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Title:
产甘油葡萄糖苷蓝细菌的筛选、种属鉴定及其合成关键基因的鉴定
Author: 钟成伟
Degree Level: 硕士
Issued Date: 2017-05
Degree Grantor: 中国科学院大学;中科院青岛生物能源与过程研究所
Place of Degree Grantor: 北京;青岛
Supervisor: 甘油葡萄糖苷 ; 蓝细菌 ; 甘油葡萄糖苷磷酸合成酶 ; 甘油葡萄糖苷磷酸酶 ; 盐耐受力
Keyword: 工学
Subject:
Alternative Title: 生物工程
Major: 蓝细菌是一种光合自养的原核微生物,可以利用太阳能将二氧化碳和水在单一细胞内转化成多种重要的代谢产物,加之蓝细菌具有生长迅速、遗传背景清晰等优势,使得蓝细菌成为了生产包括生物燃料在内的多种重要化学品的有潜力的光合细胞工厂。甘油葡萄糖苷(glucosylglcerol;GG)是蓝细菌在盐胁迫条件下产生的一种用于维持胞内外渗透压平衡的相容性物质;同时也是一种可用于化妆品的添加剂、人体保健和商业酶稳定剂的高附加值化学品。本团队之前开展了代谢工程改造模式蓝细菌合成GG的研究工作,显示了蓝细菌合成GG的巨大潜力,同时也发现模式蓝细菌集胞藻PCC 6803用于生物合成GG的不足之处,如环境耐受力差、副产物甘油和蔗糖积累。因此,为了进一步丰富蓝细菌GG生产的菌株和基因资源多样性,本研究开展一系列研究工作并取得了以下结果: 本研究从团队已搜集的非模式蓝细菌藻株出发,筛选和比较了不同藻株耐盐能力、生长速率和相容性物质合成的能力,发现粘球藻FACHB-400具有所筛选藻株中最高的耐盐能力,而海绵状念珠藻FACHB-130是耐盐能力最差的藻株。分离自运城盐湖的1B1藻株能够在600和900 mM NaCl浓度下缓慢生长,且细胞出现聚集的现象。发现生长速度最快的藻株是浅黄席藻FACHB-238;而包括盐湖藻1B1的其他蓝细菌藻株的生长速率基本都与集胞藻PCC6803相当。在蔗糖合成方面,中华植生藻 FACHB-800蔗糖合成能力最强;而1B1藻株是GG合成能力最强的藻株;而且它们分别都只合成蔗糖或GG。1B1的这些特性预示着它有可能是比集胞藻PCC 6803更佳的GG生产菌株。 基于光学显微镜、透射电镜和扫描电镜观察表征了1B1藻细胞形态特征,发现其具有与文献报道的Cyanobacterium aponinum 藻株非常相似的形态特征。进一步基于16S rRNA系统发育分析发现,1B1藻株16S rRNA序列与Cyanobacterium aponinum PCC 10605藻株的16S rRNA序列一致性(identity)高达100%;而且两者在系统发育进化树上与其他Cyanobacterium属藻株聚为紧密姐妹群。这些形态和分子证据支持将1B1藻株种属地位定为Cyanobacterium 属aponinum 种亚株。针对1B1藻株全基因组测序数据的拼接,本研究比较了四种不同软件组合的拼接效果,发现k-mer值是决定基因组拼接效果的重要因素。而分析全基因组注释结果发现,1B1基因组共有3659个蛋白编码基因和45个RNA。 通过序列同源性比对,获得了1B1基因组编码的GG合成候选基因ggpS和ggpP序列;通过克隆并在集胞藻PCC6803 GG合成缺失突变株中异源表达这些候选基因,以及分析互补藻株盐耐受能力和GG合成能力,发现导入候选基因后集胞藻突变株恢复了合成GG的能力和盐胁迫耐受能力,证明了1B1这两个候选基因编码产物就是甘油葡萄糖苷磷酸合成酶(GgpS)和甘油葡萄糖苷磷酸酶(GgpP)。
Abstract: Cyanobacteria are a kind of prokaryotes which can ultilize solar energy and convert carbon dioxide and water to biomass. With photosynthetic ability, fast growth rates and clear genetic backgrounds, cyanobacteria have been developed as a promising photo-bioreactor which can produce different biofuels and bio-chemicals by metabolic engineering. Glucosylglycerols (GGs) are known as compatible solutes accumulated by some bacteria including cyanobacteria for their adaptations to salt stresses. It was proved that GG can be used as cosmetic additives, protein staberlizers and in human healthcare. Previously, this group has reported their works on photosynthetic production of GG by metabolic engineering of the model cyanobacterium. This work proves the great potential of cyanobacterial photosynthetic production. It also pointed out the drawbacks of this novel route, namely, low environmental tolerances of model Synechocystis sp. PCC 6803, and accumulation of glycerol as byproducts. For exploring the resources of strains and genes which can be used in cyanobacterial GG production, we here performed systematic works and showed some interesting