Construction T-DNA Binary Vector Of Phya Gene And Transformation In Ganoderma Lucidum Protoplasts

Phytic acid is the main form of phosphorus exists in plant feed. Because the digestive system of mono-gastric animal does not contain phytase, so it is very difficult to use phytate phosphorus directly. The development and application of phytase could solve this problems effectively. Ganoderma is an important edible and medicinal fungus, it has a strong physiological activity and pharmacological activity. As a large fungus, it has a very high value. However, the development of Ganoderma in molecular biology level has not yet formed as a perfect system, and its safe transformation system and efficient expression system is still in the initial stage. In this study we first used Ganoderma mycelium as bioreactor system, establish Ganoderma mycelium co-transformation system and efficient expression system. We first constructed the fungal-specific T-DNA binary vector system and pSB130 NG vector by fungal-specific promoter GPD and terminator NOS, then phyA was cloned and inserted into pSB130 NG. Furthermore, we further transferded phyA to Ganoderma protoplast by Agrobacterium-mediated transformation method. And the phyA transgenic Ganoderma mycelium was finally obtained. The main results were as follows:1.The fungal-specific T-DNA binary vector system and pSB130 NG vector were successfully constructed with fungal-specific promoter GPD and terminator NOS by Pst I, Sac I and EcoR I.2.PhyA was successfully cloned and recombined into T-DNA binary vector pSB130 NG by Xba I.3. Protoplasts of Ganoderma were extracted by lywallzyme, the activity of the protoplasts was tested by FDA dyeing method, the survival rate of Ganoderma protoplasts was 90%, the size and the shape of Ganoderma protoplasts were normal.4.The recombined plasmid pSB130NG-phyA was transformed into Agrobacterium EHA105, Ganoderma protoplasts were transformed by Agrobacterium transformation method, and phyA was successfully transformed into Ganoderma protoplasts.5.Transformed Ganoderma protoplasts were regenerated on MYG medium containing hygromycin. Then obtained the transgenic Ganoderma mycelium, the transformation rate was 2/107.