溶藻弧菌kdpD基因敲除突变株的构建及其表型特征

Construction and characterization of the kdpD gene knock-out mutant of Vibrio alginolyticus

摘要:

【目的】探究kdpD基因对溶藻弧菌生物学特性的影响。【方法】采用Overlap PCR和同源重组技术，结合正负向筛选，构建kdpD基因无标记基因框内敲除突变株，比较kdpD突变株和野生株HY9901在生长速率、胞外蛋白酶活性以及毒力等方面的差异。【结果】成功构建溶藻弧菌kdpD基因敲除突变株。体外实验表明，kdpD的缺失对溶藻弧菌的生长曲线和胞外蛋白酶活性的影响不明显，但是突变株的泳动能力和生物被膜形成能力出现下降。斑马鱼致病性试验结果显示，突变株毒力下降了8.84倍。【结论】kdpD基因参与调控溶藻弧菌的泳动能力、被膜形成和毒力，但不影响生长速率和胞外蛋白酶活性。

关键词:溶藻弧菌，kdpD，基因敲除，表型

Abstract:

[Objective] To investigate the role of kdpD gene in Vibrio alginolyticus, a kdpD gene deletion mutant was generated. [Methods] Combining with chloramphenicol (Cm) and sucrose screening, we constructed an in-frame deletion mutant of kdpD using overlap PCR and homologous recombination technology. We then investigated the effect of the mutant on its growth, ECPase activity and virulence in fish. [Results] The mutant ΔkdpD was successfully constructed. The mutant ΔkdpD showed no differences of ECPase activity and growth from the wild-type strain. But a decrease in swarming ability and biofilm formation was found. Moreover, virulence assays with zebrafish model confirmed that virulence of the mutant declined by 8.84 times. [Conclusion] The kdpD gene could regulate swarming ability, biofilm formation and virulence, but it has no effect on growth and ECPase activity.

Key words:Vibrio alginolyticus, kdpD, Gene knock-out, Biological characteristics