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题名: 滑桃树内生菌宏基因组文库的构建与maytansinoids生物合成基因簇的筛选
作者: 王浩鑫
学位类别: 博士
答辩日期: 2007-12-29
授予单位: 中国科学院昆明植物研究所
授予地点: 昆明植物研究所
导师: 郝小江
关键词: 滑桃树 ; 宏基因组文库 ; 植物内生菌 ; 内生菌富集 ; 美登木素生物合成 ; 天麻抗真菌蛋白 ; 原位杂交
学位专业: 植物学
中文摘要: 本论文以“高等植物中的maytansinoids I 型聚酮骨架是由植物内生菌产生的”这一合理假说为前提,以大戟科滑桃树属植物Trewia nudiflora L.为实验材料,尝试通过不依赖于纯培养的宏基因组学(metagenomics)研究方法,构建滑桃树茎皮内生菌的宏基因组文库,并从该文库中直接筛选获得滑桃树中maytansinoids的生物合成基因簇,进一步鉴定其功能,为高等植物maytansinoids 的微生物起源提供直接的证据。 植物内生菌宏基因组研究包括内生菌的富集,宏基因组DNA 的提取,文库的构建与评价,文库的筛选四个主要步骤。内生菌的富集是整个工作的基础。植物内生菌分布于植物组织细胞间隙或内部,基因组与生物量相对植物来说非常微小,这给植物内生菌的富集带来了很大困难,这可能是植物内生菌宏基因组研究至今未能广泛开展的主要原因。通过不断地摸索与尝试,我们建立了一套行之有效的滑桃树茎皮内生菌富集方法,包括机械匀浆、低浓度SDS/NaCl 选择性沉淀与裂解和差速离心等。该方法可以一次性处理大量植物材料,快速简便地富集植物内生菌,通过扫描电镜观察与基于16S rDNA 的富集效果检测,表明该方法得到的富集样品无明显的植物细胞器残留,内生菌多样性丰富,符合构建内生菌宏基因组文库的要求。用上述方法,对1.6 公斤滑桃树茎皮材料进行内生菌的富集,用无色肽酶和溶菌酶对富集沉淀预处理,用蛋白酶K 法提取DNA,所得DNA 用UNIQ-10 柱纯化、浓缩,得“metagenomic DNA”。16S rDNA 的ARDRA 分析与测序结果表明:“metagenomic DNA”中绝大多数为微生物来源,在187 个16S rDNA 克隆中只有4 个来源于滑桃树叶绿体,其余则来源于Actinobacteria, Proteobacteria,Gemmatimonadetes,Firmicutes,Bacteroidetes,Deinococcus-Thermus,Planctomycetes等多个门的微生物,其中Actinobacteria 和Proteobacteria 门的微生物种类最丰富,总共约占80%。 用“metagenomic DNA”构建了CopyControl Fosmid 宏基因组文库,文库约含1.37×106 个包装颗粒,平均插入片段大小约为34.5kb。通过对约200 个随机Fosmid 的末端测序来评价文库质量,结果表明约88.8%的文库插入片段来源于原核生物,1.6%的插入片段来源于真核生物,其余9.6%的插入片段来源无法判断。 用地高辛标记的AHBA(3-氨基-5-羟基苯甲酸,maytansinoids 生物合成起始单元)合酶基因为探针对文库进行杂交筛选,从约75000 个文库克隆中得到一个阳性克隆509D8,随后的PCR 检测表明该克隆同时含有I 型PKS 基因、AHBA合成酶基因,对这个fosmid 插入片段的shotgun 全测序和序列分析表明整个插入片段长约34.9kb,含有22 个完整的ORF 和一个部分的ORF,包含了3 个PKS基因、7 个AHBA 合成相关基因、一个负责大环内酰胺键形成的酰胺合酶以及一个可能催化C-C 键形成滑桃树maytansinoids 特有的第二个大环的基因,说明该克隆具有合成滑桃树maytansinoids 的潜力,进一步的表达与功能鉴定工作目前正在进行中。应用多种简并引物对文库的PCR 筛选结果表明,文库中具有很多PKS 基因、卤代酶基因和萜类合成相关基因,表明植物内生菌可能是聚酮类化合物、萜类和其它多种天然产物的潜在来源。 我们通过不依赖于纯培养的方法构建了植物内生菌宏基因组文库并筛选得到了一个部分的生物合成基因簇,极有可能是maytansinoids 生物合成基因簇的一部分。本研究证明了宏基因组学方法在植物内生菌研究中的可行性,是对植物中广泛存在的未培养微生物进行研究的有益尝试,并初步显示了植物内生菌在基因资源和天然产物资源发现方面的巨大潜力。 论文最后一章是天麻抗真菌蛋白(GAFP, Gastrodianin)基因的mRNA 原位杂交分析,通过地高辛标记的mRNA 探针,对乌天麻不同组织进行非放射性mRNA原位杂交。结果表明,Gastrodianin 在天麻次生球茎中的表达呈现明显的外周表达模式,可能是天麻在地下抵御蜜环菌入侵球茎皮层内部的防卫机制之一。Gastrodianin 在地上部分具有很强的转录信号,表达量明显高于地下的球茎,提示除了抗蜜环菌的功能外,Gastrodianin 可能具有其它的生理功能。
英文摘要: This study is based on the hypothesis that the polyketide backbone of maytansinoids isolated from higher plant is produced by endophytes. In order to apply the direct proof for this theory, we try to adopt metagenomic approach on Trewia nudiflora L. (Euphorbiaceae) to isolate the gene cluster responsible for maytansinoids biosynthesis. Metagenomic study of endophytes in plant is comprised of enrichment of endophytes, isolation of metagenomic DNA, construction, evaluation and screening of metagenomic library. Enrichment of endophytes is critical to the whole study. Endophytes in plant dispersed in intercellular and intracellular, and have a minor genome and biomass compared to host plant. The metagenomic study of plant endophytes is still in its infancy nowadays because it is difficult to eradicate plant contaminant from endophytes enrichment. After two years’ endeavor, we developed an effective and practical method to enrich the endophytes in Trewia nudiflora which combined homogenization, differential centrifugation and selective lysis & precipitation using SDS and NaCl. The method could deal with large amount of plant material each treat, and is low-costing and time-saving. The Scanning electron microscopy and 16S rDNA analysis showed this method could produce endophytes pellet without apparent plant organelles contamination, which was qualified for construction of metagenomic library of endophytes. We enriched endophytes from 1.6 kilogram stem bark of Trewia nudiflora. The enrichment pretreated with achromopeptidase and lysozyme was used for DNA isolation by means