青色荧光蛋白标记的禾谷镰孢转化子的构建

Construction of CFP-labeled Fusarium graminearum transformants

摘要:

【背景】近年来玉米茎腐病在我国大部分玉米产区普遍重度发生，其中镰孢菌茎腐病不仅造成了重大的经济损失，而且镰孢菌产生的毒素给人体和动物的健康也带来严重威胁。【目的】玉米茎腐病的病原组成复杂，禾谷镰孢是其中的主要病原之一，该病原菌侵染寄主导致发病的机制急需深入研究。【方法】以pCAMBIA1300质粒为骨架，利用重叠PCR的方法构建表达青色荧光蛋白的质粒pCAMBIA1300-CFP-Kan，通过农杆菌介导的遗传转化技术，将青色荧光蛋白的编码基因整合到禾谷镰孢基因组中。【结果】经过PCR鉴定和荧光显微观察，确定获得了31株青色荧光标记的禾谷镰孢菌。【结论】侵染试验结果显示，激光共聚焦显微镜下禾谷镰孢在玉米茎秆组织中的定殖位置清晰可见，该结果为进一步研究不同镰孢菌在寄主中的定殖规律奠定了基础。

Abstract:

[Background] In recent years, maize stalk rot occurs widely and seriously in most corn-growing areas in China. Fusarium stalk rot not only causes an important economic loss of corn, but also is a serious threat to human and animal health because of the toxins produced by Fusarium species. [Objective] Among the complicated pathogenic fungi of corn stalk rot, Fusarium graminearum is one of the major pathogens. The infection mechanism of F. graminearum still remains an urgent research topic. [Methods] The plasmid pCAMBIA1300 was taken as the backbone, and the plasmid pCAMBIA1300-CFP-Kan, expressing the cyan fluorescent protein, was constructed using the overlapping PCR. Then the CFP gene was transformed to the genome of F. graminearum by Agrobacterium-mediated genetic transformation technology. [Results] After PCR identification and fluorescence microscopy, 31 strains labeled with CFP were acquired. [Conclusion] The pathogenicity test results suggested that F. graminearum successfully colonized in corn tissue under laser confocal observation. These results lay a solid foundation for study of the colonization of Fusarium species.