Cloning And Functional Analysis Of Homologous Protein Gene Fmo

The flavin monooxygenase(flavin-containing monooxygenase, FMO) have the function of Catalytic oxidation of containing nitrogen, sulfur, phosphorus, selenium and other dear nuclear xenobiotic compounds and drug. In addition, the existence of fmo is also found in yeast, shark, rainbow trout, Arabidopsis and other species, but fmo gene structure and function in filamentous fungi is not clear, the laboratory was found a sequence homologous with fmo, in the early construction EST Library of Metarhizium anisopliae infected locust haemolymph, named Mafmo. Preliminary studies have demonstrated that Mafmo gene may affect virulence of M. acridum, growth and sporulation. In order to elucidate the gene structure and function of Mafmo,This paper takes the M. acridum as experimental materials,clone full-length Mafmo gene, construct Mafmo complementary strains, detection of Mafmo expression level in different developmental stages of Metarhizium anisopliae, analyzed the effect of Mafmo on growth and stress resistance function of M. acridum, Observation of the type of blood cells in phagocytosis after M.acridum infected host locust. The results are as follows:1. Cloning of Mafmo gene and Construcing of Mafmo complementary strains.(1) Using rapid amplification of cDNA ends(rapid amplification using of cDNA ends,RACE), and designing primer based on the known EST sequence of Mafmo, dubbed CJY1439, length 710 bp,to get the 3 ’end and the 5’ end sequence, successfully amplified the full-length Mafmo gene 4792 bp by PCR.(2) Mafmo complementary vector was constructed successfully by connect the Mafmo full length DNA and pK2-sur-egfp vector; Have gotten Mafmo complementary strains by Agrobacterium mediated transformation method.2. Effect of Mafmo on the growth and development of M. acridumInoculated WT and Δ Mafmo on 1/4SDA agar culture medium, observation and comparison of growth and development of M. acridum.every 2 hours. Found that the spore germination rate and cell cycle were not different,but the time of spore structure formed were differernt, growth to 14 h WT has a sporogenous structure formation,but ΔMafmo does not have,the results suggest that Mafmo may be involved in Spore formation of M.acridum.3. Mafmo does not affect the germination rate and sporulation of M. acridum.Measurement and statistics the sporulation and germination rate of M. acridum,Analysis of date by SPSS.The results show that there were no significant differences between WT and ΔMafmo.4.Effect of Mafmo on the biomass accumulation of M. acridum.By weighing the dry weight of mycelium of WT、ΔMafmo and CP on the third day.Found that the biomass accumulation ofΔMafmo is 50% more than WT and CP. Analysis of date by SPSS,that the difference was significant. The results show that Mafmo is involved in the the metabolism of M. acridum.5. Effect of Mafmo on the heat shock sensitivity of M. acridum.To investigate the resistance of the Mafmo, We measured the germination rate of spore in the UV-B and heat shock conditions, Among them, WT was similar to ΔMafmo in the UV-B condition,but in the heat shock condition,WT have a significant difference in the rate of germination compared with ΔMafmo. The results showed that Mafmo Effect of the heat shock sensitivity of M. acridum.6. Analysis of the expression level of MafmoExtraction of M. acridum RNA in various developmental stages.Through RT-PCR and qRT-PCR analysis of the expression of Mafmo during mycelium, conidium, 24 h conidium appressorium and hyphal body development period. The results show that, the highest expression level of Mafmo is hyphal body development period, the lowest level of expression of Mafmo is spore development period.7. Observation of phagocytosis of M. acridum infected locusts blood cellsTo observe the locusts blood cells, found that the amount and type of blood cells have Great changes afte be M. acridum infection, by Wright-Giemsa staining method.