【作者】 徐舟影；

【导师】 唐明；

【作者基本信息】 西北农林科技大学 ， 微生物学， 2013， 博士

【摘要】 本文采用变性梯度凝胶电泳（DGGE）技术，研究了陕西凤县铅硐山铅锌矿区优势植物狼牙刺（Sophora viciifolia Hance.）根际细菌和丛枝菌根（Arbuscular mycorrhizal，AM）真菌群落结构。利用透射电镜（TEM）、X射线能量散布分析仪（EDS）、实时荧光定量PCR（qRT-PCR）等技术，从球囊霉素螯合重金属、AM超微结构Pb定位、Pb胁迫下AM真菌对宿主植物络合素合酶基因表达的影响等方面探讨了AM真菌提高狼牙刺耐Pb性能的机制。主要结果如下：1.铅锌矿区狼牙刺根际细菌群落结构陕西凤县铅硐山铅锌矿区不同Pb污染程度的5个样地狼牙刺根际细菌多样性随着土壤污染程度的加剧而降低，DGGE图谱上的条带数从22个（污染程度最轻样地）降至13个（污染程度最严重样地）。土壤总Pb、有效Pb和细菌群落的香侬指数（H’）极显著和显著负相关。Redundancy analysis（RDA）分析显示，土壤有效氮、有效Pb和有效磷对细菌群落结构影响较大，特征值分别为21.5%、17%和16.5%。从细菌DGGE胶上回收的15个条带中，11个属于变形菌门（Proteobacteria）中的不动杆菌属（Acinetobacter）、沙雷氏菌属（Serratia）和单胞菌属（Sphingomonas），3个属于厚壁菌门（Firmicutes）中的气球菌属（Aerococcus），1个属于放线菌属（Actinobacteria），优势细菌为变型菌门（Proteobacteria）。2.铅锌矿区狼牙刺根际AM真菌群落结构及球囊霉素螯合重金属含量不同Pb污染程度5个样地狼牙刺根系的AM真菌侵染率随着土壤Pb浓度的升高而降低。DGGE图谱上的条带数随着污染程度的加剧从31个降到13个。土壤中有效Pb含量与AM真菌的H’极显著负相关。RDA分析表明土壤总Pb、有效磷、有效氮和pH对AM真菌群落结构影响较大，特征值分别达到22.5%、20.1%、18.5%和6.2%。优势AM真菌为球囊霉科（Glomeraceae）球囊霉属（Glomus）和多孢囊霉科（Diversisporaceae）多孢囊霉属（Diversispora），在鉴定的13个序列中，10个属于球囊霉属（Glomus），2个属于多孢囊霉属（Diversispora），1个属于子囊菌门（Ascomycota）（非AM真菌）。随着土壤Pb浓度的升高（57.5~6295.2mg/kg），总球囊霉素（GRSP）含量由2.9增加至6.8mg/g风干土，螯合的Pb含量为3.29~172.46mg/kg，占土壤Pb含量的2.74%~5.72%。GRSP螯合的Pb含量与土壤中总Pb、有效Pb含量分别极显著和显著正相关。球囊霉素在土壤中螯合Pb离子是AM真菌提高狼牙刺耐Pb的机制之一。3. AM真菌对狼牙刺生长与吸Pb的影响及Pb在菌根超微结构中的定位接种AM真菌摩西球囊霉（Glomus mosseae）于狼牙刺实生苗，结果显示，低浓度Pb（50μg/g）能够促进狼牙刺幼苗生长，高浓度Pb（500和1000μg/g）则抑制狼牙刺幼苗生长。接种G. mosseae降低了狼牙刺地上和地下部的Pb浓度，其中1000μg/gPb胁迫时，接种幼苗地上和地下部Pb浓度与对照相比降低了61.0%和15.2%。1000μg/g Pb胁迫时，接种幼苗的根长、根分叉数、根尖数、根表面积和根体积分别比对照提高220%、219%、157%、225%和278%。Pb胁迫下接种G. mosseae显著降低了直径0~0.2mm根长占总根长的百分比，而提高了直径0.4~0.6、0.6~0.8和0.8~1.0mm根长占总根长的百分比，即Pb胁迫条件下接种AM真菌有使根系变粗的趋势。通过TEM和EDS技术对接种处理狼牙刺根系进行Pb的AM超微结构定位，发现Pb不仅能够沉积在狼牙刺根细胞内，还能沉积在AM真菌根内菌丝的细胞壁和空泡中，表明AM真菌在亚细胞水平上通过将重金属螯合在菌体细胞内从而增加狼牙刺的耐Pb能力。4.狼牙刺植物络合素合酶及内参基因的克隆通过Rapid-amplification of cDNA ends（RACE）方法克隆狼牙刺的植物络合素合酶基因（PCS1）及内参基因（Actin）。SvPCS1cDNA全长为2098bp，含有1503bp的开放阅读框（ORF），编码501个氨基酸，预测分子量为55.46kDa，等电点为7.13。SvPCS1编码的氨基酸序列与LjPCS1（百脉根，AAT80342）和GmPCS1（大豆，AAL78384）的同源性分别达到84.63%和85.43%。SvActin cDNA全长为1679bp，ORF为1134bp，编码378个氨基酸，预测分子量为41.65kDa，等电点为5.05。SvActin编码的氨基酸序列与RcActin（蓖麻，AAR15174）和TpActin（三叶草，AAQ74875）的同源性分别达到98.67%和98.42%。SvPCS1与SvActin的NCBI基因登录号分别为JQ780609和JQ780610。5. Pb胁迫下AM真菌对狼牙刺植物络合素合酶基因表达的影响通过Real-time quantitative PCR（qRT-PCR）技术，研究0、50和200μMPb(NO3)2胁迫1、3和7d后，接种G. mosseae对狼牙刺叶片叶绿素荧光参数、根系植物络合素合酶基因（SvPCS1）表达量和植物络合素（PCs）合成量的影响。结果显示，Pb胁迫后不接种处理狼牙刺叶片叶绿素荧光参数Fv/Fm、Fv/Fo、qP和Y(Ⅱ)随着Pb浓度的升高逐渐降低，NPQ随着Pb浓度的升高逐渐增加；接种处理各参数的变化趋势与不接种处理相同，但是变化幅度较小，说明接种G. mosseae缓解了植物所受Pb毒害。Pb胁迫1d和3d时，接种G. mosseae增加了狼牙刺根系SvPCS1的表达量和PCs的合成量，当胁迫7d时，0和50μM的Pb胁迫下接种处理SvPCS1的表达量和PCs的合成量仍然高于对照，200μM的Pb胁迫下低于对照，表明Pb胁迫一定时间和一定浓度内接种G. mosseae可以增加SvPCS1的表达量和PCs的合成量。更多还原

