茄科雷尔氏菌脂酰-CoA合成酶的功能鉴定

【目的】茄科雷尔氏菌是一种常见的农作物致病菌,引起植物青枯病。研究其脂肪酸代谢途径将有助于寻找新的抗菌药物靶点,为防治青枯病害提供新的思路。【方法】利用大肠杆菌FadD序列,进行同源比对发现茄科雷尔氏菌GMI1000中RSc2857(RsFadD)具有较高的相似性,推测其具有脂酰-CoA合成酶活性。采用PCR扩增方法获得RsfadD基因,连入表达载体pBAD24M后互补大肠杆菌fadD突变株,并检测转化子的生长情况。RsfadD与pET-28b连接后,在大肠杆菌BL(DE3)中表达,并利用Ni-NTA纯化获得带有组氨酸标签的RsFadD,体外测定RsFadD的活性。利用同源重组方法,获得RsfadD敲除突变株,分析突变株的生长性状。【结果】RsfadD异体互补大肠杆菌fadD突变株,恢复突变株在以脂肪酸为碳源的基础培养基上生长。体外活性测定RsFadD具有脂酰-CoA合成酶活性,对不同链长的脂肪酸都具有活性,但活性低于大肠杆菌FadD。RsfadD突变株在添加不同链长脂肪酸的基础培养上仅能微弱生长,而在丰富培养基上生长无差异。【结论】茄科雷尔氏菌中RsfadD编码脂酰-CoA合成酶,在脂肪酸利用过程中发挥重要作用。但RsfadD突变株在基础培养基上微弱生长,说明茄科雷尔氏菌基因组中还有其他的脂酰-CoA合成酶基因。以上研究结果为进一步研究茄科雷尔氏菌中脂酰-CoA合成酶以及脂肪酸利用机制奠定了基础。

Identification and function research of the fatty acyl-CoA synthetase in Ralstonia solanacearum

Objective] Ralstonia solanacearum, a devastating, soil-borne plant pathogen, causes a bacterial wilt disease in diverse plants. Studies on the metabolic mechanisms of fatty acids will facilitate the discovery of novel methods or biopesticides to efficiently control the bacterial wilt disease. Methods] RSc2857 (RsfadD) gene was found in the genome of Ralstonia solanacearum GMI1000 through sequence alignment with Escherichia coli FadD, which was annotated to encode a fatty acyl-CoA synthetase (FACS). For complementation analysis, RsfadD gene was amplified by PCR, and was ligated into an expression vector pBAD24M, which was subsequently transferred into an E. coli fadD mutant JW1794. The growth of transformant was analyzed. RsfadD was also fused in-frame to pET-28b, and expressed in E. coli BL21(DE3). The hexahistidine-tagged RsFadD was purified by Ni-NTA, and the activity was analyzed in vitro. RsfadD deletion mutant was obtained by homologous recombination, and the mutant growth was also analyzed. Results] RsfadD conferred the E. coli fadD mutant to grow on the minimal medium with fatty acids as the sole carbon source. In vitro enzymatic analysis demonstrated that RsFadD has FACS activity, and could utilize fatty acids of different chain lengths as substrates to form fatty acyl-CoAs. While the activity of RsFadD was lower than that of E. coli FadD. RsfadD deletion mutant grew well on nutritional medium, but grew weak on the minimal medium with fatty acids as the sole carbon source. Conclusion] All of above suggested that RsfadD encodes a FACS, which plays an important role in fatty acids utilization. The weak growth of RsfadD deletion mutant on the minimal medium indicated other genes encoding FACS may exist in the genome. This study will contribute to further research about FACS and fatty acids utilizing mechanism in R. solanacearum.