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类萌发素蛋白(germin-like proteins,GLPs)是普遍存在于植物体内的具有多种功能的糖蛋白,属于cupins蛋白家族。岷江百合类萌发素蛋白基因LrGLP2响应病原真菌尖孢镰刀菌(Fusarium oxysporum)的入侵。为了验证该基因的功能,本文构建了LrGLP2的植物超表达载体并转入烟草中,PCR筛选获得了35棵阳性转基因烟草。Southern杂交和qRT-PCR分析显示,LrGLP2基因已成功整合到转基因烟草基因组中并稳定表达。体外抑菌实验表明LrGLP2转基因烟草叶片粗蛋白能够不同程度地抑制葡萄座腔菌(Botryosphaeria dothidea)、尖孢镰刀菌和灰葡萄孢(Botrytis cinerea)菌丝的生长。活体接种结果显示,转基因烟草具有很强的抗尖孢镰刀菌和灰葡萄孢侵染的能力。此外,尖孢镰刀菌侵染前后,与野生型烟草相比,T_2代转基因烟草中超氧化物歧化酶、谷胱甘肽硫转移酶和草酸氧化酶酶活性均显著增强。本文的研究结果表明岷江百合类萌发素蛋白基因LrGLP2是一个抗真菌功能基因,在烟草中超表达增强了T_2代转基因烟草对几种病原真菌的抗性。
Germin-like proteins (GLPs) are ubiquitous glycoproteins that have multiple functions in plants and belong to the cupins family of proteins. Infection of Minjiang Lily Sprout Protein Gene LrGLP2 in Response to Fusarium Oxysporum Pathogen. In order to verify the function of this gene, we constructed a plant overexpression vector of LrGLP2 and transferred it into tobacco plants. 35 positive transgenic tobacco plants were obtained by PCR screening. Southern hybridization and qRT-PCR analysis showed that the LrGLP2 gene has been successfully integrated into the genome of transgenic tobacco and stably expressed. In vitro antibacterial experiments showed that the crude protein of LrGLP2 transgenic tobacco could inhibit the growth of Botryosphaeria dothidea, Fusarium oxysporum and Botrytis cinerea to varying degrees. In vivo vaccination results showed that the transgenic tobacco has a strong resistance to Fusarium oxysporum and Botrytis cinerea infection. In addition, the activities of superoxide dismutase, glutathione S-transferase and oxalate oxidase in T 2 transgenic tobacco were significantly increased compared with wild-type tobacco before and after infection with Fusarium oxysporum. The results of this study indicate that LrGLP2 is an antifungal gene, and overexpression enhances the resistance of T_2 transgenic tobacco to several pathogenic fungi.