【Abstract】 The arbuscular mycorrhizal (AM) fungal and bacterial community structures in therhizospere of the dominant plant Sophora viciifolia Hance. grown at Qiandongshan lead andzinc mine were investigated using PCR-DGGE. Using the technologies of transmissionelectron microscope (TEM), energy dispersive X-ray spectroscopy (EDS) and qutitativereverse transcription polymerase chain reaction (qRT-PCR), we discussed the mechnisms ofAM fungi improving S. viciifolia’s Pb resistance ability in the aspects of ultrastructurallocalization of Pb in AM, Pb chelation by glomalin related soil protein (GRSP) and the S.viciifolia phytochelatin synthase gene expression pattern impacted by AM fungi under Pbthreat. The main results are as follows:1. The bacterial community structure in the rhizospere of S. viciifolia grown at fiveincreasing Pb concentration sitesIn the five sites with increasing soil Pb concentration, the diversity of bacteria declinedwith increasing Pb concentration, the band number in DGGE profile decreased from22to13.Bacterial community H′was negatively correlated with the total and available Pbconcentrations in the soil. RDA analysis revealed that the available nitrogen, the available Pb,the available P affected the bacterial community mostly, with eigenvalues of21.5%,17%and16.5%, respectively. Among the fifteen species cloned from the gel, eleven wereAcinetobacter, Serratia and Sphingomonas in Proteobacteria; three were Aerococcus inFirmicutes and the other one was Actinobacteria in Actinobacteria.2. The AM fungi community structure in the rhizospere of S. viciifolia grown at fiveincreasing Pb concentration sites and the heavy metal concentration chelated by glomalinrelated soil proteinAM root colonization decreased from30.5%to9.6%with the increase of the Pbconcentration in soil. The band number in the DGGE gel decreased from31to13. The H′ofthe AM fungal community showed a significant negative correlation with the available Pbconcentration The total soil Pb concentration, the available P, the available N and pH affectedthe AM fungi community structure mostly. The dominant AM fungi is Glomus in Glomeraceae and Diversispora in Diversisporaceae, among the thirteen sequences clonedfrom the gel, ten were Glomeraceae; two were Diversisporaceae and the other one was non-AM fungi.Total GRSP (2.9~6.8mg/g dry soil) increased with the increase of the Pb concentration(57.5~6295.2mg/kg) in soil. The amount of Pb bound to GRSP varied from3.3to172.5mg/kg, which positively correlated with total and available soil Pb concentration, thus reducingthe bioavailability of Pb.3. The influence of AM fungi inoculation on the growth and Pb uptake of S. viciifoliaand the Pb ultrastructure localizationThe seedlings of S. viciifolia were inoculated with Glomus mosseae under different Pbappication levels, results showed low Pbconcentration (50μg/g) promoted the growth of S.viciifolia seedlings, but higher (500and1000μg/g) inhibited. Compared with non-inoculationtreatment, G. mosseae inoculation decreased both the Pb concentrations of aboveground andbelowground, the Pb concentrations of aboveground and belowground of the mycorrhizal