findings as follows: First, we screened cyanobacterial strains before collected by their salt tolerances, growth rates and abilities to synthesize compatible solutes. And it was found that Gloeocapsa alpicola FACHB-400 and Nostoc spongiaeforme FACHB-130 show the highest and lowest tolerances to salt. 1B1,which was isolated from the salt lake in Yuncheng, Shanxi Province, showed the similar salt tolerance to Synechocystis sp. PCC 6803. Richelia sinica FACHB-800 and 1B1 were found to be best producers for sucrose and GG respectively. In addition, both of them synthesized only sucrose or GG, respectively. Second, we analysized the morphorical and molecular characteriatics of the 1B1 strain by light microscope,transmission electron microscope and scanning electron microscope analyses and 16 S rRNA-based phylogenetic analysis.1B1 showed the similar mophorical characteriatics to the reported Cyanobacterium aponinum strain. And the 16S rRNA sequences of 1B1 are exactly same as that of Cyanobacterium aponinum PCC 10605. The neighbor-joining phylogenetic trees revealed that the 1B1 strain clustered with Cyanobacterium aponinum PCC 10605. These taxa were sister group to the clade including some other Cyanobacterium strains. The above results lead us to consider the 1B1 strain as a new species of Cyanobacterium and propose for its name as Cyanobacterium aponinum 1B1. For better assembling the genome sequences of the 1B1, we tried four different workflows and evaluated their assemblies. It was found that the k-mer value is the most important factor affecting the short reads assembly. And the draft genome of the 1B1 strain encodes 3659 proteins and 45 RNAs. To confirm the genes responsible for GG biosynthesis, we obtained the candidate genes encoding glucosylglycerol phosphate synthase and glucosylglycerol phosphate phosphatase, expressed them in the corresponsing mutants of Synechocystis sp. PCC 6803, analyzed the salt tolerances and GG synthesis abilities of the resulting strains. It was found that both the ∆ggpS and ∆ggpP mutants were endowed tolerances to salts and abilities to synthesize GG by introducing the candidate genes of ggpS and ggpP from the 1B1 genome. The results confirmed the functions of the candidate genes from the 1B1 genome.
English Abstract: 中文;英语
Language: 中文
Department: 微生物代谢工程团队
Available Date: 2022-07-01
DOC Type: 学位论文
Content Type: 学位论文
URI: http://ir.qibebt.ac.cn/handle/337004/9986
Appears in Collections:微生物代谢工程团队_学位论文

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description.institution: 中国科学院大学;中科院青岛生物能源与过程研究所

Recommended Citation:
钟成伟. 产甘油葡萄糖苷蓝细菌的筛选、种属鉴定及其合成关键基因的鉴定[D]. 北京;青岛. 中国科学院大学;中科院青岛生物能源与过程研究所. 2017.
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