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题名: 植物相关微生物的富集方法及其多样性的分子特征
作者: 焦军影
学位类别: 硕士
答辩日期: 2005
授予单位: 中国科学院昆明植物研究所
授予地点: 中国科学院昆明植物研究所
导师: 曾英
关键词: 美登木 ; 微生物富集 ; 滑桃树 ; 相关细菌 ; 多样性
中文摘要: 本论文探索了植物相关微生物(包括内生和表生,可培养的和不可培养的)的富集方法。对于可培养微生物,通过特定的培养基进行选择性培养,从而达到富集效果;而如何从植物体内富集不可培养微生物,尚无报道。我们以美登木(Maytenus serrata)叶片为试验材料,通过纤维素酶和离析酶等酶解以及差速离心的方法富集了美登木叶片相关微生物。利用PcR扩增植物核基因ITS序列,原核微生物的16S-23S转录单元内间隔区和叶绿体trnL-trnF基因来初略估计植物核基因组、叶绿体基因组和原核微生物基因组在各级沉淀中提取的总DNA相对丰度;运用165 rDNA技术,建立原核微生物165 rDNA文库分析富集沉淀的细菌生物多样性检测富集效率。将液氮研磨好的材料悬浮于纤维素酶和离析酶酶反应液中(1320mg/LCaCl2·2H2O,88.3mg/L KH2PO4,0.7mol/L甘露醇,1g/L 2-N-吗琳乙烷磺酸,10g/L纤维素酶,1g/L离析酶),搅拌均匀,于140r/min,28℃的条件下反应12小时。然后在15℃的条件下逐级离心,去500g离心力下的沉淀,收取3000g离心力下的沉淀(500g离心15min。3000g离心20min)。在3000g沉淀中,通过16SrDNA克隆测序分析,挑取的114个阳性克隆中,有103个克隆属于细菌1 6S rDNA克隆,而微生物未经富集的对照实验中,挑取的110个阳性克隆中,只有2个克隆属于细菌16S rDNA克隆。说明此方法有效地富集了美登木叶片相关微生物。我们探索到植物相关微生物富集的有效方法,为研究植物相关微生物多样性排除植物DNA干扰提供了有效途径;为建立植物相关微生物宏基因组文库打下基础。本论文还利用16S rDNA技术,建立微生物16S rDNA基因文库,通过富集滑桃树(Trewia nudiflora)茎皮、根皮、幼嫩种子和成熟种子相关细菌和不富集两种途径比较滑桃树茎皮、根皮、幼嫩种子和成熟种子相关细菌生物多样性。再次证明了我们探索的植物相关微生物富集方法是有效的。在我们挑取到的100多个茎皮、根皮和成熟种子相关细菌16SrDNA克隆里都含有Gammaproteobacteria16SrDNA克隆,而且占有优势。幼嫩种子得到两个细菌16SrDNA克隆属于不可培养的细菌;茎皮112个细菌16S rDNA克隆都属于Gammaproteobacteria;根皮113个细菌16S rDNA克隆大部分属于Gammaproteobacteria,此外还含有Alphaproteobacteria、放线菌(Actinobacteria)和不可培养或分类地位不明确的细菌;成熟种子91个细菌16S rDNA克隆除了含有Gammaproteobacteria、Alphaproteobacteria和不可培养或分类地位不明确的细菌,还含有Betaproteobacteria、杆菌(Bacilli)和梭状芽胞杆菌(clostridia)。可见,成熟种子含有丰富的相关细菌,根皮次之,幼嫩种子最少。
英文摘要: The enrichment for microbes associated with plant tissues was studied in this thesis. The microbes include the epiphytic and endophytic microbes, the cultured and uncultured microbes. The cultured microbes were enriched by selective media for cultivation of the microbes. There is no report about how to enrich for uncultured microbes associated with the plant tissues. We enriched the microbes associated with the leaves of Maytenus hookeri by enzyme treatment with cellulase and macerozyme as well as differential centrifugation. Theoretically, the DNA isolated from plant tissues may derive from the plant nuclei, the plastids, the mitochondria and if any, the plant-associated microbes. Three marker genes including the entire ITS (nuclei-specific), the trnL-trnF spacer (plastid-specific) and 16S-23S gene spacer (bacterium-specific) were amplified by PCR using an Eppendorf Mastercycler gradient, in order to estimate qualitatively the relative abundance of the DNA from different genomes. Enriching efficiency is estimated on the proportion of bacterium-derived clones and their RPLP types as detected by 16S rDNA techniques. The plant tissue was ground, frozen in liquid nitrogen, added in the cellulase and macerozyme buffer(1320mg/L CaCl2'2H20, 88.3mg/L KH2PO4, 0.7mol/L mannitol, 1 g/L MES, 1 Og/L cellulase, 1 g/L macerozyme) and incubated at 28 °C for 12 hours at 140r/min, then centrifugated at 500g for 15 min to sediment the plant cell, nucleus and relics. The supernatant was centrifugated at 3000g for 20 min to sediment the bacteria. There were 103 clones fallen into the bacterium in 114 clones derived from the pellet at 3000g, but there were only 2 clones fallen into the bacterium in 110 clones derived from the leaves (no enriching) .This method is effective to enrichment for microbes associated with plant tissues, which offers an effective approach for the study of the diversity of the endophytes by decreasing the chloroplast DNA and lays solid foundations for construction of the metagenomic library of the endophytes. The enriching method was successfully applied in the study of microbial diversity of Trewia nudiflora stem barks, root barks and seeds. Based on the 16S rDNA analyses, most of clones fall into the Gammaproteobacteria in over 300 clones derived from those organs. Two clones derived from the young seed fall into the unculturable bacteria. 112 clones derived from the stem bark all showed high similarity with members of the Gammaproteobacteria, whereas 113 clones derived from the root bark were highly related to the Alphaproteobacteria and to the Actinobacteria except the Gammaproteobacteria which was majority. 91 clones derived from the mature seeds fall into the Alphaproteobacteria, Betaproteobacteria, Bacilli, Clostridia, unculturable bacteria and unclassified bacteria. It is obvious that the mature seeds possessed a significantly higher diversity of plant-associated bacteria, compared to the young seeds.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.kib.ac.cn/handle/151853/810
Appears in Collections:昆明植物所硕博研究生毕业学位论文_学位论文

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植物相关微生物的富集方法及其多样性的分子特征.焦军影[d].中国科学院昆明植物研究所,2005.20-25
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