王沛譞^{1, 3,},
徐焱²,
宋亚娜^1,,
1.福建省农业科学院生物技术研究所 福州 350003
2.福州时代中学 福州 350007
3.福建师范大学附中福州 350007
基金项目: 福建省自然科学基金项目2015J01105
福建省公益类科研院所专项2015R1019-12


详细信息

作者简介:王沛譞, 主要从事土壤微生物分子生态学研究。E-mail:1092546064@qq.com

通讯作者:宋亚娜, 主要从事土壤微生物分子生态学研究。E-mail:syana@sina.com

中图分类号:S154.3

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收稿日期:2017-02-13
录用日期:2017-05-18
刊出日期:2018-01-01

Effect of transgenic rice straw return to soil on nitrification and denitrification microbial community

WANG Peixuan^{1, 3,},
XU Yan²,
SONG Yana^1,,
1. Institute of Biological Technology, Fujian Academy of Agricultural Sciences, Fuzhou 350003, China
2. Fuzhou Shidai Middle School, Fuzhou 350007, China
3. Affiliated High School of Fujian Normal University, Fuzhou 350007, China
Funds: the Natural Science Foundation of Fujian Province2015J01105
the Public Welfare Foundation of Fujian Province2015R1019-12


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Corresponding author:Song Yanan,E-mail:syana@sina.com

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摘要
摘要:转基因作物可能通过根系分泌物和植株残体组成的改变及外源基因的转移释放令土壤微生物群落产生变化，影响土壤微生物的生态功能。氨氧化细菌和反硝化细菌是驱动土壤硝化和反硝化过程的关键微生物，其群落结构的变化直接关系土壤氮素的转化与利用。本研究利用荧光定量PCR和PCR-DGGE技术分析了转cry1Ac/cpti双价抗虫基因水稻‘Kf8’秸秆还田降解过程中，土壤氨氧化细菌和反硝化细菌群落丰度与组成的变化，探讨转基因水稻是否存在影响稻田土壤氮素转化与N₂O排放的可能。结果显示：无论是氨氧化细菌amoA基因还是反硝化细菌nirS基因，其丰度在转基因水稻‘Kf8’与非转基因水稻‘Mh86’的秸秆还田土壤中都没有显著差异；转基因水稻‘Kf8’和非转基因水稻‘Mh86’秸秆还田降解过程中0~10 cm土层中的amoA基因丰度均显著高于10~20 cm及20~30 cm土层（P < 0.05）；各深度土层中的nirS基因丰度均存在随秸秆还田时间延长而增加的趋势。水稻秸秆还田降解过程中，转基因水稻‘Kf8’的土壤氨氧化细菌和反硝化细菌的群落多样性指数及组成，均与非转基因水稻‘Mh86’没有显著差异。相关分析结果表明土壤氨氧化细菌和反硝化细菌群落组成均与水稻秸秆还田时间存在显著相关性（P=0.002），反硝化细菌群落组成还与土层深度显著相关（P=0.024）。本研究表明转cry1Ac/cpti抗虫基因水稻秸秆还田对稻田土壤硝化和反硝化关键微生物群落不会产生明显影响。就土壤微生物群落而言，转cry1Ac/cpti抗虫基因水稻秸秆还田不存在影响土壤氮素转化与N₂O排放的可能。
关键词:转基因水稻/
秸秆还田/
氨氧化细菌/
amoA基因/
反硝化细菌/
nirS基因/
群落结构
Abstract:The cultivation of genetically modified plants may have the potential to alter microbial community structure and function in soils through root exudates and plant residues. Ammonia-oxidizing bacteria or denitrifying bacteria are the key microbes for nitrification or denitrification. A change in community structure of ammonia-oxidizing bacteria or denitrifying bacteria can affect the conversion and utilization of nitrogen in soil. The purpose of this study was to explore the possibility of transgenic rice to induce change in nitrogen transformation and N₂O emission in paddy soils. In the study, the abundance and composition of ammonia-oxidizing bacteria or denitrifying bacteria in paddy soils under straw return to soil under cry1Ac/cpti transgenic gene rice 'Kf8' or non-transgenic rice 'Mh86' were analyzed by real-time PCR and denaturing gradient gel electrophoresis (DGGE) based on amoA gene or nirS gene. The results showed that there were no differences in the abundance of amoA gene or nirS gene in the soil with returned transgenic rice 'Kf8' straw and non-transgenic rice 'Mh86' straw. The abundance of amoA gene in the 0-10 cm soil layer was significantly (P < 0.05) higher than that in the 10-20 cm and 20-30 cm soil layers under degraded transgenic rice 'Kf8' straw or non-transgenic rice 'Mh86' straw. The abundance of nirS gene in the soil increased with the time of returned straw to soil for either transgenic rice 'Kf8' or no-transgenic rice 'Mh86'. At the same time, the Shannon-Weiner index and composition of ammonia-oxidizing bacteria or denitrifying bacteria in the soil under degraded transgenic rice 'Kf8' straw were similar to those under degraded non-transgenic rice 'Mh86' straw. The composition of ammonia-oxidizing bacteria in the soil was significantly correlated with the time of rice straw return (P=0.002). Also the abundance of denitrifying bacteria was significantly correlated with the time of rice straw return (P=0.002) and depth of soil (P=0.024). The findings demonstrated that there was no significant effect of returned cry1Ac/cpti transgenic rice straw to soil on key microbial com-munities for nitrification or denitrification in soil. In terms of soil microbial community, there was no signiifanct effect of returned cry1Ac/cpti transgenic rice straw to soil on nitrogen transformation and N₂O emission in paddy soils.
Key words:Transgenic rice/
Straw return/
Ammonia-oxidizing bacteria/
amoA gene/
Denitrifying bacteria/
nirS gene/
Community structure

