DNA-SIP鉴定甘蔗//大豆间作土壤~(15)N-DNA富集位置的氮循环功能基因qPCR方法
|
摘要
为筛选有效鉴定甘蔗//大豆间作系统的稳定性同位素核酸探针技术(DNA-SIP)中超高速离心后15N-DNA富集位置的指示功能基因,利用实时荧光定量PCR技术(qPCR),检测6个氮素循环功能基因在不同浮力密度离心液DNA中的相对丰度分布,通过对氮素循环功能基因相对丰度作图分析,nifH和amoA基因在甘蔗//大豆间作和大豆单作种植模式中15N标记组与对照组基因丰度峰发生偏移,chiA基因丰度峰仅在大豆单作种植模式下存在偏移,而nirS、nirK、nosZ等3个基因的丰度峰值在两种种植模式下均不发生偏移。结果表明nifH和amoA基因可作为指示基因,能够有效鉴定甘蔗//大豆间作系统中DNA-SIP技术15N-DNA位置。
Using DNA stable isotope probes(DNA-SIPs)is a reliable, new technique for studying the nitrogen cycle. To identify indicator function genes of15N-DNA enrichment in ultra-high-speed centrifugation in a sugarcane-soybean intercropping system for DNA-SIPs, the relative abundance distributions of six nitrogen cycling functional genes in the DNA of different buoyant density centrifugation fluids were detected via real-time PCR(qPCR). Through the analysis of the relative abundance of nitrogen cycling functional genes, the gene abundance peaks of the nifH and amoA genes in sugarcane-soybean intercropping and soybean monoculture were shifted in the15 N marker group and the control group, the gene abundance peaks of chiA were only shifted in soybean monocropping mode, and the abundance peaks of nirS, nirK, and nosZ did not shift with either planting pattern. The results showed that the nifH and amoA genes could be used as indicator genes to effectively identify DNA-SIP technology15N-DNA positions in sugarcane-soybean intercropping systems.
Using DNA stable isotope probes(DNA-SIPs)is a reliable, new technique for studying the nitrogen cycle. To identify indicator function genes of15N-DNA enrichment in ultra-high-speed centrifugation in a sugarcane-soybean intercropping system for DNA-SIPs, the relative abundance distributions of six nitrogen cycling functional genes in the DNA of different buoyant density centrifugation fluids were detected via real-time PCR(qPCR). Through the analysis of the relative abundance of nitrogen cycling functional genes, the gene abundance peaks of the nifH and amoA genes in sugarcane-soybean intercropping and soybean monoculture were shifted in the15 N marker group and the control group, the gene abundance peaks of chiA were only shifted in soybean monocropping mode, and the abundance peaks of nirS, nirK, and nosZ did not shift with either planting pattern. The results showed that the nifH and amoA genes could be used as indicator genes to effectively identify DNA-SIP technology15N-DNA positions in sugarcane-soybean intercropping systems.
引文
[1] Chu G X, Shen Q R, Cao J L. Nitrogen fixation and N transfer from peanut to rice cultivated in aerobic soil in an intercropping system and its effect on soil N fertility[J]. Plant&Soil, 2004, 263(1):17-27.
[2] Li Y Y, Yu C B, Cheng X, et al. Intercropping alleviates the inhibitory effect of N fertilization on nodulation and symbiotic N2fixation of faba bean[J]. Plant and Soil, 2009, 323(1/2):295-308.
[3] Fan F, Zhang F, Song Y, et al. Nitrogen fixation of faba bean(Vicia faba L.)interacting with a non-legume in two contrasting intercropping systems[J]. Plant&Soil, 2006, 283(1/2):275-286.
[4] H?gh-Jensen H. The nitrogen transfer between plants:An important but difficult flux to quantify[J]. Plant&Soil, 2006, 282(1/2):1-5.
[5] Schipanski M E, Drinkwater L E, Russelle M P. Understanding the variability in soybean nitrogen fixation across agroecosystems[J]. Plant&Soil, 2010, 329(1/2):379-397.
[6]雍太文,杨文钰,向达兵,等.小麦/玉米/大豆和小麦/玉米/甘薯套作对土壤氮素含量及氮素转移的影响[J].作物学报, 2012, 38(1):148-158.YONG Tai-wen, YANG Wen-yu, XIANG Da-bing, et al. Effect of wheat/maize/soybean and wheat/maize/sweet potato relay strip intercropping on soil nitrogen content and nitrogen transfer[J]. Acta Agronomica Sinica, 2012, 38(1):148-158.
[7]贺纪正,张丽梅.土壤氮素转化的关键微生物过程及机制[J].微生物学通报, 2013, 40(1):98-108.HE Ji-zheng, ZHANG Li-mei. Key processes and microbial mechanisms of soil nitrogen transformation[J]. Microbiology China, 2013, 40(1):98-108.
