柱花草甲壳质酶基因SgGH19-1的克隆表达及酶学性质分析

甲壳质是真菌细胞壁、昆虫外骨骼和甲壳类动物外壳的主要成分。甲壳质的降解主要依赖甲壳质酶的水解作用。诱导PR-3类甲壳质酶蛋白积累是植物增强对真菌性病害抗性的适应性机制。柱花草是一种重要的热带豆科牧草,由胶孢炭疽菌引起的炭疽病是危及柱花草生产的主要真菌性病害。但柱花草甲壳质酶对炭疽菌侵染的响应仍不清楚。本研究旨在鉴定柱花草PR-3类甲壳质酶基因,并对其表达模式、编码蛋白的生化酶学性质进行分析。结果表明：通过同源克隆获得了1个柱花草PR-3类甲壳质酶基因,其编码区序列全长984 bp,属于糖苷水解酶19家族的ClassⅠ亚族,将其命名为SgGH19-1。荧光定量PCR分析表明,接种炭疽菌诱导SgGH19-1在柱花草叶片中显著增强表达,并伴随着甲壳质酶活性的提高。随后,在大肠杆菌中表达纯化了SgGH19-1重组蛋白,生化酶学性质表明,SgGH19-1蛋白兼具甲壳质内切酶与外切酶活性,但内切酶活性比外切酶活性高9.1倍。SgGH19-1的最适pH为5.0,最适温度为40 ℃。综上所述,SgGH19-1基因参与柱花草对炭疽菌侵染的响应,其编码的蛋白具有甲壳质酶活性,可作为柱花草抗炭疽病育种的分子标记。

Cloning and Expression of a Chitinase Gene SgGH19-1 from Stylosanthes and Its Enzymatic Properties Analysis

Chitin is a major component of the cell wall of fungi, exoskeleton of insects, and crustacean shells. Chitinase is one of the main hydrolase that catalyzes the degradation of chitin. Induction of PR-3 chitinase protein accumulation is an adaptive mechanism for plants to enhance the resistance to fungal diseases. Stylo (Stylosanthes spp.) is an important tropical forage legume. Although anthracnose caused by Colletotrichum gloeosporioides is one of the most destructive fungal diseases of stylo, little information is available regarding the responses of stylo chitinase during the pathogen infection. In the study, the PR-3 chitinase of stylo was characterized, and its expression pattern and biochemical properties were also analyzed. The results showed that a chitinase gene of PR-3 group in stylo was the cloned by homologous gene sequence method, and its coding region was 984 bp. The gene belongs to the Class I chitinase of the glycoside hydrolase 19 family, and was named SgGH19-1. Quantitative PCR analysis showed that the expression level of SgGH19-1 gene in the leaves of stylo was significantly increased after C. gloeosporioides infection, and with the increase of chitinase activity. Subsequently, the recombinant protein of SgGH19-1 was expressed and purified in Escherichia coli. Biochemical properties of SgGH19-1 showed both exochitinase and endochitinase activities, and the activities of endochitinase was 9.1 fold higher than that of exochitinase. Furthermore, the optimum pH and temperature for SgGH19-1 activity was 5.0 and 40 ℃ respectively. Taken together, SgGH19-1 is a chitinase that involved in the response of stylo to C. gloeosporioides attack. These results herein suggest that SgGH19-1 could potentially be employed as a new marker gene for developing cultivars of stylo with great tolerance to anthracnose.