万顺铅锌矿区蜈蚣草内生细菌砷氧化基因(aox B)的多样性分析
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摘要
【背景】近年来,由于金属矿的开采和冶炼、砷产品的加工与使用、煤的燃烧等各种因素,导致土壤环境中的砷污染越来越严重,导致许多人暴露于极度危险的砷毒毒害之下。【目的】研究四川的万顺铅锌矿区蜈蚣草根组织内生细菌aoxB基因的多样性,为提高土壤重金属污染生态修复效率提供理论依据。【方法】利用实时荧光定量PCR (Real-time quantitative PCR,qPCR)和限制性片段长度多态性(Restriction fragment length polymorphism,RFLP)技术,对四川省汉源县万顺铅锌矿区蜈蚣草根组织内生细菌aoxB基因表达量及多样性进行研究。【结果】qPCR结果表明,不同采样点间的蜈蚣草根组织内生细菌aoxB基因表达量存在明显差异,表现为选矿区>进山口>弃渣场>尾矿区>矿口。酶切图谱结果表明,不同采样点蜈蚣草根组织内生细菌aoxB基因多样性存在明显差异,多样性指数表现为尾矿>矿口>弃渣场>进山口>选矿区。Pearson相关分析显示,aoxB基因的表达量与重金属As之间呈显著负相关(P<0.05),多样性指数则与重金属Pb和As之间呈极显著正相关(P<0.01)。系统发育分析显示,aoxB基因的优势菌群为α-变形菌门(Alphaproteobacteria)。【结论】蜈蚣草根组织中存在丰富的含aoxB基因内生细菌种群,这些内生细菌表现出潜在的应用价值。
[Background] In recent years, due to the mining and smelting of metal ores, the processing and use of arsenic products, the burning of coal and other factors, arsenic pollution in the soil environment has become more and more serious, causing many people to be exposed to extremely dangerous arsenic poisoning. [Objective] To study the diversity of endophytic bacteria aoxB gene in Pteris vittata root of Wanshun Pb-Zn mining area in Sichuan, and provide a theoretical basis for improving the efficiency of soil heavy metal pollution ecological restoration. [Methods] Real-time quantitative PCR(qPCR) and restriction fragment length polymorphism(RFLP) were used to study the abundance and diversity of arsenic oxidation gene(aoxB) of the endophytic bacteria in Pteris vittata root which were isolated from a Pb-Zn mining area in Hanyuan of Sichuan province, China. [Results] The results of QPCR showed that there was a significant difference in the expression of aoxB genes between the different sampling sites. The abundance aoxB gene were in the order of dressing area>entrance to the mountain>spoil area>mine tailings>mine mouth. The RFLP results showed that there were significant differences in the diversity index of the aoxB gene of endophytic bacteria among different sampling sites, and the diversity index of the species were in the order of mine tailings>mine mouth>spoil area>outside mine area>mine mouth. Pearson correlation analysis showed that the abundance of aoxB genes was negatively correlated with concentration of As(P<0.05), and the diversity index was positively correlated with concentration of Pb and As(P<0.01). Phylogenetic analysis showed that aoxB containing endophytic bacteria in the Pteris vittata were mainly belonged to Alphaproteobacteria. [Conclusion] The results showed that there were abundant endophytic bacterial populations containing aoxB gene in Pteris vittata root, and these endophytic bacteria showed potential application value.
[Background] In recent years, due to the mining and smelting of metal ores, the processing and use of arsenic products, the burning of coal and other factors, arsenic pollution in the soil environment has become more and more serious, causing many people to be exposed to extremely dangerous arsenic poisoning. [Objective] To study the diversity of endophytic bacteria aoxB gene in Pteris vittata root of Wanshun Pb-Zn mining area in Sichuan, and provide a theoretical basis for improving the efficiency of soil heavy metal pollution ecological restoration. [Methods] Real-time quantitative PCR(qPCR) and restriction fragment length polymorphism(RFLP) were used to study the abundance and diversity of arsenic oxidation gene(aoxB) of the endophytic bacteria in Pteris vittata root which were isolated from a Pb-Zn mining area in Hanyuan of Sichuan province, China. [Results] The results of QPCR showed that there was a significant difference in the expression of aoxB genes between the different sampling sites. The abundance aoxB gene were in the order of dressing area>entrance to the mountain>spoil area>mine tailings>mine mouth. The RFLP results showed that there were significant differences in the diversity index of the aoxB gene of endophytic bacteria among different sampling sites, and the diversity index of the species were in the order of mine tailings>mine mouth>spoil area>outside mine area>mine mouth. Pearson correlation analysis showed that the abundance of aoxB genes was negatively correlated with concentration of As(P<0.05), and the diversity index was positively correlated with concentration of Pb and As(P<0.01). Phylogenetic analysis showed that aoxB containing endophytic bacteria in the Pteris vittata were mainly belonged to Alphaproteobacteria. [Conclusion] The results showed that there were abundant endophytic bacterial populations containing aoxB gene in Pteris vittata root, and these endophytic bacteria showed potential application value.
