Construction Of Agrobacterium-mediated Transformation System In Lentinula Edodes

Lentinula edodes is a kind of widely cultivated edible fungi. With its genome sequencing has completed, functional genomics research is gradually developed. As a genetic function research foundation, genetic transformations has been widely used in edible fungus researches, in which the Agrobacterium-mediated transformation method has the characteristics of few copies, stable and effective. An Agrobacterium-mediated genetic transformation system of L. edodes was constructed in this study.First, using cloned gpd promoter of L. edodes W1to replace the promoter CaMV35S which before the HYG gene from the plasmid pCAMBIA1301constructs a new transformation vector pCAMBIA1301-gpd. Second, vectors pCAMBIA1301-gpd and pCAMBIA1301are imported into Agrobacterium EHA105respectively. Under acetosyringone (AS), Agrobacterium EHA105are co-cultured with L. edodes Wl, L205mycelium for3days. After the ddH2O mycelium washing of L. edodes, hygromycin, the antibiotics are used in resistance selection, then PCR is choosed to rule out the false positive interference. The last, the transformants are successfully obtained. In the end, the transformants, which are subcultured in malt extract medium (MYG) after5rounds, still has a stable growth in the hygromycin resistance medium.The different screening efficiency under hygromycin B of promoter to different receptor strains shows that, when vector pCAMBIA1301-gpd transforms L. edodes W1, the highest screening efficiency under hygromycin B reaches about30%. Among them the screening efficiency under hygromycin B with gpd promoter is twice as likely as CaMV35S promoter. Those implies that constitutive endogenous promoter for expression gene in L. edodes has a strong promotion effect. The screening efficiency under hygromycin B of strain Wl is higher than strain L205, this may has relation to the genetic background of receptor strains.At the same time, this study optimizes the co-cultured conditions from four aspects, Agrobacterium infection time, co-cultured temperature, co-cultured time, and whether to add AS in the process of co-cultured. The results show that the influence of screening efficiency under hygromycin B with Agrobacterium among the infection time of10min,20min and30min are not obvious; the optimum co-cultured temperature is25℃; screening efficiency under hygromycin B between the co-cultured time for3days and4days has no significant difference, and co-cultured for2days can not complete the transformation; adding AS during the co-cultured is necessary for the growth of transformants.The genetic transformation system of L. edodes constructed in this study, is to lay the foundation for the future study of gene recombination, gene silencing, gene expression and insertional mutagenesis in L. edodes. What’s more, the system can provide an important techonical support of growth mechanism research in L. edodes.