S.viciifolia were118.48and47.49μg/g when exposed to1000μg/g Pb, decreased by61.0%and15.2%. The root length, root forks, root tips, root surface and root volumn of mycorrhizalS. viciifolia were higher than corresponding non-mycorrhizal plants. Compared with non-mycorrhizal plant, these parameters of mycorrhizal plants increased by220%、219%、157%、225%和278%when exposed to1000μg/g Pb(NO3)2. The ratio of root length whosediameters were between0~0.2mm to the total root length significantly increased with theincreasing Pb additions, and G. mosseae inoculation significantly reduced the ratio. Under Pbstress, G. mosseae increased the ratios of root length with0.4~0.6,0.6~0.8and0.8~1.0mmdiameters to the total root length, indicating that AM fungi inoculation thicken the roots with0.4~1.0mm diameter under Pb additions.The combination data of TEM and EDS indicated that Pb can deposited not only in plantcells, but also the cell walls and vacuoles of the AM fungi intracellular hyphae, whichrevealling that the subcellular level mechanism of AM fungi alleviated the Pb toxicity to host.4. Cloning the genes of SvPCS1and SvActinThe full length of SvPCS1cDNA is2098bp, containing a1503bp length ORF, coding501amino acids, predicted molecular weight is55.46kDa and the isoelectric point is7.13.The deduced amino acids of SvPCS1showed84.63%homology with LjPCS1(Lotusjaponicas, AAT80342), and85.43%with GmPCS1(Glycine max, AAL78384). The fulllength of SvActin cDNA is1679bp, including an1134bp length ORF, coding378aminoacids, predicted molecular weight is41.65kDa and the isoelectric point is5.05. Thehomology of SvActin with RcActin (Ricinus communis, AAR15174) and TpActin (Trifolium pretense, AAQ74875) reached98.67%and98.42%, respectively. The NCBI gene bankaccessing numbers of SvPCS1and SvActin are JQ780609and JQ780610, respectively.5. The influence of AM fungi inoculation on SvPCS1expressionThe response of SvPCS1expression, chlorophyll fluorescence parameters and phyto-chelatins (PCs) to G. mosseae inoculation under Pb stress (0,50and200μM Pb(NO3)2) atdifferent durations (1,3and7day) were studied. The chlorophyll fluorescence parametersFv/Fm, Fv/Fo, qP and Y(Ⅱ) of non-mycorrhizal S. viciifolia decreased and NPQ rose with theincreasing Pb concentration, all the parameters of mycorrhizal S. viciifolia showed the samechange patterns with non-mycorrhizal, but smaller change, indicating that mycorrhizalsymbiosis alleviated the Pb toxicity to plants. When S. viciifolia was exposed to0,50and200μM Pb for1and3d, G. mosseae inoculation promoted the expression of SvPCS1andsynthesis of PCs; when the duration increased to7d, SvPCS1expressions and PCs productionexposed to0and50μM Pb were still higher than controls, but lower in the S. viciifoliaexposed to200μM Pb, indicating that G. mosseae inoculation promoted SvPCS1expressionand SvPCs production under Pb threat for a certain period of time.更多还原