球形芽胞杆菌C3-41磷酸果糖激酶的克隆、表达及基本生物活性

Cloning, expression and elementary characterization of phosphofructokinase from Bacillus sphaericus C3-41

摘要:

【目的】球形芽孢杆菌缺乏EMP、HMP、ED途径的关键酶，如磷酸果糖激酶等被认为是其不能以糖类物质进行生长的主要原因。杀蚊球形芽孢杆菌C3-41全基因组序列分析表明，在染色体DNA上存在的磷酸果糖激酶基因pfk，为了进一步分析球形芽孢杆菌糖酵解途径，进一步确定磷酸果糖激酶在糖酵解途径中的功能。【方法】通过pfk基因在球形芽孢杆菌菌株中的Southern-blot拷贝数鉴定，在C3-41 pfk基因克隆的基础上进行pfk基因在大肠杆菌中的融合表达、序列分析和序列比对等方法进行研究。【结果】证明了球形芽孢杆菌pfk基因由960 bp核苷酸组成，表达42 kDa的PFK融合蛋白，有保守的底物结合域和ATP结合域，同时pfk基因重组表达质粒可以回复大肠杆菌pfk缺陷型菌株DF1020代谢糖的能力。【结论】杀蚊球形芽孢杆菌C3-41的pfk表达产物具有磷酸果糖激酶活性，为今后深入研究球形芽孢杆菌产能代谢机理奠定了基础。

关键词:球形芽胞杆菌;糖酵解;磷酸果糖激酶

Abstract:

[Objective] Bacillus sphaericus is unable to use hexose and pentoses as the sole carbon source, due to the lack of key enzymes in Embden-Meyerhof-Parnas pathway (EMP), Hexose Monophophate Pathway (HMP) and Entner-Doudoroff (ED) pathway, such as phosphofructokinase (PFK). Based on the genome sequence annotation results of B. sphaericus C3-41, the phosphofructokinase gene pfk was verified with a single copy on chromosome, the aim of this research is to analysis the EMP pathway in B. sphaericus further, and confirm the function of phosphofructokinase. [Methods] The methods of southern-blot of pfk gene among different B. sphaericus strains, pfk ORF cloning from C3-41 and expressing in Escherichia coli, the corresponding sequence analysis and anlignment were used. [Results] The pfk ORF of B. sphaericus was composed of 960 bp nucleitides encoding a protein about 42 kDa, and the PFK sequence analysis showed it had the conservative amino acids-binding sites and an ATP-binding domain. The expression of pfk in recombinant E. coli strain could complement the PFK activity of a pfk mutated E. coli strain DF1020. [Conclusion] The expressed PFK had the conventional phosphofructokinase activity, and settled the foundation for the further research of catabolism of B. sphaericus.

Key words:Bacillus sphaericus; glycolysis; phosphofructokinase