[8] Peoples M B, Chalk P M, Unkovich M J, et al. Can differences in15N natural abundance be used to quantify the transfer of nitrogen from legumes to neighbouring non-legume plant species?[J]. Soil Biology&Biochemistry, 2015, 87:97-109.
[9]贾仲君.稳定性同位素核酸探针技术DNA-SIP原理与应用[J].微生物学报, 2011, 51(12):1585-1594.JIA Zhong-jun. Principle and application of DNA-based stable isotope probing[J]. Acta Microbiologica Sinica, 2011, 51(12):1585-1594.
[10] Dumont M G, Murrell J C. Stable isotope probing-linking microbial identity to function[J]. Nat Rev Microbiol, 2005, 3(6):499-504.
[11] Bernard L, Mougel C, Maron P A, et al. Dynamics and identification of soil microbial populations actively assimilating carbon from13C-labelled wheat residue as estimated by DNA-and RNA-SIP techniques.[J]. Environmental Microbiology, 2007, 9(3):752-764.
[12] Venter Z S, Scott S L, Strauss J, et al. Increasing crop diversity increased soil microbial activity, nitrogen-sourcing and crop nitrogen,but not soil microbial diversity[J]. South African Journal of Plant&Soil, 2017, 34:1-8.
[13] Chen S C, Duan G L, Ding K, et al. DNA stable-isotope probing identifies uncultivated members of Pseudonocardia associated with biodegradation of pyrene in agricultural soil[J]. Fems Microbiology Ecology,2018, 94(3):1-10.
[14] Feng Y, Lin X, Zhu J, et al. A phototrophy-driven microbial food web in a rice soil[J]. Journal of Soils&Sediments Protection Risk Assessment&Rem, 2011, 11(2):301-311.
[15]钱明媚,肖永良,彭文涛,等.免耕水稻土固定CO2自养微生物多样性[J].中国环境科学, 2015, 35(12):3754-3761.QIAN Ming-mei, XIAO Yong-liang, PENG Wen-tao, et al. Diversities of autotrophic CO2-fixing microbes in no-tillage paddy soils[J].China Environmental Science, 2015, 35(12):3754-3761.
[16]郑燕,贾仲君.基于核酸DNA/RNA同位素示踪技术的水稻土甲烷氧化微生物研究[J].土壤学报, 2016, 53(2):490-501.ZHENG Yan, JIA Zhong-jun. The application of biomarker genes for DNA/RNA-stable isotope probing of active methaneotrophs responsible for aerobic methane oxidation in six paddy soils[J]. Acta Pedologica Sinica, 2016, 53(2):490-501.
[17] Meng L, Zhang A, Wang F, et al. Arbuscular mycorrhizal fungi and rhizobium facilitate nitrogen uptake and transfer in soybean/maize intercropping system[J]. Frontiers in Plant Science, 2015, 6:339.
[18] Frey B, Schuepp H. A role of vesicular-arbuscular(VA)mycorrhizal fungi in facilitating interplant nitrogen transfer[J]. Soil Biology&Biochemistry, 1993, 25(6):651-658.
[19]肖焱波.豆科/禾本科间作体系中养分竞争和氮素转移研究[D].北京:中国农业大学, 2003.XIAO Yan-bo. Interspecific competition for nutriens and nitrogen transfer between the intercropped legume and cereal[D]. Beijing:China Agriculture University, 2003.
[20] Tran-Nguyen L T T, Schneider B. Cesium chloride-bisbenzimide gradients for separation of phytoplasma and plant DNA[J]. Methods Mol Biol, 2013, 938(938):381-393.
[21] Neufeld J D, Vohra J, Dumont M G, et al. DNA stable-isotope probing.[J]. Nature Protocols, 2007, 2(4):860-866.
[22] Gore N R, Wray J L. Leucine:tRNA Ligase from cultured cells of nicotiana tabacum var. Xanthi:Evidence for de novo synthesis and for loss of functional enzyme molecules[J]. Plant Physiology, 1978, 61(1):20-24.
[23] R?sch C, Mergel A, Bothe H. Biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid forest soil[J]. Applied&Environmental Microbiology, 2002, 68(8):3818-3829.
[24] Williamson N, Brian P, Wellington E M H. Molecular detection of bacterial and streptomycete chitinases in the environment[J]. Antonie Van Leeuwenhoek, 2000, 78(3):315-321.
[25] Rotthauwe J H, Witzel K P, Liesack W. The ammonia monooxygenase structural gene amoA as a functional marker:Molecular fine-scale analysis of natural ammonia-oxidizing populations[J]. Applied&Environmental Microbiology, 1997, 63(12):4704-4712.
