Abstract:Blakeslea trispora (B. trispora), an important filamentous fungus, is used industrially in the mass production of carotenoids. The lack of a deep understanding of the genetic background of B. trispora and the shortage of genetic manipulation tools have hampered the genetic studies of B.trispora and the biological studies of carotenoid synthesis. For B. trispora, protoplast transformation rate is low and the spore cell wall is thick, which cause difficulty in introducing exogenous vectors into the cell. To solve this problem, this study constructed a transformation system mediated by Agrobacterium tumefaciens (A. tumefaciens) infecting protoplast, and cloned ku80 gene which was related with the non-homologous end joining repair pathways at the same time, and then applied the transformation system in the knockout of ku80. A recombinant knockout vector pDH-85H3 was constructed by inserting ku80 knockout frame into the binary vector pDHt-sk. And then the vector pDH-85H3 was introduced into A. tumefaciens LBA4404 by chemical transformation. Finally, the protoplast of B. trispora was transformed by A. tumefaciens-mediated method. By hygromycin screening and PCR analysis, there were 18 transformants in which the knockout box was inserted into the genome out of 20 transformants, and the transformation rate was 90%. Further analysis indicated that there were 2 transformants in which homologous recombination occurred and the knockout rate was 10%. The result indicated that A. tumefaciens mediated protoplast transformation in B. trispora was feasible.
