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研究生:王譽臻
研究生(外文):Yu-ChenWang
論文名稱:非生物逆境下白背芒根部關聯菌之多源基因體解析
論文名稱(外文):Deciphering root-associated microbiomes in Miscanthus sinensis var. glaber under abiotic stresses with metagenomics
指導教授:蔣鎮宇蔣鎮宇引用關係
指導教授(外文):Tzen-Yuh Chiang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:57
中文關鍵詞:芒屬多源基因體學根內微生物根際微生物16S rRNA轉錄體非生物逆境
外文關鍵詞:Miscanthus sinensisMetagenomicsEndosphereRhizosphere16SrRNAAbiotic stress
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台灣(Taiwan)複雜的天然地形塑造多樣的生態系,少數生物具有廣泛的適應能力而得以出現在不同的環境中,經常可見的芒草(Miscanthus)便是其中的代表。芒草在台灣分成兩種芒草複合群,五節芒(Miscanthus floridulus)和中國芒(Misanthus sinensis),其中中國芒複合群顯現多元的環境適應性,特別是分布最廣的白背芒(M. sinensis var. glaber),具備重金屬耐性、生長快速、遺傳歧異度高與糧食作物同為禾本科的遺傳背景更顯示芒草具有促進經濟發展的可應用性,植物的微生物群使植物提高對逆境的耐受性並增加個體的代謝功能,而芒草的根內和根際微生物間的交互作用是如何適應重金屬逆境目前仍在剖析中。
因此在本研究在台灣新店區獅子頭山(XD)、士林區小油坑(YMmS)和瑞芳區金瓜石(JFCu)採集,運用16S分子標籤和次世代定序解構芒草根內與根際的共生微生物相組成。NMDS結果發現在根內和根際間YMmS和JFCu兩種逆境與XD一般環境都存在差異。相較其他根際樣點,JFCu中放線菌綱(Actinobacteria)、硝化螺旋菌綱(Nitrospira)和熱原體綱(Thermoplasmata)的相對比例最多,反之酸桿菌綱(Acidobacteriia)比例最低,其菌綱已知在酸性環境下具有高耐受性且在重金屬逆境下會和金屬螯合而保護植物。在相關性分析部分,在根際樣本的物種多樣性和土壤酸鹼值有高度相關性,顯示土壤酸鹼值為影響芒草根際微生物組成的重要因子,核心菌群分析發現,在根內樣本中找到710個OTU(佔整體8.9%),根際樣本中找到1693個OTU(佔整體14%),其中最大宗的為變形菌門且特別在JFCu根際中α-、β-和δ-變形綱的比例最高而γ-變形綱則比例最低,透過本研究發現白背芒可能透過變形菌綱、放線菌綱、硝化螺旋菌綱和熱原體綱的協助,而提高其在銅逆境與硫逆境的適應能力。
Owing to the industrialization, environmental contamination has been getting worse year by year all around the world. Adaption of plants, coupled with co-existing microbes against contaminants may have evolved under various stresses. Here, at diverse habitats under sulfur-rich and copper-rich stresses, investigating endophytes and soil microbiomes in Miscanthus sinensis by a 16S metagenomic approach and transcript analysis. In endosphere, ecological factors contributed to the bacterial composition about 75-91% of the comprising bacteria derived from the common ancestor, and figured out the communication between endosphere and rhizosphere, by NMDS, HCA analysis and taxonomical composition all indicated diverse microbial floras specially in plants growing in copper-rich soil. Soil microbiomes associated with heavy metals were dominated by bacrteria of Proteobacteria, Acidobacteriia Actinobacteria, Nitrospira and Thermoplasmata. In core microbiomes analysis, consisted more α-, β-, δ-proteobacteria and fewer γ-proteobacteria in copper rich stress.
In conclusion, soil microbiomes, which was variant and significant than endophytes, was substantially involved in the adaptation of M. sinensis.
摘要 I
Extended abstract II
致謝 V
目錄 VI
表目錄 VIII
圖目錄 VIII
壹、 緒論 1
一、 芒屬植物 (Miscanthus) 1
二、 根內共生菌與根際微生物(Endophyte and rhizosphere microbiomes) 2
三、 DNA分子條碼(DNA barcode)和多源基因體學(metagenomics) 4
四、 轉錄體學(Transcriptomics) 6
五、 非生物逆境(Abiotic stress) 6
六、 研究動機與目的 8
貳、 材料與方法 9
一、 樣本的採集 9
二、 根際與內微生物之 DNA萃取 9
三、 測量土壤基本化學性質 11
四、 聚合酶連鎖反應(Polymerase chain reaction; PCR) 11
五、 純化擴增子(Amplicons) 12
六、 基因選殖(DNA cloning)、藍白菌篩選(transformation)和序列前測 12
七、 DNA濃縮和定序 14
八、 序列合併和標準化(Normalization) 15
九、 重取樣分析(rarefaction analysis) 15
十、 非度量多維度分析(Non-metric multidimensional scaling) 15
十一、 核心微生物群(Core microbiomes) 16
十二、 階層分群分析(Hierarchical clustering analysis) 16
十三、 多源基因體多樣性分析 16
參、 結果 17
一、 各樣本讀序數 (reads)資料和Greengene基因庫比對資料 17
A. 讀序數 17
B. OTU數 17
二、 根內和根際共生微生物相與豐富度 18
A. 非度量多維度分析(NMDS) 18
B. 階層分群分析(HCA) 19
C. 土壤的基本性質 19
D. 物種多樣性 20
E. 根內與際微生物相之菌群分布 20
三、 根部共生菌差異分布分析 23
A. 一般環境之微生物相差異 23
B. 重金屬逆境之微生物相差異 23
C. 核心微生物 23
四、 熱點圖(Heatmaps) 25
肆、 討論 26
一、 各樣本間的關係 26
二、 芒草根部的微生物群 26
三、 重金屬逆境下的微生物群 27
伍、 結論 30
陸、 參考文獻 31
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