荧光蛋白在双色蜡蘑中的构建与表达

菌根是真菌与植物之间形成的互惠互利的营养共生体，对生态环境有重大的意义。外生菌根真菌与植物互作机制以及真菌基因功能的深入研究都需要对菌根真菌进行遗传转化，本研究以外生菌根真菌模式生物双色蜡蘑(Laccaria bicolor)为研究对象，选择细胞核中的核小体蛋白H₂B基因为目的基因，以pCEBN为表达载体，融合红色荧光蛋白，最终构建在真菌中表达的双元载体，使用根瘤农杆菌介导转化法转化双色蜡蘑菌丝，随后利用PCR对真菌转化子进行验证后，通过激光共聚焦显微镜观察到菌丝细胞核中的红色荧光，成功将融合荧光蛋白转化菌根真菌，为后续研究菌根真菌中基因的亚细胞定位提供了实验平台。结果表明，利用双元载体和农杆菌转化方法，建立了高效的双色蜡蘑转化体系(93.33%),在激光共聚焦显微镜下观察到菌丝细胞核中红色荧光信号，验证了融合荧光蛋白在双色蜡蘑中的成功表达。本研究成功地构建了菌根真菌中的核小体蛋白和红色荧光蛋白融合表达的真菌转化体系。

Construction and Expression of Fluorescent Proteins in Laccaria bicolor

Mycorrhiza is a mutually beneficial nutritional symbionts formed between fungi and plants, which play key role in ecological environment. The research of the interaction mechanism between ectomycorrhizal fungi and plants and the function of fungal genes require the establishment of genetic transformation of ectomycorrhizal fungi. In this study, ectomycorrhizal fungal mode of Laccaria bicolor were chosen as a reaserch object, and selected nucleosome protein H₂B gene of nucleus as target gene, and take pCEBN as expression vector fused with the red fluorescent protein, and finally constructed a binary vector in fungus, and mediated with Agrobacterium tumefaciens to transform L.bicolor mycelium, and after verified the fungal trnasfomant with PCR, the red fluorescence in mycelial nucleus was observed using laser confocal microscope, thus successfully the fused fluorescent protein transformed the mycorrhizal fungi. And provided an experiment platform for subsequent study on subcellular localization of mycorrhizal fungi. The results showed that high efficient L.bicolor transformant system was established (93.33%) using binary vector and A. tumefaciens transformation method. The signal of hyphae with red fluorescence in nucleus was observed under laser confocal microscope, thus verified that the fusion fluorescent protein was successfully expressed in L.bicolor. In a word, this study the fusion expressed fungal transformant system of nucleosome protein and red fluorescent protein in mycorrhizal fungi was successfully established.