【作者】 高经纬；

【导师】 谢志雄；

【作者基本信息】 武汉大学 ， 微生物学， 2013， 博士

【摘要】 假单胞菌属(Pseudomonas)是隶属于Y-变形杆菌(y-Proteobacteria)亚纲的专性需氧革兰氏阴性杆菌。该属成员在自然界中分布广泛,代谢途径及产物多样,在工业、农业和医疗等领域有重要的科研价值和应用价值。本实验室从环境中分离获得一株高产铁载体假单胞菌株HYS,其特殊的产铁载体能力暗示其具有相对特殊的铁离子代谢体系,并且具有在生物防治领域应用的潜能。本实验室曾采用革兰氏染色、BIOLOG生理生化检测鉴定以及16S核糖体DNA序列测序分析等方法对其进行鉴定,但无法将此菌株正确分类至种。此菌株遗传背景未知,也无法从相近菌株获得遗传信息,对深入研究此菌株相关的分子机理和生物学效应造成了很大的障碍。为获得此菌株的遗传信息,本研究首先对此菌株进行了基因组测序,并结合生物信息学方法和生理生化方法对此菌株的分类地位进行了分析。首先对菌株HYS基因组进行测序。测序采用Solexa方法进行,获得有效Reads数据长度为519.18Mb。利用SOAPdenovo组装软件对有效数据进行组装,获得长度大于200bp且不含有任何不确定碱基N的contigs共231个,序列总长度为5,639,475bp,测序深度为92×。根据reads比对计算reads利用率,推算出拼接的序列结果对真实基因组的覆盖度为91.06%。根据reads末端配对信息,使用paired-end mapping的方式,可以将已获得的231个contigs拼接成为64个scaffolds。测序所获得的基因组序列已上传至NCBI whole genome shotgun数据库,此项目的NCBI接入号为AJJP00000000,当前版本的接入号为AJJP01000000.基于已获得的基因组序列信息,结合生理生化方法,从三个方面对菌株HYS的分类进行了分析。首先,以基因组序列平均核苷酸同一性(average nucleotide identity, ANI)作为指示菌株基因组序列相似度的参数,将菌株HYS基因组序列与NCBI已公布的33个来源于9个不同的种的假单胞菌属菌株的基因组进行比对。这9个种分属于2个属内世系,及6个不同的类群,具有很好的代表性。比对结果显示,菌株HYS与恶臭假单胞类群的杀虫假单胞菌和恶臭假单胞菌亲缘关系最近,但不属于其中任何一个种。随后,采用核酸比对和rRNAmmer软件分析确定16SrDNA序列；采用核酸比对和蛋白质比对的方法确定看家基因DNA促旋酶B亚基编码基因gyrB和RNA聚合酶σ因子编码基因rpoD序列。利用三个保守序列分子分别构建分子进化树,结果都显示菌株HYS与P. vranovesis处于同一进化分支,但16S rDNA分子进化树显示两者属于恶臭假单胞菌类群,而gyrB和rpoD分子进化树则显示两者与恶臭假单胞类群亲缘关系较近,为相对独立的分枝。最后,采用醌型分析,脂肪酸分析及明胶液化实验等方法对菌株HYS和恶臭假单胞菌模式菌株以及P. vranovensis进行了对比。醌型分析确定此菌株确实具有恶臭假单胞菌类群醌型特征。脂肪酸分析和明胶液化则确定此菌株具有不同与恶臭假单胞菌及P. vranovensis的脂肪酸组成和明胶液化能力。综合以上结果确定,菌株HYS和P. vranovesis是与恶臭假单胞菌类群亲缘关系较近的、进化地位相对原始的、独立进化的分支。菌株HYS生理生化特征和P. vranovesis种的特征不尽相相同,可能是P. vranovesis中具有新的特征的菌株,也可能是一个新种。参考植物益生菌基因组相关特征对菌株HYS基因组进行全面分析,结果显示此菌株HYS具有适应植物根际环境的能力但不具备成为植物病原菌的植物侵染系统,具有竞争性吸收环境中铁元素抑制有害真菌的体系,包含编码可能杀死线虫的毒性分子的基因簇。基因组分析所显示出的此菌株对植物和微生物的生物学效应由本实验室其他成员检测验证。本研究主要利用模式生物秀丽隐杆线虫作为研究对象,在实验室条件下检测此菌株是否具有杀死线虫的能力。本研究检测了菌株HYS对秀丽隐杆线虫胚胎及成虫的影响。菌株HYS培养胚胎将导致胚胎死亡。胚胎接触菌株HYS后,发育进程减缓,细胞自噬标记基因lgg-1表达量升高,且呈现出无规律的表达模式。菌株HYS喂食秀丽隐杆线虫成虫将导致群体半数致死时间由12.8d显著降低至3.6d。由于秀丽隐杆线虫胚胎的发育过程在线虫动物门中具有保守性,且寄生性植物病原线虫的胚胎在土壤中孵化,具有接触到菌株HYS的可能,因此菌株HYS导致秀丽隐杆线虫胚胎死亡的作用也可能是此菌株作为生物防治菌株应用的主要功效。微生物导致秀丽隐杆线虫发育进程改变的现象此前从未报道过,菌株HYS杀死秀丽隐杆线虫胚胎可能涉及新的效应分子或机理。本实验室构建了菌株HYS转座子随机插入突变文库,并从中筛选出了多株产铁载体缺陷株。选择不同类型的产铁载体缺陷株进行秀丽隐杆线虫胚胎孵化影响实验,证实菌株HYS杀死秀丽隐杆线虫胚胎与其高产铁载体能力无关,并发现双组分调控系统GacS/GacA可能参与调控毒性基因表达。采用基因定点敲除和遗传回补的方法,确定基因gacS和gacA对毒性基因表达具有正调控作用,并确定rsmA基因对毒性基因表达具有阻遏作用。基于这一结果,可采用免疫共沉淀或蛋白表达谱等高通量分析方法对毒性相关基因进行筛选,为后续研究提供了新的思路和方法。综上所述,本研究为研究菌株HYS提供了所需要的遗传信息,并确定了菌株HYS属于新的进化分支,可能是P. vranovesis种中具有新特征的菌株,或者是一个新的种。基于生物学分析确定此菌株具有成为生物防治菌株的可能性,并以秀丽隐杆线虫作为研究对象证实了此菌株具有杀线虫能力,建立了新的微生物对秀丽隐杆线虫胚胎致死形式研究模型。通过确定GacS/GacA和RsmA参与相关基因调控,为后续筛选相关基因提供了思路和方法。更多还原