[26] Braker G, Fesefeldt A, Witzel K P. Development of PCR primer systems for amplification of nitrite reductase genes(nirK and nirS)to detect denitrifying bacteria in environmental samples[J]. Appl Environ Microbiol, 1998, 64(10):3769-3775.
[27] Michotey V, Méjean V, Bonin P. Comparison of methods for quantification of cytochrome cd 1-denitrifying bacteria in environmental marine samples[J]. Applied and Environmental Microbiology, 2000, 66(4):1564-1571.
[28] Kloos K, Mergel A, R?sch C, et al. Denitrification within the genus Azospirillum and other associative bacteria[J]. Australian Journal of Plant Physiology, 2001, 28(9):991-998.
[29] Throb?ck I N, Enwall K, Jarvis A, et al. Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE[J]. Fems Microbiology Ecology, 2004, 49(3):401-417.
[30] Vaitomaa J, Rantala A, Halinen K, et al. Quantitative Real-Time PCR for determination of microcystin synthetase E copy numbers for microcystis and anabaena in lakes[J]. Applied&Environmental Microbiology, 2003, 69(12):7289-7297.
[31] Zhang L M, Hu H W, Shen J P, et al. Ammonia-oxidizing archaea have more important role than ammonia-oxidizing bacteria in ammonia oxidation of strongly acidic soils[J]. Isme Journal, 2012, 6(5):1032-1045.
[32] Buckley D H, Huangyutitham V, Hsu S F, et al.15N2-DNA-stable isotope probing of diazotrophic methanotrophs in soil[J]. Soil Biology&Biochemistry, 2008, 40(6):1272-1283.
[2] Li Y Y, Yu C B, Cheng X, et al. Intercropping alleviates the inhibitory effect of N fertilization on nodulation and symbiotic N2fixation of faba bean[J]. Plant and Soil, 2009, 323(1/2):295-308.
[3] Fan F, Zhang F, Song Y, et al. Nitrogen fixation of faba bean(Vicia faba L.)interacting with a non-legume in two contrasting intercropping systems[J]. Plant&Soil, 2006, 283(1/2):275-286.
[4] H?gh-Jensen H. The nitrogen transfer between plants:An important but difficult flux to quantify[J]. Plant&Soil, 2006, 282(1/2):1-5.
[5] Schipanski M E, Drinkwater L E, Russelle M P. Understanding the variability in soybean nitrogen fixation across agroecosystems[J]. Plant&Soil, 2010, 329(1/2):379-397.
[6]雍太文,杨文钰,向达兵,等.小麦/玉米/大豆和小麦/玉米/甘薯套作对土壤氮素含量及氮素转移的影响[J].作物学报, 2012, 38(1):148-158.YONG Tai-wen, YANG Wen-yu, XIANG Da-bing, et al. Effect of wheat/maize/soybean and wheat/maize/sweet potato relay strip intercropping on soil nitrogen content and nitrogen transfer[J]. Acta Agronomica Sinica, 2012, 38(1):148-158.
[7]贺纪正,张丽梅.土壤氮素转化的关键微生物过程及机制[J].微生物学通报, 2013, 40(1):98-108.HE Ji-zheng, ZHANG Li-mei. Key processes and microbial mechanisms of soil nitrogen transformation[J]. Microbiology China, 2013, 40(1):98-108.
[8] Peoples M B, Chalk P M, Unkovich M J, et al. Can differences in15N natural abundance be used to quantify the transfer of nitrogen from legumes to neighbouring non-legume plant species?[J]. Soil Biology&Biochemistry, 2015, 87:97-109.
[9]贾仲君.稳定性同位素核酸探针技术DNA-SIP原理与应用[J].微生物学报, 2011, 51(12):1585-1594.JIA Zhong-jun. Principle and application of DNA-based stable isotope probing[J]. Acta Microbiologica Sinica, 2011, 51(12):1585-1594.
[10] Dumont M G, Murrell J C. Stable isotope probing-linking microbial identity to function[J]. Nat Rev Microbiol, 2005, 3(6):499-504.
[11] Bernard L, Mougel C, Maron P A, et al. Dynamics and identification of soil microbial populations actively assimilating carbon from13C-labelled wheat residue as estimated by DNA-and RNA-SIP techniques.[J]. Environmental Microbiology, 2007, 9(3):752-764.
[12] Venter Z S, Scott S L, Strauss J, et al. Increasing crop diversity increased soil microbial activity, nitrogen-sourcing and crop nitrogen,but not soil microbial diversity[J]. South African Journal of Plant&Soil, 2017, 34:1-8.
[13] Chen S C, Duan G L, Ding K, et al. DNA stable-isotope probing identifies uncultivated members of Pseudonocardia associated with biodegradation of pyrene in agricultural soil[J]. Fems Microbiology Ecology,2018, 94(3):1-10.