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图1转基因水稻‘Kf8’和常规水稻‘Mh86’秸秆还田后不同时间不同深度土壤氨氧化细菌amoA基因(A)和反硝化细菌nirS基因(B)丰度的变化
同一深度土层不同字母表示差异显著(P < 0.05)。
Figure1.Changes in abundances of ammonia-oxidizing bacterial amoA gene (A) and denitrifying bacteria nirS gene (B) in paddy soil at different depths after different times of straw incorporation of transgenic rice 'Kf8' and no-transgenic rice 'Mh86'
Values of the same depth of soil in the same time followed by different letters are significantly different (P < 0.05).


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图2转基因水稻‘Kf8’和常规水稻‘Mh86’秸秆还田后不同时间不同深度土壤氨氧化细菌(A)和反硝化细菌(B)多样性指数的变化
同一深度土层不同字母表示差异显著(P < 0.05)。
Figure2.Changes in Shannon-Weiner indexes of ammonia-oxidizing bacteria (A) and denitrifying bacteria (B) in paddy soil at different depths after different times of straw incorporation of transgenic rice 'Kf8' and no-transgenic rice 'Mh86'
Values of the same depth of soil in the same time followed by different letters are significantly different (P < 0.05).


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图3转基因水稻‘Kf8’和常规水稻‘Mh86’秸秆还田后不同时间不同深度土壤氨氧化细菌(a)和反硝化细菌(b)群落组成的主成分分析
Figure3.PCA analysis of the community composition of ammonia-oxidizing bacteria (a) and denitrifying bacteria (b) in paddy soil at different depths after different times of straw incorporation of transgenic rice 'Kf8' and no-transgenic rice 'Mh86'


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表1PCR引物及反应条件
Table1.Primers and reaction conditions for PCR

