甘蔗中的侵染定殖情况,分别在接菌后60、90和120 d,调查甘蔗的株高、叶绿素含量及鲜重,并测定氮代谢关键酶活性指标。【结果】获得了稳定表达的DX120E-gfp接合子菌株;接种后第7 d,在3个甘蔗品种的根部和叶片中均检测出nif H基因表达,片段大小约为360 bp。荧光显微镜观察结果表明,接种后第7 d,固氮菌定殖在甘蔗的根毛区、新生侧根处、根断裂伤口处及叶片横切处。盆栽试验结果表明,接种固氮菌DX120E对3个甘蔗品种有明显促生作用,与不接菌对照相比,接菌后120 d,ROC22、B8和GT21的株高分别增加23%、20%和14%,地上部分鲜重分别增加25%、31%和11%,地下部分鲜重分别增加40%、34%和30%。且3个甘蔗品种叶片的谷氨酰胺合成酶和硝酸还原酶活性总体上高于对照。【结论】采用菌液浸根法接种甘蔗,固氮菌DX120E能在甘蔗根部定殖并向地上部分转移定殖。采用该方法处理甘蔗,对甘蔗有明显的促生效应,但不同品种间存在一定差异。
【Objective】The infection and colonization of nitrogen-fixing bacteria DX120 E in sugarcane seedlings and the effects on the growth and physiology in different sugarcane varieties were studied to provide reference for further analysis of the interaction mechanism between nitrogen-fixing bacteria and sugarcane.【Method】Sugarcane varieties ROC22,GT21 and B8 were used as test materials. After DX120 E was labeled with gfp gene,sugarcane was inoculated by rootsoaking method,and infection and colonization of DX120 E in sugarcane were observed. The plant height,chlorophyll content and fresh weight of sugarcane were investigated at 60,90 and 120 d after inoculation,and the key enzyme activities of nitrogen metabolism were measured.【Result】Stably expressed DX120 E-gfp zygote strains was obtained. On day 7 after inoculation,nif H gene expression was detected in the roots and leaves of three sugarcane varieties,and the fragment size was approximately 360 bp. The results of fluorescence microscopy showed that on day 7 after inoculation,nitrogenfixing bacteria was colonized in the root hair zone,the neonatal lateral roots,the root fracture wounds and the cross-sections of the leaves of sugarcane. Pot experiment showed that compared with the control of inoculation with no nitrogen-fixing bacteria,the sugarcane inoculated with nitrogen-fixing bacteria DX120 E could effectively promote the growth of the three varieties. And 120 d after inoculation,the plant heights of ROC22,B8 and GT21 respectively increased by 23%,20%and 14%;the fresh weight of aboveground increased by 25%,31% and 11% respectively;the fresh weight of underground part increased by 40%,34% and 30% respectively. The activity of glutamine synthetase and nitrate reductase in leaves of the three sugarcane varieties was also higher than that of the control.【Conclusion】By using the root-soaking method to inoculate sugarcane,the nitrogen-fixing bacteria strain DX120 E can colonize the roots and transfer to the leaves of sugarcane. Using this method to treat sugarcane has obvious promoting effects on sugarcane,but there are some differences between different varieties.
【Objective】The infection and colonization of nitrogen-fixing bacteria DX120 E in sugarcane seedlings and the effects on the growth and physiology in different sugarcane varieties were studied to provide reference for further analysis of the interaction mechanism between nitrogen-fixing bacteria and sugarcane.【Method】Sugarcane varieties ROC22,GT21 and B8 were used as test materials. After DX120 E was labeled with gfp gene,sugarcane was inoculated by rootsoaking method,and infection and colonization of DX120 E in sugarcane were observed. The plant height,chlorophyll content and fresh weight of sugarcane were investigated at 60,90 and 120 d after inoculation,and the key enzyme activities of nitrogen metabolism were measured.【Result】Stably expressed DX120 E-gfp zygote strains was obtained. On day 7 after inoculation,nif H gene expression was detected in the roots and leaves of three sugarcane varieties,and the fragment size was approximately 360 bp. The results of fluorescence microscopy showed that on day 7 after inoculation,nitrogenfixing bacteria was colonized in the root hair zone,the neonatal lateral roots,the root fracture wounds and the cross-sections of the leaves of sugarcane. Pot experiment showed that compared with the control of inoculation with no nitrogen-fixing bacteria,the sugarcane inoculated with nitrogen-fixing bacteria DX120 E could effectively promote the growth of the three varieties. And 120 d after inoculation,the plant heights of ROC22,B8 and GT21 respectively increased by 23%,20%and 14%;the fresh weight of aboveground increased by 25%,31% and 11% respectively;the fresh weight of underground part increased by 40%,34% and 30% respectively. The activity of glutamine synthetase and nitrate reductase in leaves of the three sugarcane varieties was also higher than that of the control.【Conclusion】By using the root-soaking method to inoculate sugarcane,the nitrogen-fixing bacteria strain DX120 E can colonize the roots and transfer to the leaves of sugarcane. Using this method to treat sugarcane has obvious promoting effects on sugarcane,but there are some differences between different varieties.
