茄科雷尔氏菌脂酰-CoA合成酶的功能鉴定

发布时间：2019-05-16 13:35

【摘要】：【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。
[Abstract]:Objective: Solanaceae is a common pathogen of crop, which causes plant bacterial wilt. The study of fatty acid metabolism pathway will help to find new antibacterial drug targets and provide a new way of thinking for the prevention and treatment of youth and blight diseases. [methods] using E. coli FadD sequence, It was found that RSc2857 (RsFadD) in GMI1000 of Solanaria solanacearum had high similarity, and it was speculated that it had the activity of acyl-CoA synthase. The RsfadD gene was obtained by PCR amplification and supplemented with E. coli fadD mutant after being inserted into the expression vector pBAD24M, and the growth of the transformant was detected. RsfadD was expressed in E. coli BL (DE3) after it was ligated with pET-28b. RsFadD, with histidine label was purified by Ni-NTA to determine the activity of RsFadD in vitro. The RsfadD knockout mutant was obtained by homologous recombination method, and the growth traits of the mutant were analyzed. [results] RsfadD allogenic complementary Escherichia coli fadD mutant grew on the basic medium with fatty acid as carbon source. In vitro, RsFadD had the activity of acyl-CoA synthase, which was active against fatty acids with different chain lengths, but the activity was lower than that of E. coli FadD.RsfadD mutants on the basis of adding fatty acids with different chain lengths. However, there was no difference in growth on rich medium. [conclusion] RsfadD encodes acyl-CoA synthase in Solanaria solanacearum and plays an important role in fatty acid utilization. However, the RsfadD mutant grew weakly on the basic medium, which indicated that there were other acyl-CoA synthase genes in the genome of Corell solanum. The above results laid a foundation for further study of acyl-CoA synthase and fatty acid utilization mechanism in Corell solanum.
【作者单位】：广东食品药品职业学院;华南农业大学生命科学学院广东省农业生物蛋白质功能与调控重点实验室;河南省三门峡市卢氏县第一高级中学;
【基金】：国家自然科学基金项目(No.31601601,31671987) 广东食品药品职业学院院级课题资助项目(No.2015YZ006)~~
【分类号】：S432.4

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