Construction Of Agrobacterium-mediated Transformation System In Macrocybe Gigantea

Abstract:Macrocybe gigantea（Massee）Pegler&Lodge is a rare and nutritious wild edible fungus in the tropical regions.It has been successfully domesticated and cultivated.The fruit body of M.gigantea can be stored at 8℃～12℃for 30 days without color changes.Studying its anti-browning mechanism is of great significance for the storage and preservation of edible fungi.With the completion of its genome and transcriptome sequencing,functional genomics research will be the focus of future research.The genetic transformation system is the basis for the study of gene functions.Agrobacterium-mediated transformation has been successfully applied to a variety of edible fungi due to its wide selection of transforming recipient materials,high transformation efficiency,and simple operation procedures.Nevertheless,few studies have been conducted to construct genetic transformation system in M.gigantea.In this study,an efficient system for genetic transformation of M.gigantea was constructed.Various parameters were optimized to obtained successful Agrobacterium-mediated transformation.At the same time,the gene function of TYR7523 was initially investigated by overexpression technology.The main results are as follows:1.The hygromycin resistance gene Hyg was used as a screening marker.The lethal concentration of hygromycin in M.gigantea mycelium was determined to be 70μg/m L by the hygromycin sensitivity test,and it was used as the screening concentration of transformants.The inhibitory concentration was 300μg/m L,based on the toxicity test of cefotaxime to M.gigantea mycelium and Agrobacterium tumefaciens.2.The Agrobacterium mediated transformation system of M.gigantea was established by transforming the binary vector plasmamid4 into Agrobacterium tumefaciens EHA105,using M.gigantea mycelium as the receptor material,hyg as the screening marker and e GFP as the reporter gene.The transformation efficiency was the highest when the co-cultivation temperature was 25℃and the co-cultivation time was 36 h.3.PCR identification and green fluorescence analysis of randomly selected transformants were carried out,and the target bands of hyg were successfully amplified,and obvious green fluorescence was observed under the fluorescence microscope,indicating that hyg was successfully integrated into the genomic DNA of M.gigantea,and e GFP gene was expressed in M.gigantea.In order to verify the mitotic stability of the transformants,they were subcultured for five generations,and the green fluorescence was still observed in the fluorescence microscope,indicating that the exogenous gene e GFP could be inherited stably in M.gigantea.4.In order to study the gene function of TYR7523,a tyrosinase gene selected by transcriptome sequencing,specific primers were designed based on its sequence,and a 381 bp target fragment was cloned,encoding a protein of 126 amino acids.The domain of Cu B was highly conserved according to the predicted amino acid sequence,combining with three histidine residues,the structure of H_{Cu B}xxx H_{Cu B}x（n）Fxx HH_{Cu B}was formed,but there was no domain of Cu A.In addition,there is"tyrosine motif",i.e.Y/Fx Y motif,which was very important for the overall structural integrity of N-terminal domain.5.The specific primers were designed based on the 1000 bp upstream sequence from the start codon（ATG）of gpd gene,and a 1019 bp gpd promoter sequence was cloned.An TYR7523 overexpression vector,plasmid4-GT,was constructed.