淡紫灰链霉菌gCLA4坏死诱导蛋白基因的克隆表达及功能

Expression and characterization of necrosis-inducing protein from Streptomyces lavendulae gCLA4

摘要:

【背景】淡紫灰链霉菌(Streptomyces lavendulae) gCLA4是从黄瓜中分离到的一株放线菌，研究表明该菌对多种植物病原菌均有很好的拮抗作用，具有潜在的生防价值。【目的】深入研究Streptomyces lavendulae gCLA4中坏死诱导蛋白(necrosis-inducing protein) 4955的功能，明确其提高植物抗性的作用机制。【方法】对坏死诱导蛋白4955基因进行克隆，于Escherichia coli BL21(DE3)中表达，并以烟草为材料检测该蛋白的活性和稳定性；使用Protparam、PredictProtein、NCBI CDD、SWISS-MODEL分析蛋白的基本性质和三维结构；检测该蛋白对烟草防御反应相关酶活(CAT、SOD、POD、PAL)和防御相关基因(NPR1、PR1-b、PAL、LOX、PR1-a)表达量的影响。【结果】蛋白4955的耐受温度达40 °C，耐受pH 6.0?10.0。该蛋白分子量为24 491.12 Da，由225个氨基酸组成，其等电点为5.96，经氨基酸序列比对含保守NPP1结构域。蛋白4955处理烟草2 d时，烟草CAT、SOD、PAL酶活增加，POD酶活无显著变化。该蛋白处理烟草第1、3、5天时，基因PR1-b、LOX表达量提高；在第4天时，基因PAL的表达量提高。【结论】Streptomyces lavendulae gCLA4中的坏死诱导蛋白4955确实能诱导烟草中的植物防御反应。

Abstract:

[Background] Streptomyces lavendulae gCLA4 is an actinomycete strain isolated from cucumber. Preliminary researches have showed that the strain was prominent in antagonism against a variety of pathogenic bacteria, and has potential biocontrol value. [Objective] In-depth study of the function of necrosis-inducing protein 4955 in Streptomyces lavendulae gCLA4, and to clarify its mechanism of action in improving plant resistance. [Methods] The necrosis-inducing protein 4955 gene was cloned and expressed in Escherichia coli BL21(DE3). Activity and stability of the protein were tested in tobacco. Then, the basic properties and tertiary structure of the protein were analyzed with Protparam, PredictProtein, NCBI CDD, and SWISS-MODEL. Tobacco defense-related enzymatic activities (CAT, SOD, POD, PAL) were detected after treatment with protein 4955. Additionally, altered gene expression (NPR1, PR1-b, PAL, LOX, PR1-a) was detected by qPCR. [Results] The protein 4955 retained its activity at temperatures up to 40 °C and pH 6.0 to 10.0. The protein has a molecular weight of 24 491.12 Da and consists of 225 amino acids. Its isoelectric point is 5.96, and the amino acid sequence alignment contains a conserved NPP1 domain. The activity of CAT, SOD and PAL increased in tobacco after two days of treatment with 4955, and the activity of POD did not change significantly. The expression of PR1-b and LOX genes was increased 1, 3, and 5 days after treatment with protein 4955, and the expression of PAL was up-regulated on the 4th day. [Conclusion] The necrosis-inducing protein 4955 in Streptomyces lavendulae gCLA4 does induce plant defense response in tobacco.