《植物生理学报》 2015, 51(12): 2223-2230

超表达岷江百合类萌发素蛋白基因LrGLP2增强烟草对几种病原真菌的抗性

韩青, 杨野, 陈瑞, 葛锋, 陈朝银, 刘迪秋^*

昆明理工大学生命科学与技术学院, 昆明650500

通信作者：刘迪秋；E-mail: diqiuliu@126.com；Tel: 0871-65920621

摘　要：

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, T₂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T₂代转基因烟草对几种病原真菌的抗性。

关键词：岷江百合; 类萌发素蛋白; 尖孢镰刀菌; 病原真菌抗性

收稿：2015-05-15   修定：2015-10-09

资助：国家自然科学基金(31160401)。

Overexpression of Germin-Like Protein Gene from Lilium regale Enhance Resistance to Several Pathogenic Fungi in Transgenic Tobacco Plants

HAN Qing, YANG Ye, CHEN Rui, GE Feng, CHEN Chao-Yin, LIU Di-Qiu^*

Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China

Corresponding author: LIU Di-Qiu; E-mail: diqiuliu@126.com; Tel: 0871-65920621

Abstract:

Germin-like proteins (GLPs) are ubiquitous plant glycoproteins and belong to the cupin super family. The GLPs possess diverse functional roles in plants. Previously, a GLP gene from Lilium regale Wilson, Lr-GLP2, responded to the infection of Fusarium oxysporum f. sp. lilii. In order to verify the function of LrGLP2, the constitutive plant expression vector of LrGLP2 was constructed and transferred into tobacco (Nicotiana tabacum L. cv Xanthi). Totally, there were 35 positive transgenic tobacco screened out through the polymerase chain reaction (PCR) analysis. The LrGLP2 gene was successfully integrated into the genome of the transgenic tobacco lines and expressed as expected in the transformants through Southern blotting and quantitative reverse transcription-PCR (qRT-PCR) analysis. The antifungal activity of LrGLP2 was detected in vitro plates, and the crude protein extract of transgenic tobacco lines inhibited the hyphal growth of the B. dothidea, F. oxysporum, and B. cinerea, respectively. Furthermore, in vivo inoculation assay indicated that the transgenic tobacco lines showed strong resistance to the infection of F. oxysporum and B. cinerea stress, respectively. Simultaneously, the SOD, GST and OXO showed significantly higher activities in the T₂ transgenic lines than in wild type under normal conditions or after inoculation with F. oxysporum. In conclusion, overexpression of LrGLP2 from L. regale enhanced resistance to several pathogenic fungi in the T₂ transgenic tobacco plants.

Key words: Lilium regale; germin-like protein; Fusarium oxysporum; resistance to fungal pathogen