目标基因 Target gene	引物序列 Primer sequence	PCR-DGGE反应条件 Reaction conditions for PCR-DGGE	荧光定量PCR反应条件 Reaction conditions for real-time PCR
amoA基因 amoA gene	amoA1F*:GGGGTTTCTACTGGTGGT amoA2R:CCCCTCKGSAAAGCCTTCTTC amoA1F-GC*	95 ℃预变性5 min, 94 ℃变性30 s, 58 ℃退火45 s, 72 ℃延伸45 s, 35个循环, 72 ℃ 10 min 95 ℃ 5 min, 94 ℃ 30 s, 58 ℃ 45 s, 72 ℃ 45 s, 35 cycles, 72 ℃ 10 min	95 ℃预变性30 s, 94 ℃变性30 s, 58 ℃退火30 s, 72 ℃延伸40 s, 40个循环95 ℃ 30 s, 94 ℃ 30 s, 58 ℃ 30 s, 72℃ 40 s, 40 cycles
nirS基因nirS gene	nirS4F*:TTCRTCAAGACSCAYCCGAA nirS6R:CGTTGAACTTSCCGGT nirS4F-GC*	95 ℃预变性5 min, 95 ℃变性30 s, 45 ℃ (-0.5 ℃/循环)退火40 s, 72 ℃延伸40 s, 10个循环, 95 ℃变性30 s, 43 ℃退火40 s, 72 ℃延伸40 s, 25个循环, 72 ℃ 10 min 95 ℃ 5 min, 95 ℃ 30 s, 45 ℃ (-0.5 ℃/cycle) 40 s, 72 ℃ 40 s, 10 cycles, 95 ℃ 30 s, 43 ℃ 40 s, 72 ℃ 40 s, 25 cycles, 72 ℃ 10 min	95 ℃预变性30 s, 94 ℃变性30 s, 45 ℃退火40 s, 72 ℃延伸40 s, 40个循环95 ℃ 30 s, 94 ℃ 30 s, 45 ℃ 40 s, 72 ℃ 40 s, 40 cycles
*:分别在amoA1F和nirS4F引物序列前加一个GC夹序列(CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGG)用于DGGE的PCR扩增。*: A GC clamp (CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGG) was attached to the sequence of amoA1F or nirS4F for PCR of DGGE.

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参考文献(24)

