The Function Analysis Of ATP-dependent Lon Protease In Magnaporthe Oryzae And Thermomyces Lanuginosus

Magnaporthe oryzae, which is a world-wide plant pathogenic fungi, brings to the greatest loss of rice production every year in China. MAP1-a homologous Lon protease in Magnaporthe oryzae, has been proved to have a relationship with the pathogenicity of M. oryzae. In this research, we submitted the MAP1 amino acid sequence to the M. oryzae databases and analyzed by BLAST and got another homologous lon protease-MGG₀7123, named as MAP2.When analysed the sequence between MAP1 and MAP2 by Clustal 2.0 software, we found that the sequence similarity was 23.49%. Furthermore, to know the specific function of MAP2, we constructed the knockout vector by using the method of homologous recombination and gained a knocked-out mutant using the method of ATMT. According to further study of the mutant, we demonstrated that the mutant did not only affect the growth and development, appressorium formation, and pathogenicity of M. oryzae, but also did not participate in abiotic responses.Thermomyces lanuginosus, which belongs to a theomophilic fungi, is a wide-ranging filamentous fungi and it takes a close relation to our life. At present, it can be widely used in fermentation industry, brewing industry, medicine, waste treatment, and so on, so, the researchers focused their studies on the physiological and biochemical of lipase, xylanase, chitin decomposition enzyme, but there is no study on other genes about genetic function aspect. In this study, we submitted the MAPI amino acid sequence to T.lanuginosus databases and analyzed by BLAST and got two homologous lon protease-Thela2p4₀05149 and Thela2p4₀06664. Aligned by Clustal 2.0 software, it found that the sequence similarity was 64.61% beween MAP1 and Thela2p4₀05149, and the sequence similarity was 24.96% beween MAP1 and Thela2p4₀06664, but the sequence similarity was only 23.80% beween Thela2p4₀05149 and Thela2p4₀06664. Next, according to the analysis of prediction of protein subcellular locations, and identified two Lon proteases located in mitochondria and peroxisomes, which were designated as Mlon and Plon, respectively.We sumitted the Mlon and Plon amino acid sequence to each bioinformatics website, the results showed that Mlon contains 1092 amino acids and belongs to unstable and hydrophilic protein and Plon contains 914 amino acids belonging to stable and hydrophilic protein; the main secondary structure of Mlon and Plon is β-sheet and random coil respectively, and the occupancy is 57.6% and 51.53%; at SWISS-MODEL online service, we acquired the homologous modeling of Mlon and Plon, and then did the Three-dimensional structure of both of them, the results showed that the Mlon had a compact structure but Plon had a relatively loose structure.To further study the function of Mlon and Plon, we constructed the knockout vector and complementary/subcellular localization vector and gained the knocked-out and complementary/subcellular localization mutant of Mlon and Plon - ΔMlon, ΔPlon, ΔMlon/Mlon, ΔPlon/Plon using the method of ATMT and PEG-mediated transformation of protoplast. The results illuminated that both of ΔMlon and ΔPlon grew slower than that of wild strain; the aerial hyphae of ΔMlon was much denser and thicker than that of wild strain, but ΔPlon owned much fewer aerial hyphae than that of wild strain; the dry weight of hypha of ΔMlon was much heavier than that of wild and complementary strain, but the ΔPlon had the opposite result; the spores of ΔMlon are one third of that of wild and complementary strain, but the spores of ΔPlon are three times of that of wild and complementary strain; the wild strain cultured at 37℃ stimulated the relative expression of Mlon and Plon gene and the relative expression of Mlon was higher than that of Plon gene; and the relative expression of Plon gene was higher than that of Mlon gene when the wild strain cultured at 50℃ for 1 and 2 days, but the relative expression of Plon gene was lower than that of Mlon gene when under the same condition; the spore germination number of ΔMlon was more than that of wild and complementary strain, but the ΔPlon strain had a contrary consequence.We also did the H2O2 stress test, the results as follows:Both of ΔMlon and ΔPlon mutants were sensitive to H2O2 stress and the growth of AMlon mutant was significantly inhibited when the medium added 5mM H2O2, but when the medium added 2.5mM H2O2, the growth of APlon mutant has been significantly inhibited; when the hypha of WT, ΔMlon and ΔPlon were stained with DAB for 8h, only little hypha of ΔMlon turned sepia compare to WT, but much hypha of ΔPlon turned sepia; and the concentration of H2O2 in WT, ΔMlon and ΔPlon is 9.7μ mol/g,14.6μ mol/g, and 17.4μ mol/g respectively; when added 1 mM H2O2 in medium, both of the relative expression of Mlon and Plon was higher than the control; comparison to wild type strain, both of Mlon and Plon were partly related to cell wall integrity, but both of them were not related to osmotic pressure sensitivity.Above all, the study was on the basis of a homologous Lon protease in M.oryzae, and through amino acid sequence alignment in M.oryzae and T. lanuginosus database, we got three homologous Lon protease-MAP2, Mlon and Plon. Furthermore, via to delete the MAP2, Mlon and Plon gene, we gained the knocked-out mutant-ΔMAP2, ΔMlon and ΔPlon. The latter experiments verified that the MAP2 gene of M.oryzae did not participate in abiotic responses and was no related to pathogenicity of M.oryzae, the Mlon gene in T. lanuginosus involved in the regulation of procreation growth of T. lanuginosus, the Plon gene in T. lanuginosus involved in the regulation of vegetative growth of T. lanuginosus, and both of Mlon and Plon were related to H2O2 stress response. And through the function analysis of ATP-dependent Lon Protease in M. oryzae and T. lanuginosus, we can further study the functional difference between the proteins within the same family in different/same species, and we can also provide some theoretical and practical basis for T. lanuginosus and other thermophilic fungi to break the thermophilic mechanism of thermophilic fungi.