烟草PR10蛋白生物活性及赤星病菌Alternaria alternata诱导下的表达分析
Biological activity of tobacco PR10 protein and expression analysis induced by Alternaria alternata

为深入研究植物PR10蛋白的生物学功能，以烟草PR10蛋白（NtPR10）为研究对象，采用冷休克蛋白表达载体pCold Ⅱ，构建烟草PR10融合蛋白原核表达系统，优化表达及纯化条件，获得高纯度NtPR10融合蛋白；分别采用底物法和滤纸片法体外分析其核酸酶活性及抑菌活性；并利用荧光定量PCR方法分析了烟草赤星病菌Alternaria alternata侵染下，NtPR10基因在抗、感烟草品种内不同时间点的表达差异。结果表明，15℃、0.1 mmol/L IPTG过夜条件下可诱导获得大量可溶性目的蛋白，50、100 mmol/L咪唑缓冲液洗脱能够获得较高纯度的目的蛋白；纯化后的NtPR10蛋白能够降解烟草总RNA，具有核酸酶活性，且1.0、0.5、0.25 μg/μL的NtPR10蛋白溶液均对烟草赤星病菌的菌丝生长具有显著的抑制作用，但随着蛋白浓度的降低，抑菌作用减弱；荧光定量PCR结果显示，接种烟草赤星病菌后，NtPR10基因在感病品种和抗病品种中均显著上调表达，但其在抗病品种的响应速度和表达量显著高于感病品种，表明NtPR10应答了烟草赤星病菌的侵染过程。
In order to investigate the biological function of the plant pathogenesis-related protein PR10, the tobacco PR10 protein (NtPR10) was studied. The cold shock protein expression vector pCold Ⅱ was used to construct the prokaryotic expression system of NtPR10 by the method of seamless cloning; the expression and purification conditions were optimized and the nuclease activity and antifungal activity was analyzed by using substrate method and filter paper method, respectively. The expression pattern of gene NtPR10 in the resistant and susceptible cultivars was studied by RT-qPCR. The results showed that soluble protein was obtained under the conditions of 15℃ and 0.1 mmol/L IPTG induction overnight; the pure target protein was obtained by using 50, 100 mmol/L imidazole buffer elution. The activity analysis showed that NtPR10 could degrade tobacco total RNA and inhibit Alternaria alternata growth, indicating that NtPR10 had ribonuclease activity and antifungal activity. The gene NtPR10 was significantly up-regulated in both resistant and susceptible cultivars after A. alternata infection. However, in resistant cultivar, the response rate and expression level were significantly higher than in susceptible cultivar, suggesting that the NtPR10 might have an important function in the process of A. alternata infection.