of SDS and proteinase K treatment. The metagenomic DNA preparation was concentrated and purified with UNIQ-10 column. The refined metagenomic DNA was subjected to ARDRA (amplified ribosomal DNA restriction analysis) and sequencing. The results showed that most of the metagenomic DNA came from eubacteria rather than plant host. In one hundred and eighty seven 16S rDNA clones, only four clones was originated from chloroplast, and the others came from bacteria belonging to phylum Actinobacteria, Proteobacteria, Gemmatimonadetes, Firmicutes, Bacteroidetes, Deinococcus-Thermus and Planctomycetes. CopyControl Fosmid library was constructed using metagenomic DNA. The metagenomic library contains 1.37×106 phage particles, and the average size of inserts is about 34.5 kb. The quality of the metagenomic library was evaluated by end-sequencing of 200 fosmid chosen randomly. The BLASTX search results indicated that 88.8% of the inserts in metagenomic library were originated from prokaryotes, 1.6% from eukaryotes and the other 9.6% were not assignable. 3-amino-5-hydroxybenzoic acid (AHBA) synthase is a key enzyme for biosynthesis of AHBA starter unit of maytansinoids, and DIG labeled AHBA gene fragment was used for screening biosynthesis gene cluster in metagenomic library. One positive fosmid clone named 509D8 was recovered from 75000 clones. Following PCR analysis showed this fosmid contains not only nearly all the genes required for the biosynthesis of AHBA but also type I PKS genes, which indicated the insert of this fosmid could be involved in the biosynthesis of maytansinoids. The shotgun sequencing and ORF predicting show the 34.9kb insert contains 22 full and 1 partial ORF. Besides two full and one partial type I PKS genes, seven genes involved in the AHBA synthesis, one amide synthase gene responsible for the formation of intramolecular amide bond and one gene possibly catalyzing the formation of unusual second large ring in maytansinoids of Trewia nudiflora were identified.The extensive study on expression and function is still on-going. We found that the metagenomic library harbors many PKS genes, halogenases and genes related to biosynthesis of terpenoids by PCR screening using degenerated primers, which implied the plant endophytes might be the potential resources of polyketides, terpenoids and other natural products. Harnessing culture independent method, we constructed and screened a metagenomic library of plant endophytes in Trewia nudiflora. A fosmid clone containing partial gene cluster, that is very likely responsible for the biosynthesis of maytansinoids was identified by hybridization. Our work showed it is feasible to apply metagenomic approach into field of plant endophytes. Our study is a beneficial attempt to exploit uncultured endophytes of plant, and showed the great potential of searching for new genes and natural products in plant endophytes. The last chapter is the mRNA in situ hybridization of Gastrodia antifungal protein(GAFP, Gastrodianin) gene. Nonradioactive mRNA in situ hybridization was conducted by DIG labeled GAFP gene probe in Gastrodia elata Bl. f. glauca S.chow. From the peripheral expression pattern in corms, the gastrodianin is considered to play a defensive role against the fungus Armillaria. Take into account the apparently abundant expression in the above-ground parts of the plant, however, gastrodianin seems unlikely to be limited to such a role.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.kib.ac.cn/handle/151853/150
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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Recommended Citation:
滑桃树内生菌宏基因组文库的构建与maytansinoids生物合成基因簇的筛选.王浩鑫[d].中国科学院昆明植物研究所,2007.20-25
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