[1]	MASOERO F, MOSCHINI M, ROSSI F, et al. Nutritive value, mycotoxin contamination and in vitro rumen fermentation of normal and genetically modified corn (Cry1Ab) grown in northern Italy[J]. Maydica, 1999, 44(3):205-209 https://s3-eu-west-1.amazonaws.com/pfigshare-u-files/4994056/plos_one_data_availability.xlsx
[2]	CAPONE D G. Methane, nitrogen oxides and halomethanes[M]//Rodgers J E, Whitman W B. Microbial Consumption of Greenhouse Gases. Washington, DC:American Society for Microbiology, 1991:225-275
[3]	STEPHEN J R, McCAIG A E, SMITH Z, et al. Molecular diversity of soil and marine 16S rRNA gene sequences related to β-subgroup ammonia-oxidizing bacteria[J]. Applied and Environmental Microbiology, 1996, 62(11):4147-4154 http://www.academia.edu/7169374/Molecular_Diversity_of_Soil_and_Marine_16S_rRNA_Gene_Sequences_Related_to_b-Subgroup_Ammonia-Oxidizing_Bacteria
[4]	WOESE C R, WEISBURG W G, HAHN C M, et al. The phylogeny of purple bacteria:The gamma subdivision[J]. Systematic and Applied Microbiology, 1985, 6(1):25-33 doi: 10.1016/S0723-2020(85)80007-2
[5]	ZUMFT W G. Cell biology and molecular basis of denitrification[J]. Microbiology and Molecular Biology Reviews, 1997, 61(4):533-616 http://www.doc88.com/p-1156630897554.html
[6]	宋亚娜, 林智敏, 林艳.氮肥对稻田土壤反硝化细菌群落结构和丰度的影响[J].中国生态农业学报, 2012, 20(1):7-12 http://www.ecoagri.ac.cn/zgstny/ch/reader/view_abstract.aspx?file_no=20120102&flag=1 SONG Y N, LIN Z M, LIN Y. Response of denitrifying bacteria community structure and abundance to nitrogen in paddy fields[J]. Chinese Journal of Eco-Agriculture, 2012, 20(1):7-12 http://www.ecoagri.ac.cn/zgstny/ch/reader/view_abstract.aspx?file_no=20120102&flag=1
[7]	LU H H, WU W X, CHEN Y X, et al. Decomposition of Bt transgenic rice residues and response of soil microbial community in rapeseed-rice cropping system[J]. Plant and Soil, 2010, 336(1/2):279-290 https://www.researchgate.net/profile/Janice_Thies/publication/226968948_Decomposition_of_Bt_transgenic_rice_residues_and_response_of_soil_microbial_community_in_rapeseed-rice_cropping_system/links/02e7e51b88b0c72735000000.pdf?origin=publication_detail
[8]	WU W X, YE Q F, MIN H, et al. Bt-transgenic rice straw affects the culturable microbiota and dehydrogenase and phosphatase activities in a flooded paddy soil[J]. Soil Biology and Biochemistry, 2004, 36(2):289-295 doi: 10.1016/j.soilbio.2003.09.014
[9]	宋亚娜, 陈在杰, 林智敏.水稻生育期内红壤稻田氨氧化微生物数量和硝化势的变化[J].中国生态农业学报, 2010, 18(5):954-958 http://www.ecoagri.ac.cn/zgstny/ch/reader/view_abstract.aspx?file_no=20105954&flag=1 SONG Y N, CHEN Z J, LIN Z M. Abundance of ammonia-oxidizer and potential nitrification rate of quaternary red-clay paddy soil during rice growth[J]. Chinese Journal of Eco-Agriculture, 2010, 18(5):954-958 http://www.ecoagri.ac.cn/zgstny/ch/reader/view_abstract.aspx?file_no=20105954&flag=1
[10]	袁飞, 冉炜, 胡江, 等.变性梯度凝胶电泳法研究我国不同土壤氨氧化细菌群落组成及活性[J].生态学报, 2005, 25(6):1318-1324 http://www.wenkuxiazai.com/doc/eca264ad2af90242a995e528-2.html YUAN F, RAN W, HU J, et al. Ammonia-oxidizing bacteria communities and their influence on the nitrification potential of Chinese soils measured by denaturing gradient gel electrophoresis (DGGE)[J]. Acta Ecologica Sinica, 2005, 25(6):1318-1324 http://www.wenkuxiazai.com/doc/eca264ad2af90242a995e528-2.html
[11]	WEI X D, ZOU H L, CHU L M, et al. Field released transgenic papaya effect on soil microbial communities and enzyme activities[J]. Journal of Environmental Sciences, 2006, 18(4):734-740 https://content.iospress.com/articles/journal-of-environmental-sciences/jes18-4-19
