Effect Of Polyketide Stnthase Gene Silencing On CheA Synthesis In Chaetomium Globosum

Fungi can synthesize a large number of secondary metabolites which have different chemical structures and biological activies. Meanwhile, fungal metabolites played important roles in drug research and biocontrol. Chaetomium spp. is a large family of fungi genus which can produce a variety of secondary metabolites as described before. In this experiment, we used Chaetomium globosum W7 as the wild type. We used the RNAi to find out the influences of polyketide synthase gene in chaetoglobosin A(Che A) biosynthetic pathway.In this paper, we analyzed the genome of C. globosum in NCBI database. Then we found the polyketide synthase gene and figured out the conserved domain in the sequence. According to what we found, we amplified the conserved sequence by PCR amplification. We used the cloned sequences to construct Gene Silencing Vector named p S-pks for drown-Regulated the pks gene in the wild type C. globosum W7. We obtained totally 22 transformants by PEG-mediated protoplast transformation. Then we divided the transformants into three categories. The first type were transformants 0、9、10、11、12、16; the second type were transformants △1、△2、△3、△5、△6、△7、3、5、18、25、27、28、29; the third type were transformants △4、15、31. Comprehensive consideration, we chose transformants 0、△7、31 to undertake the further experiments.Then we also found a good way to extract Che A from fermentation. The results were as follows. The extraction solvent was acetone. The mobile phase was the ratio of acetonitrile to water equals 0.82(45:55). Detection wavelength was 227 nm. Detection time was 40 min. Retention time of Che A was about 20 min with no miscellaneous peak.Finally, we studied on the characteristics of three transformants and wild type in three aspects. 1. We detected the expressions of pks gene in transformants and wild type by using real-time quantitative PCR. The results showed that expressions of the three transformants of pks gene were decreased at different degrees. The expression of the transformants 0、△7、31 were 68%、49%、92% lower than the wild type. 2. We detected fermentation products of the transformants and wild type by HPLC. The yields of Che A in transformants 0、△7、31 were 2.57 μg/m L、3.62 μg/m L、0 μg/m L. The yield of the wild type was up to 57.08 μg/m L. It was much higher than all the chosen transformants which means it had the same result with real-time quantitative PCR. 3.Inhibition effects of transformants 0、△7、31 and wild type were against Rhizoctonia solan by dual culture on potato dextrose agar Petri plates. The results showed that decline of Che A production of each transformants hsd the decreases in bacteriostatic abilities.For C. globosum, Che A production was significant in biocontrol.This research proved that the expression of pks gene was important in the growth processes of C. globosum. Pks gene was one of the synthetic gene in polyketide assembly of Che A. Meanwhile, pks gene had the crucial role in fungal biocontrol.