Abstract
During sexual reproduction of Closterium psl. complex, the mt- cells secrete a pheromone PR-IP Inducer and then mt+ cells secrete PR-IP in response to the Inducer. Several reproduction-related and sex-specific genes have also been identified. To investigate function of such genes, we have developed a gene introduction system using particle bombardment. Here, we report the evaluation of stable transformants, as a first step of construction for over-expression system in C. psl. complex.
A novel vector, both for the selection by phleomycin and for the over-expression of a gene encoding PR-IP Inducer (CpPI), was constructed and introduced into the mt+ cells, in which expression of CpPI hardly occurred irrespective of the culture condition. Several transformants could be obtained and showed ~18-fold expression of CpPI compared with WT mt+ cells. The expression level of PR-IP gene (Cp19KSU) was also elevated ~80-fold in these cells. These results suggest that the active PR-IP Inducer can be produced in mt+ cells and can induce the expression of PR-IP gene in the same cells. This method would be applicable to the studies based on reverse genetics of unknown genes in the C. psl. complex.
