向日葵锈菌胞外金属蛋白酶的特性分析

为进一步了解锈菌侵染向日葵的致病机制，对向日葵锈菌(Puccinia helianthi)分泌的一种新型的金属蛋白酶(metalloproteinase,Mep)水解活性和生物学特性进行了表征。使用偶氮酪蛋白法测定蛋白酶的活性，在波长280 nm下测吸光度。结果表明，该蛋白酶对偶氮酪蛋白具有水解活性，纯酶最适温度和pH分别是50℃和7.0，在pH 5.0～9.0和温度50℃以下保持稳定。蛋白酶受到抑制剂乙二胺四乙酸二钠(EDTA)、乙二醇双(2-氨基乙基醚)四乙酸(EGTA)、1,10-菲咯啉的显著抑制；Ca²⁺、Zn²⁺、Mg²⁺可以显著提高该蛋白酶的活性，表明该酶为金属蛋白酶。另外，利用qRT-PCR试验测定了基因Mep在锈菌侵染向日葵的过程中的表达情况，结果显示，在接菌48 h时，基因表达量达到高峰，后逐渐降低，表明该蛋白酶参与了锈菌对向日葵的早期侵染过程。

Characterization of extracellular metalloproteinase from Puccinia helianthi

To further understand the pathogenic mechanism of sunflower infected by rust, the hydrolytic and biological activity of a novel metalloproteinase (Mep) secreted by Puccinia helianthi were characterized and analized. Protease activity was determined by azocasein method, and its absorbance was measured at 280 nm. Results showed that the protease had hydrolytic activity to azocasein, and the optimum temperature and pH of pure enzyme were 50℃ and 7.0 respectively. It remained stable at pH 5.0-9.0 and below 50℃. The activity of Mep was significantly inhibited by ethylene diamine tetraacetic acid disodium (EDTA), ethylene glycol (2-aminoethyl ether) tetraacetic acid (EGTA) and 1,10-phenanthroline. Ions Ca²⁺, Zn²⁺ and Mg²⁺ could significantly increase the activity of Mep. The results indicated that the enzyme was a metalloproteinase. In addition, qRT-PCR results showed the expression of Mep gene in the process of rust infection of sunflower. The gene expression reached a peak at 48 h after inoculation, and then gradually decreased. It could be proved that Mep was involved in the early infection process of P. helianthi to sunflower.