【作者】 程静；

【导师】 杨永华； 陆桂华；

【作者基本信息】 南京大学 ， 微生物与生化药学， 2017， 硕士

【摘要】 随着转基因技术的快速发展,转基因作物也因此越来越普及,至2015年全球转基因作物累计种植面积已达到20亿公顷,其中转基因大豆全球种植总面积最大。转基因大豆在给社会带来巨大经济效益同时,其对环境与生态的安全性问题包括对土壤微生物多样性的影响也逐渐引起人们的重视。在土壤和植物及其互作系统中,一方面,土壤根际微生物无疑起着十分重要又不可替代的作用,另一方面,植物通过根系分泌物也影响着根际微生物的多样性。转基因作物的外源基因及其表达产物在土壤生态系统中的累积很可能会对土壤中的微生物产生一定程度的影响,因此在对转基因大豆进行环境安全性评价时,研究其对土壤微生物群落多样性变化的影响,特别是对土壤固氮相关微生物群落的影响是十分必要的。本研究选取的种植点及大豆品种分别为:北京市顺义实验基地种植点清水处理的转EPSPS和GAT基因大豆品种中黄10-6(ZH10-6)和受体品种中黄10号以及喷洒一定剂量草甘膦的转基因品种中黄10-6剂量。研究内容:测定不同大豆样品的根围土壤理化性质;大豆植株、根围土的碳、氮含量;根围土壤碳、氮循环相关酶活;根际土可培养固氮菌数量;土壤固氮菌群的结瘤效应以及nifH基因丰度表征的土壤固氮菌群总体丰度变化等指标,分析了转EPSPS和GAT基因抗除草剂大豆对土壤固氮菌群的影响。同时还对受体品种和转基因品种的根围土、根际土和根部微生物总DNA进行16S rDNA(V4区)扩增子进行高通量测序,深入分析了转EPSPS和GAT基因抗除草剂大豆对土壤微生物群落多样性的影响。主要研究结果:1.实验数据表明,不同处理的转EPSPS和GAT基因抗除草剂大豆ZH10-6与受体品种中黄10号相比,在土壤和植株的C、N含量、土壤固氮酶活性、可培养固氮菌数量均没有显著性差异。此外,nifH基因实时定量PCR实验结果表明,与受体品种相比,不同处理的转EPSPS和GAT基因抗除草剂大豆的根际固氮菌群的相对丰度无显著差异,仅在花期根围土的固氮菌群的相对丰度有显著差异。2.16S rDNA(V4区)扩增子高通量测序及分析结果表明,与受体品种相比,不同处理的转EPSPS和GAT基因抗除草剂大豆的根围土壤细菌、根际细菌和大豆根内生细菌的Alpha多样性各指数在大豆的苗期、花期没有显著差异,仅在鼓粒期有显著差异。根据PCA、PCoA、NMDS分析,与受体品种相比,不同处理的转EPSPS和GAT基因抗除草剂大豆的根围土壤细菌、根际细菌和大豆根内生细菌的Beta多样性在大豆的苗期、花期、鼓粒期没有显著差异。结论:综合以上数据分析可知,在这一生长季节中,种植转EPSPS和GAT基因抗除草剂大豆中黄10-6(ZH10-6)或者对其喷洒单次的草甘膦基本上对根际土壤原核微生物群落不产生显著影响,另由于田间管理欠佳,导致三种处理的大豆在不同生长阶段长势较差,以上结论还需要多年多点进一步的实验验证。更多还原

【Abstract】 With the development of plant transgenic technology,more and more genetically modified(GM)crops have been.The cumulative area of global GM crops has reached 2 billion hectares,in which the cultivation area of GM soybean is the largest.Although the transgenic soybean varieties have brought great economic and social benefits to society,its environmental problems including the impact on the diversity of soil microbes have gradually attracted worldwide attention.In the soil and plants interaction system,on one side,the soil rhizosphere microorganisms undoubtedly play a very important and irreplaceable role.on other side,plant species strongly influence the diversity of rhizosphere microorganisms.Therefore,it is necessary to research the impact of the exogenous genes and expression products of GM soybean on rhizosphere microbial community,including soil nitrogen fixationbacteria.Experimental field and tested soybeans:The EPSPS&GAT transgenic herbicide-tolerant soybeans ZH10-6,its acceptor soybeans Zhonghuangl0(or simply ZH10),were planted at the Shungyi experimental base in BeiJing.Research contents:We measured soil physical and chemical properties,nitrogen content,soil carbon and nitrogen cycling-related enzyme activity of surrounding soil,we also measured soil nitrogen-fixing bacteria diversity,nodulation effect of the nitrogen-fixing bacteria group,and nitrogen-fixing bacteria abundance of different soybean rhizosphere soil via quantitave PCR of nifH.Besides,high-throughput sequencing of 16S rDNA(V4)amplicons has been performed for clarifying the impact of the EPSPS&GAT transgenic herbicide-tolerant soybeans ZH10-6 on the rhizosphere bacterial diversity compared to its acceptor soybeans ZH10.Main results:1.Our experimental data showed that there were no significant differences in the C or N content of soil and plants,soil nitrogenase activity,nodule nitrogenase activity,and the diversity of culturable nitrogen fixing bacteria.Additionally,no significant difference in the relative abundance of rhizosphere nitrogen-fixing bacteria,which was characterized by quantitative real-time PCR of nifH gene,between the EPSPS&GAT transgenic herbicide-tolerant soybeans ZH10-6 and its acceptor soybean ZH10at different developmental stages,except that there was a significant difference in the relative abundance of nitrogen-fixing bacteria in the surrounding soil between the transgenic ZH10-6 and its acceptor ZH10 at flowering stage.2.Analysis of 16S rDNA(V4 region)amplicons high-throughput sequencing data showed there were no significant differences in most of indices of alpha diveristy between the surrounding soil,rhizosphere soil and roots endophyte of the transgenic line ZH10-6 treated by water or glyphosate herbicide and those of its acceptor ZH10 treated by water only at seedlings,flowering,and pod-setting stage except the simposon index at pod-setting stage.Furthermore,based on the results of PCA,PCoA,and NMDS,there were also no significant differences in beta diveristy between the surrounding soil,rhizosphere soil and roots endophyte of the transgenic line ZH10-6 treated by water or glyphosate herbicide and those of its acceptor ZH10 treated by water at there developmental stage.Conclusion:Based on the above data analysis,the cultivation of the EPSPS&GAT transgenic herbicide-tolerant soybeans or treated once by glyphosate basically had no significant impact on soil rhizosphere microbial communities during the single growth season.In addition,the above conclusion need to be confirmed by more field experiment at different location due to poor field management in this year.更多还原