引文
黄杏,杨丽涛,李杨瑞.2009.固氮菌接种对甘蔗根系生理特性的影响[J].广西农业科学,40(3):233-237.[Huang X,Yang L T,Li Y R.2009.Effects of inoculating nitrogen fixation bacteria on physiological characteristics of roots in sugarcane[J].Guangxi Agricultural Sciences,40(3):233-237.]
李海碧.2017.甘蔗根际假单胞菌的分离鉴定及其对甘蔗的促生作用[D].南宁:广西大学.[Li H B.2017.Isolation and characterization of Pseudomonas species from sugarcane rhizospheric soil and their influence on the growth of sugarcane[D].Nanning:Guangxi University.]
李合生.2000.植物生理生化实验原理和技术[M].北京:高等教育出版社:123-124.[Li H S.2000.Experimental Principle and Technology of Plant Physiology and Biochemistry[M].Beijing:Higer Education Press:123-124.]
李杨瑞,杨丽涛,谭宏伟,朱秋珍,王维瓒.2014.广西甘蔗栽培技术的发展进步[J].南方农业学报,45(10):1770-1775.[Li Y R,Yang L T,Tan H W,Zhu Q Z,Wang WZ.2014.Development and progress of sugarcane farming technologies in Guangxi,China[J].Journal of Southern Agriculture,45(10):1770-1775.]
林丽.2011.联合广西新台糖22号甘蔗固氮菌的筛选和对菌株Klebsiella sp.DX120E、Burkholderia sp.CZ152S和Micrebacterium sp.16SH的研究[D].南宁:广西大学.[Lin L.2011.Screening of nitrogen-fixing bacteria associated with ROC22 sugarcane plants cultivated in Guangxi and studies on klebsiella sp.DX120E,Burkholderia sp.CZ152S and Micrebacterium sp.16SH strains[D].Nanning:Guangxi University.]
罗霆,欧阳雪庆,杨丽涛,李杨瑞.2010.15N同位素稀释法研究固氮菌接种对甘蔗生物固氮的影响[J].核农学报,24(5):1026-1031.[Luo T,Ouyang X Q,Yang L T,Li Y R.2010.Effects of nitrogen-fixing bacteria inoculation on biological nitrogen fixation in sugarcane by15N isotope dilution technique[J].Journal of Nuclear Agricultural Sciences,24(5):1026-1031.]
吕泽勋,李久蒂,朱至清.2001.用绿色荧光蛋白基因(gfp)标记产酸克雷伯氏菌SG-11研究其在水稻苗期根部的定殖[J].农业生物技术学报,9(1):13-18.[LüZ X,Li J D,Zhu Z Q.2001.Klebsiellaoxytoca SG-11 labeling by green fluorescent protein gene(gfp)and its colonization in rice seedling roots[J].Journal of Agricultural Biotechnology,9(1):13-18.]
王伦旺,李杨瑞,杨荣仲,李翔,黄家雍,方锋学.2010.不同甘蔗基因型对低氮胁迫的响应[J].西南农业学报,23(2):508-514.[Wang L W,Li Y R,Yang R Z,Li X,Huang JY,Fang F X.2010.Response of low nitrogen stress in different sugarcane genotypes[J].Southwest China Journal of Agricultural Sciences,23(2):508-514.]
魏春燕,邢永秀,林丽,杨丽涛,李杨瑞,胡春锦.2014.接种变栖克雷伯氏菌DX120E对不同甘蔗品种的促生效应[J].应用生态学报,25(7):2085-2092.[Wei C Y,Xing Y X,Lin L,Yang L T,Li Y R,Hu C J.2014.Growth-promoting effect of inoculating Klebsiellavariicola DX120E on different sugarcane cultivars[J].Chinese Journal of Applied Ecology,25(7):2085-2092.]
韦莉萍.2005.不同钼水平对甘蔗固氮生理生化的影响[D].南宁:广西大学.[Wei L P.2005.Effects of different levels of molybdenum on the physiological and biochemical characteristic in relation to nitrogen fixation in sugarcane[D].Nanning:Guangxi University.]
邢永秀.2006.甘蔗内生固氮菌的分离、鉴定和生长特性[D].南宁:广西大学.[Xing Y X.2006.Isolation and identification of endophytic nitrogen fixation in sugarcane and growth characteristics[D].Nanning:Guangxi University.]
