Construction Of Agrobacterium-Mediated Transformation System In Hypsizygus Marmoreus

Hypsizygus marmoreus is a special rare mushroom in north temperate regions and one of the main mushrooms which has been achieved year-round industrial cultivation in our country. Nevertheless, H. marmoreus functional genomics research is still relatively weak currently. The lack of a stable and efficient exogenous gene transformation system is an important limiting factor. In recent years, Agrobacterium tumefaciens-mediated transformation (ATMT) in edible fungi has made some progress, but the transformation system of H. marmoreus has not yet been reported. In our study, we try to build a stable and efficient genetic transformation system of H. marmoreus using ATMT as a tool. The main results of this paper are as follows:(1) After binary expression vector pYN6982 was transformed into A. tumefaciens LBA4404, we transformed H. marmoreus SIEF3133 blended vegetative dikaryotic mycelia via Agrobacterium-mediated transformation method. Using hygromycin resistance selection, PCR identification and mitotic stability test, we confirmed that hygromycin phosphotransferase (hph) gene was integrated into H. marmoreus genome. Dozens of transgenic strains of H. marmoreus with genetic stability were obtained. After the above study, we initially established a stable genetic transformation method of Hypsizygus marmoreus.(2) The relevant factors affecting H. marmoreus ATMT transformation efficiency were optimized. Optimization factors include following six aspects:Agrobacterium species, infection concentrations of A. tumefaciens, infection time, co-cultivation temperature, length of duration of co-cultivation, AS concentration. So a system of Agrobacterium-mediated transformation in H. marmoreus was established successfully. The optimal conditions for this ATMT system were as followed:Agrobacterium strains EHA105, OD600=0.5 of infection concentrations,20 min of infection time,24℃ of co-cultivation temperature,60 h of co-cultivation time,200μmol/L of AS concentration.(3) We probed egfp gene expression and T-DNA integration case of transgenic H. marmoreus strains. By fluorescence microscopy analysis of randomly selected transformants, green fluorescence can be observed. This result indicated that egfp gene has expressed in transgenic H. marmoreus. Moreover, two of eight randomly selected H. marmoreus transformants were found containing kan gene sequences from outside the T-DNA border repeats using PCR analysis. This means that DNA sequences from beyond the classically defined T-DNA border direct repeats can be transferred into the H. marmoreus mycelia.(4) We conducted colony morphology observation and fruiting test in some transgenic H. marmoreus strains. Compared to wild-type H. marmoreus strain, individual transformants were detected significant differences in colony morphology. We speculated that inserted T-DNA might damage functional gene leading to changes in transformants colony morphology. This suggests that we can use ATMT as a tool for insertional mutagenesis in H. marmoreus. In addition, we found that most of H. marmoreus transformants could normal fruiting. A clear target band ofhph gene was got from all of 11 normally fruiting H. marmoreus transformants using PCR analysis. This result further validated that T-DNA integration is highly conserved via A. tumefaciens mediated transformation.