引文
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[5] Huang YF, Chen GF, Xiong LM, et al. A review of soil heavy metal pollution status of cultivated land and analysis of phytoremediation application[J]. Journal of Anhui Agricultural Sciences, 2015, 43(26):88-89(in Chinese)黄雁飞,陈桂芬,熊柳梅,等.耕地土壤重金属污染现状及植物修复的应用[J].安徽农业科学, 2015, 43(26):88-89
[6] Shen HL, He ZY, Ma M. Advance of the mechanisms of arsenic hyperaccumulation in Pteris vittata L. and applications for arsenic-remediation[J]. Plant Physiology Journal, 2014, 50(5):591-598(in Chinese)申红玲,何振艳,麻密.蜈蚣草砷超富集机制及其在砷污染修复中的应用[J].植物生理学报, 2014, 50(5):591-598
[7] Liu WJ, Zhao FJ. A brief review of arsenic uptake and metabolism in plants[J]. Environmental Chemistry, 2011, 30(1):56-62(in Chinese)刘文菊,赵方杰.植物砷吸收与代谢的研究进展[J].环境化学, 2011, 30(1):56-62
[8] Chen TB, Wei CY, Huang ZC, et al. Arsenic hyperaccumulator Pteris vittata L. and its arsenic accumulation[J]. Chinese Science Bulletin, 2002, 47(11):902-905(in Chinese)陈同斌,韦朝阳,黄泽春,等.砷超富集植物蜈蚣草及其对砷的富集特征[J].科学通报, 2002, 47(3):207-210
[9] Wang HJ, Ning P, Zhang ZB, et al. Pretreatment of arsenic gold ore by Pteris vittata L.[J]. Journal of Wuhan University of Technology, 2010, 32(8):59-63(in Chinese)王海娟,宁平,张泽彪,等.含砷金矿的蜈蚣草除砷预处理初步研究[J].武汉理工大学学报, 2010, 32(8):59-63
[10] Hallmann J, Morgenroth W, Paulmann C, et al. Time-resolved X-ray diffraction of the photochromicα-styrylpyrylium trifluoromethanesulfonate crystal films reveals ultrafast structural switching[J]. Journal of the American Chemical Society, 2009, 131(41):15018-15025
[11] Luo MY, Lu XJ, Liao XL. Research advances on application of endophytic bacteria[J]. Modern Agricultural Sciences and Technology, 2010(7):11-12,16(in Chinese)罗茗月,路雪君,廖晓兰.植物内生细菌的应用研究进展[J].现代农业科技, 2010(7):11-12,16
[12] Liu LH, Liu SJ, Chen FM, et al. Effect of endophytic bacteria inoculation on cadmium uptake in Solanum nigrum L.[J]. Acta Scientiae Circumstantiae, 2013, 33(12):3368-3375(in Chinese)刘莉华,刘淑杰,陈福明,等.接种内生细菌对龙葵吸收积累镉的影响[J].环境科学学报, 2013, 33(12):3368-3375
[13] Zhang XC. Isolation and identification of endophytes from Sedum alfredii and the mechanisms of their enhancement on heavy metal hyperaccumulation[D]. Hangzhou:Doctoral Dissertation of Zhejiang University, 2012(in Chinese)张新成.东南景天内生菌分离鉴定及其强化重金属超积累效应与机制[D].杭州:浙江大学博士学位论文, 2012
[14] Ma Y, Rajkumar M, Luo YM, et al. Inoculation of endophytic bacteria on host and non-host plants—Effects on plant growth and Ni uptake[J]. Journal of Hazardous Materials, 2011, 195:230-237
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