[14] Feng Y, Lin X, Zhu J, et al. A phototrophy-driven microbial food web in a rice soil[J]. Journal of Soils&Sediments Protection Risk Assessment&Rem, 2011, 11(2):301-311.
[15]钱明媚,肖永良,彭文涛,等.免耕水稻土固定CO2自养微生物多样性[J].中国环境科学, 2015, 35(12):3754-3761.QIAN Ming-mei, XIAO Yong-liang, PENG Wen-tao, et al. Diversities of autotrophic CO2-fixing microbes in no-tillage paddy soils[J].China Environmental Science, 2015, 35(12):3754-3761.
[16]郑燕,贾仲君.基于核酸DNA/RNA同位素示踪技术的水稻土甲烷氧化微生物研究[J].土壤学报, 2016, 53(2):490-501.ZHENG Yan, JIA Zhong-jun. The application of biomarker genes for DNA/RNA-stable isotope probing of active methaneotrophs responsible for aerobic methane oxidation in six paddy soils[J]. Acta Pedologica Sinica, 2016, 53(2):490-501.
[17] Meng L, Zhang A, Wang F, et al. Arbuscular mycorrhizal fungi and rhizobium facilitate nitrogen uptake and transfer in soybean/maize intercropping system[J]. Frontiers in Plant Science, 2015, 6:339.
[18] Frey B, Schuepp H. A role of vesicular-arbuscular(VA)mycorrhizal fungi in facilitating interplant nitrogen transfer[J]. Soil Biology&Biochemistry, 1993, 25(6):651-658.
[19]肖焱波.豆科/禾本科间作体系中养分竞争和氮素转移研究[D].北京:中国农业大学, 2003.XIAO Yan-bo. Interspecific competition for nutriens and nitrogen transfer between the intercropped legume and cereal[D]. Beijing:China Agriculture University, 2003.
[20] Tran-Nguyen L T T, Schneider B. Cesium chloride-bisbenzimide gradients for separation of phytoplasma and plant DNA[J]. Methods Mol Biol, 2013, 938(938):381-393.
[21] Neufeld J D, Vohra J, Dumont M G, et al. DNA stable-isotope probing.[J]. Nature Protocols, 2007, 2(4):860-866.
[22] Gore N R, Wray J L. Leucine:tRNA Ligase from cultured cells of nicotiana tabacum var. Xanthi:Evidence for de novo synthesis and for loss of functional enzyme molecules[J]. Plant Physiology, 1978, 61(1):20-24.
[23] R?sch C, Mergel A, Bothe H. Biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid forest soil[J]. Applied&Environmental Microbiology, 2002, 68(8):3818-3829.
[24] Williamson N, Brian P, Wellington E M H. Molecular detection of bacterial and streptomycete chitinases in the environment[J]. Antonie Van Leeuwenhoek, 2000, 78(3):315-321.
[25] Rotthauwe J H, Witzel K P, Liesack W. The ammonia monooxygenase structural gene amoA as a functional marker:Molecular fine-scale analysis of natural ammonia-oxidizing populations[J]. Applied&Environmental Microbiology, 1997, 63(12):4704-4712.
[26] Braker G, Fesefeldt A, Witzel K P. Development of PCR primer systems for amplification of nitrite reductase genes(nirK and nirS)to detect denitrifying bacteria in environmental samples[J]. Appl Environ Microbiol, 1998, 64(10):3769-3775.
[27] Michotey V, Méjean V, Bonin P. Comparison of methods for quantification of cytochrome cd 1-denitrifying bacteria in environmental marine samples[J]. Applied and Environmental Microbiology, 2000, 66(4):1564-1571.
[28] Kloos K, Mergel A, R?sch C, et al. Denitrification within the genus Azospirillum and other associative bacteria[J]. Australian Journal of Plant Physiology, 2001, 28(9):991-998.
[29] Throb?ck I N, Enwall K, Jarvis A, et al. Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE[J]. Fems Microbiology Ecology, 2004, 49(3):401-417.
[30] Vaitomaa J, Rantala A, Halinen K, et al. Quantitative Real-Time PCR for determination of microcystin synthetase E copy numbers for microcystis and anabaena in lakes[J]. Applied&Environmental Microbiology, 2003, 69(12):7289-7297.
[31] Zhang L M, Hu H W, Shen J P, et al. Ammonia-oxidizing archaea have more important role than ammonia-oxidizing bacteria in ammonia oxidation of strongly acidic soils[J]. Isme Journal, 2012, 6(5):1032-1045.
[32] Buckley D H, Huangyutitham V, Hsu S F, et al.15N2-DNA-stable isotope probing of diazotrophic methanotrophs in soil[J]. Soil Biology&Biochemistry, 2008, 40(6):1272-1283.