Bartosik D,Baj J,Sochacka M,Piechucka E,Wlodarczyk M.2002.Molecular characterization of functional modules of plasmid p WKS1 of Paracoccus pantotrophus DSM11072[J].Microbiology,148(9):2847-2856.
Becker T W,Hirel B,Carrayol E.2000.Glutamine synthetase and glutamate dehydrogenase isoforms in maize leaves:Localization,relative proportion and their role in ammonium assimilation or nitrogen transport[J].Planta,211(6):800-806.
Carvalho T L G D,Ferreira P C G,Hemerly A S.2011.Sugarcane genetic controls involved in the association with beneficial endophytic nitrogen fixing bacteria[J].Tropical Plant Biology,4(1):31-41.
Castanheira N L,Dourado A C,Pais I,Semedo J,Scotti-Campos P,Borges N,Carvalho G,Crespo M T B,Fareleira P.2017.Colonization and beneficial effects on annual ryegrass by mixed inoculation with plant growth promoting bacteria[J].Microbiological Research,198:47-55.
D?bereiner J.1961.Nitrogen-fixing bacteria of the genus Beijerinckiaderx in the rhizosphere of sugar cane[J].Plant&Soil,15(3):211-216.
Gyaneshwar P,James E K,Mathan N,Reddy P M,ReinholdHurek B,Ladha J K.2001.Endophytic colonization of rice by a diazotrophic strain of Serratia marcescens[J].Journal of Bacteriology,183(8):2634-2645.
James E K,Olivares F L,Oliveira A L M D,Reis F B,da Silva L G.2001.Further observations on the interaction between sugar cane and Gluconacetobacter diazotrophicus under laboratory and greenhouse conditions[J].Journal of Experimental Botany,52(357):747-760.
James E K,Reis V M,Olivares F L,Baldani,Dobereiner J.1994.Infection of sugar cane by the nitrogen-fixing bacterium Acetobacter diazotrophicus[J].Journal of Experimental Botany,45(275):757-766.
Jha P N,Kumar A.2007.Endophytic colonization of Typha australis by a plant growth promoting bacterium Klebsiella oxytoca strain GR-3[J].Journal of Applied Microbiology,103(4):1311-1320.
Magnani G S,Didonet C M,Cruz L M,Picheth C F,Pedrosa F O,Souza E M.2010.Diversity of endophytic bacteria in Brazilian sugarcane[J].Genetics&Molecular Research,9(1):250-258.
Suman A,Gaur A,Shrivastava A K,Yadav R L.2005.Improving sugarcane growth and nutrient uptake by inoculating Gluconacetobacter diazotrophicus[J].Plant Growth Regulation,47(2-3):155-162.
李海碧.2017.甘蔗根际假单胞菌的分离鉴定及其对甘蔗的促生作用[D].南宁:广西大学.[Li H B.2017.Isolation and characterization of Pseudomonas species from sugarcane rhizospheric soil and their influence on the growth of sugarcane[D].Nanning:Guangxi University.]
李合生.2000.植物生理生化实验原理和技术[M].北京:高等教育出版社:123-124.[Li H S.2000.Experimental Principle and Technology of Plant Physiology and Biochemistry[M].Beijing:Higer Education Press:123-124.]
李杨瑞,杨丽涛,谭宏伟,朱秋珍,王维瓒.2014.广西甘蔗栽培技术的发展进步[J].南方农业学报,45(10):1770-1775.[Li Y R,Yang L T,Tan H W,Zhu Q Z,Wang WZ.2014.Development and progress of sugarcane farming technologies in Guangxi,China[J].Journal of Southern Agriculture,45(10):1770-1775.]
林丽.2011.联合广西新台糖22号甘蔗固氮菌的筛选和对菌株Klebsiella sp.DX120E、Burkholderia sp.CZ152S和Micrebacterium sp.16SH的研究[D].南宁:广西大学.[Lin L.2011.Screening of nitrogen-fixing bacteria associated with ROC22 sugarcane plants cultivated in Guangxi and studies on klebsiella sp.DX120E,Burkholderia sp.CZ152S and Micrebacterium sp.16SH strains[D].Nanning:Guangxi University.]
罗霆,欧阳雪庆,杨丽涛,李杨瑞.2010.15N同位素稀释法研究固氮菌接种对甘蔗生物固氮的影响[J].核农学报,24(5):1026-1031.[Luo T,Ouyang X Q,Yang L T,Li Y R.2010.Effects of nitrogen-fixing bacteria inoculation on biological nitrogen fixation in sugarcane by15N isotope dilution technique[J].Journal of Nuclear Agricultural Sciences,24(5):1026-1031.]