【Abstract】 Members of the genus Pseudomonas are Gram-negative, rod-shape, and aerobic bacteria. They colonize diverse habitats because of their remarkable metabolic and physiologic versatility, and are important for industrial, agricultural and medical applications. Siderophore high-yielding Pseudomonas strain HYS was isolated from water of East Lake of Wuhan. The special siderophore secreting ability indicates that strain HYS have a special iron acquisition or regulation system, and this strain has potential in biocontrol applications. Its unknown genetic background is a barrier for molecular and the relevant bio-effects analysis of this strain. In this study, the genomic DNA of strain HYS was sequenced with Solexa paired-end sequencing technology to obtain the genetic information.Sequencing genomic DNA of Pseudomonas sp. HYS yielded519.1838Mb effective raw paired-end sequence reads. Raw paired-ended reads were de novo assembled using the SOAPdenovo alignment tool which generated231contigs excluding contigs shorter than200bp, and then assembled into64scaffolds by paired-end mapping. The total length of the draft genome is5,639,475bp. Based on the ratio of used paired-end sequence reads, draft genome data covered91.06%of the real whole genome. These sequences have been deposited at NCBI Whole Genome Shotgun database under the accession AJJP00000000. The version described in this paper is the first version AJJPO1000000.Average nucleotide identity of the draft genome with33sequenced genomes from nine Pseudomonas species were calculated by using the BLASTN algorithm, showing that the strain is closely related to strains from P. putida group, but not belongs to any of them. Three phylogenetic trees based on sequences of ’housekeeping’genes16S rRNA, gyrB and rpoD have been constructed respectively, showing that strain HYS and P. vranovesis always located in the same branch. In the tree of16S rRNA gene, this branch is located inside of the P. putida group, but it located independently outside of the P. putida group in other two trees.Analysis of isoprenoid quinines identify strain HYS has the same type of ubiquinone as members of P. putida group. The characterization of fatty acids components is different from P. putida type strain and P. vranovesis, and also the ability to hydrolyze gelatin. According to the results above, strain HYS and P. vranovesis are independently evolutional branch, whith is closely related to P. putida group. Strain HYS is either a strain of P. vranovesis with different biochemical characteristic or a new species.Comparative genomics analysis of strain HYS with plant-promotion Pseudomonas spp. reveal that:1) it has potential colonize plant rhizosphere but lacks of plant pathogenetic system;2) it may inhibit pathogenetic fungus by competitive iron absorbtion;3) it has gene clusters encoding potential nematode killing toxins. The first two hypothesises are verified by other groups of our lab, and this study focus on the nematode killing ability of the strain HYS. Model organism Caenorhabditis elegans has been chosen to perform the nematode killing assay.Strain HYS kills Caenorhabditis elegans embryo by delaying the embryogenesis and triggers autophagy of embryo cells. Half of adult worms feed on HYS died after3.6d, while the time is12.8d if feed on normal food.C. elegans has a characteristic nematode biochemistry and shares structures found to be important to parasitic species. Since, most of the plant parasitic species lay eggs in soil and get into plant after L2stage, the embryo killing ability of HYS makes it a potential biocontrol agent to kill embryo of plant parasitic nematode.By tested siderophore defective mutant of strain HYS, confirm that siderophore is not the toxin killing embryo, and find that GacS and GacA involved in killing assay. Gene knockout and rescue assay verify this nematicide activity is positively controlled by GacS/GacA two-component regulatory system and blocked by RsmA.更多还原