论文部分内容阅读
假单胞菌株H78是本实验室从油菜根际分离得到的一株生防细菌。通过16S r RNA及藤黄绿菌素(pyoluteorin,Plt)合成基因plt C基因(编码聚酮合成酶)保守区段进行PCR测序,初步确定H78菌株可能属于P.protegens。本研究旨在进一步克隆H78菌株的Plt生物合成基因簇,分析其Plt合成特性以及H78菌株的分类地位。对假单胞菌株H78进行全基因组de novo测序;使用blast工具,分析假单胞菌株H78与P.protegens Pf-5、CHA0、P.aeruginosa M18、LESB58中Plt合成基因簇的同源性;使用KMB培养基对菌体进行发酵,测定菌体生长,对Plt合成进行HPLC测定。假单胞菌H78的Plt合成基因簇与其它4个菌株都存在保守的基因组成与布局,在Plt合成基因编码氨基酸序列上,H78与CHA0、Pf-5菌株的一致性高达99%~100%,其中与CHA0同源性更高;而与铜绿假单胞菌M18及LESB58菌株的一致性相对较低,为52%~88%。在KMB培养基中,假单胞菌株H78中累积合成Plt超过30滋g/m L。H78菌株属于P.protegens,其Plt合成基因簇与P.protegens Pf-5、CHA0具有高度同源性,而铜绿假单胞菌M18、LESB58的Plt基因簇存在不同的进化起源;H78菌株能合成较高水平的Plt,通过进一步研究改造,此菌有潜力成为安全并高效合成Plt的生防菌株。
Pseudomonas strain H78 is a biocontrol bacterium isolated from the rhizosphere of rapeseed in our laboratory. The 16S rRNA and pyoluteorin (Plt) were used to synthesize the gene fragment of plt C gene (encoding polyketide synthase) and sequenced. The results showed that the H78 strain belonged to P.protegens. The aim of this study was to further clone the Plt biosynthetic gene cluster of H78 strain and analyze its Plt synthesis characteristics and the taxonomic status of H78 strain. Whole genome de novo sequencing was performed on Pseudomonas strain H78; blast tools were used to analyze the homology of the Plt synthetic gene cluster in Pseudomonas strain H78 to P. protegens Pf-5, CHA0, P. aeruginosa M18, LESB58; KMB medium was used to ferment the mycelia, the growth of the mycelium was measured, and the HPLC analysis of Plt synthesis was carried out. Pseudomonas H78 Plt synthetic gene cluster and other four strains have a conserved genetic composition and layout, in the Plt synthetic gene coding amino acid sequence, H78 and CHA0, Pf-5 strains of the same as high as 99% to 100% , Of which the homology with CHA0 was higher, but the consistency with Pseudomonas aeruginosa M18 and LESB58 was relatively low, ranging from 52% to 88%. In KMB medium, Pichia pastoris strain H78 accumulated more than 30 μg / m L. The H78 strain belongs to P.protegens and its Plt gene cluster has high homology with P.protegens Pf-5 and CHA0, while there are different evolutionary origins of the Plt gene cluster of Pseudomonas aeruginosa M18 and LESB58. H78 strain can synthesize The higher level of Plt, through further research and modification, has the potential to be a biocontrol strain that safely and efficiently synthesizes Plt.