吕泽勋,李久蒂,朱至清.2001.用绿色荧光蛋白基因(gfp)标记产酸克雷伯氏菌SG-11研究其在水稻苗期根部的定殖[J].农业生物技术学报,9(1):13-18.[LüZ X,Li J D,Zhu Z Q.2001.Klebsiellaoxytoca SG-11 labeling by green fluorescent protein gene(gfp)and its colonization in rice seedling roots[J].Journal of Agricultural Biotechnology,9(1):13-18.]
王伦旺,李杨瑞,杨荣仲,李翔,黄家雍,方锋学.2010.不同甘蔗基因型对低氮胁迫的响应[J].西南农业学报,23(2):508-514.[Wang L W,Li Y R,Yang R Z,Li X,Huang JY,Fang F X.2010.Response of low nitrogen stress in different sugarcane genotypes[J].Southwest China Journal of Agricultural Sciences,23(2):508-514.]
魏春燕,邢永秀,林丽,杨丽涛,李杨瑞,胡春锦.2014.接种变栖克雷伯氏菌DX120E对不同甘蔗品种的促生效应[J].应用生态学报,25(7):2085-2092.[Wei C Y,Xing Y X,Lin L,Yang L T,Li Y R,Hu C J.2014.Growth-promoting effect of inoculating Klebsiellavariicola DX120E on different sugarcane cultivars[J].Chinese Journal of Applied Ecology,25(7):2085-2092.]
韦莉萍.2005.不同钼水平对甘蔗固氮生理生化的影响[D].南宁:广西大学.[Wei L P.2005.Effects of different levels of molybdenum on the physiological and biochemical characteristic in relation to nitrogen fixation in sugarcane[D].Nanning:Guangxi University.]
邢永秀.2006.甘蔗内生固氮菌的分离、鉴定和生长特性[D].南宁:广西大学.[Xing Y X.2006.Isolation and identification of endophytic nitrogen fixation in sugarcane and growth characteristics[D].Nanning:Guangxi University.]
Bartosik D,Baj J,Sochacka M,Piechucka E,Wlodarczyk M.2002.Molecular characterization of functional modules of plasmid p WKS1 of Paracoccus pantotrophus DSM11072[J].Microbiology,148(9):2847-2856.
Becker T W,Hirel B,Carrayol E.2000.Glutamine synthetase and glutamate dehydrogenase isoforms in maize leaves:Localization,relative proportion and their role in ammonium assimilation or nitrogen transport[J].Planta,211(6):800-806.
Carvalho T L G D,Ferreira P C G,Hemerly A S.2011.Sugarcane genetic controls involved in the association with beneficial endophytic nitrogen fixing bacteria[J].Tropical Plant Biology,4(1):31-41.
Castanheira N L,Dourado A C,Pais I,Semedo J,Scotti-Campos P,Borges N,Carvalho G,Crespo M T B,Fareleira P.2017.Colonization and beneficial effects on annual ryegrass by mixed inoculation with plant growth promoting bacteria[J].Microbiological Research,198:47-55.
D?bereiner J.1961.Nitrogen-fixing bacteria of the genus Beijerinckiaderx in the rhizosphere of sugar cane[J].Plant&Soil,15(3):211-216.
Gyaneshwar P,James E K,Mathan N,Reddy P M,ReinholdHurek B,Ladha J K.2001.Endophytic colonization of rice by a diazotrophic strain of Serratia marcescens[J].Journal of Bacteriology,183(8):2634-2645.
James E K,Olivares F L,Oliveira A L M D,Reis F B,da Silva L G.2001.Further observations on the interaction between sugar cane and Gluconacetobacter diazotrophicus under laboratory and greenhouse conditions[J].Journal of Experimental Botany,52(357):747-760.
James E K,Reis V M,Olivares F L,Baldani,Dobereiner J.1994.Infection of sugar cane by the nitrogen-fixing bacterium Acetobacter diazotrophicus[J].Journal of Experimental Botany,45(275):757-766.
Jha P N,Kumar A.2007.Endophytic colonization of Typha australis by a plant growth promoting bacterium Klebsiella oxytoca strain GR-3[J].Journal of Applied Microbiology,103(4):1311-1320.
Magnani G S,Didonet C M,Cruz L M,Picheth C F,Pedrosa F O,Souza E M.2010.Diversity of endophytic bacteria in Brazilian sugarcane[J].Genetics&Molecular Research,9(1):250-258.
Suman A,Gaur A,Shrivastava A K,Yadav R L.2005.Improving sugarcane growth and nutrient uptake by inoculating Gluconacetobacter diazotrophicus[J].Plant Growth Regulation,47(2-3):155-162.