[12]	GRIFFITHS B S, CAUL S, THOMPSON J, et al. A comparison of soil microbial community structure, protozoa and nematodes in field plots of conventional and genetically modified maize expressing the Bacillus thuringiensis CryIAb toxin[J]. Plant and Soil, 2005, 275(1/2):135-146 http://www.academia.edu/4385380/A_qualitative_multi-attribute_model_for_assessing_the_impact_of_cropping_systems_on_soil_quality
[13]	SONG Y N, SU J, CHEN R, et al. Diversity of microbial community in a paddy soil with cry1Ac/cpti transgenic rice[J]. Pedosphere, 2014, 24(3):349-358 doi: 10.1016/S1002-0160(14)60021-7
[14]	宋亚娜, 苏军, 林艳, 等.转cry1Ac/cpti基因水稻对土壤氨氧化细菌群落组成和丰度的影响[J].生物安全学报, 2012, 21(1):67-73 http://www.cqvip.com/QK/83505A/201201/41183677.html SONG Y N, SU J, LIN Y, et al. Effect of cry1Ac/cpti transgenic rice on community composition and abundance of ammonia-oxidizing bacteria in paddy soil[J]. Journal of Bi-osafety, 2012, 21(1):67-73 http://www.cqvip.com/QK/83505A/201201/41183677.html
[15]	王建武, 骆世明, 冯远娇, 等.转Bt基因作物Bt毒素在土壤中的环境去向及其生态效应[J].生态学报, 2003, 23(4):797-804 http://www.cqvip.com/QK/93129X/200603/21951433.html WANG J W, LUO S M, FENG Y J, et al. Environmental fate and ecological effects of Bt toxin from transgenic Bt crops in soil[J]. Acta Ecologica Sinica, 2003, 23(4):797-804 http://www.cqvip.com/QK/93129X/200603/21951433.html
[16]	ZHOU X Y, HUANG Q Y, CHEN S W, et al. Adsorption of the insecticidal protein of Bacillus thuringiensis on montmorillonite, kaolinite, silica, goethite and red soil[J]. Applied Clay Science, 2005, 30(2):87-93 doi: 10.1016/j.clay.2005.04.003
[17]	SAXENA D, FLORES S, STOTZKY G. Bt toxin is released in root exudates from 12 transgenic corn hybrids representing three transformation events[J]. Soil Biology and Biochemistry, 2002, 34(1):133-137 doi: 10.1016/S0038-0717(01)00161-4
[18]	SIMS S R, HOLDEN L R. Insect bioassay for determining soil degradation of Bacillus thuringiensis subsp. kurstaki CryIA (b) protein in corn tissue[J]. Environmental Entomology, 1996, 25(3):659-664 doi: 10.1093/ee/25.3.659
[19]	DONEGAN K, PALM C J, FIELAND V J, et al. Changes in levels, species and DNA fingerprints of soil microorganisms associated with cotton expressing the Bacillus thuringiensis var. kurstaki endotoxin[J]. Applied Soil Ecology, 1995, 2(2):111-124 doi: 10.1016/0929-1393(94)00043-7
[20]	DONEGAN K K, SCHALLER D L, STONE J K, et al. Microbial populations, fungal species diversity and plant pathogen levels in field plots of potato plants expressing the Bacillus thuringiensis var. tenebrionis endotoxin[J]. Transgenic Research, 1996, 5(1):25-35 doi: 10.1007/BF01979919
[21]	白耀宇, 蒋明星, 程家安, 等.转Bt基因作物Bt毒蛋白在土壤中的安全性研究[J].应用生态学报, 2003, 14(11):2062-2066 doi: 10.3321/j.issn:1001-9332.2003.11.056 BAI Y Y, JIANG M X, CHENG J A, et al. Advances in safetystudies of soil Bt toxin proteins released from transgenic Bt crops[J]. Chinese Journal of Applied Ecology, 2003, 14(11):2062-2066 doi: 10.3321/j.issn:1001-9332.2003.11.056
[22]	FIRESTONE M K, DAVIDSON E A. Microbial basis of NO and N2O production and consumption in soils[M]//Andreae M O, Schimel D S. Exchange of Trace Gases Between Terrestrial Ecosystems and the Atmosphere. New York:John Wiley, 1989:7-21
[23]	ZHU X, BURGER M, DOANE T A, et al. Ammonia oxidation pathways and nitrifier denitrification are significant sources of N2O and NO under low oxygen availability[J]. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(16):6328-6333 doi: 10.1073/pnas.1219993110
[24]	朱永官, 王晓辉, 杨小茹, 等.农田土壤N2O产生的关键微生物过程及减排措施[J].环境科学, 2014, 35(2):792-800 http://www.ecologica.cn/stxb/ch/html/2015/11/stxb201310082413.htm ZHU Y G, WANG X H, YANG X R, et al. Key microbial processes in nitrous oxide emissions of agricultural soil and mitigation strategies[J]. Environmental Science, 2014, 35(2):792-800 http://www.ecologica.cn/stxb/ch/html/2015/11/stxb201310082413.htm