Study On Gene Cloning And Expression Of Infectious Extracellular Proteases From Nematophagous Fungi

Every year severe damage caused by plant-parasitic nematodes to crops is more than 100 billion US dollars in the world (Sasser & Freekman, 1987). The more information about molecular background of infection mechanism we know, the more success of biological control of parasitic nematodes we can get. The key factors affecting the biocontrol efficiency is pathogenicities of nematophagous fungi. It is an effective solution to successful biocontrol of parasitic nematodes that molecular cloning and expression of specific genes encoding fungal extracellular enzymes related to infection process. The study is of scientific importance and broad applications for good understanding the recognition (interaction) between fungi and nematodes, the molecular mechanism of fungal infection to host, and practical application in biocontrol strategy. Typical experimental materials with different infection mode were selected for further research about key enzymes during the infection process of target nematodes. The flowchart, containing bioassays (nematodes-immobilized experiment in vitro) as indicator for infectious enzymes with high activity, was established. Separation, chromatographic purification, molecular characterization and N-terminal analysis of infectious extracellular protease were completed. Among them, one novel cDNA gene encoding fungal infectious proteases was first report. A recombinant alkaline protease was successfully expressed in a high level heterologous expression system and the expression protein was characterized by western blotting and functional analysis. This study primarily reveals fungal infection mechanism and enriches the knowledge of properties & gene structure of infectious extracellular serine proteases as virulence factors. These experimental data laid a good foundation for construction genetic strains with high pathogenicity in biocontrol practice. Main results were achieved as following. (i) To purify and characterize three fungal proteases (accession number in SWISS-PROT) and corresponding two cDNA genes encoding cuticle-degrading protease were cloned (Genbank No.). P83290. The protease was purified from culture filtrate of Arthrobotrys oligospora by ammonium sulfate precipitation, ion exchange chromatography and gel filtration. The purified neutral protease(designated Aoz1) showed a molecular mass of 38kDa, pI of pH 4.9, optimum temperature of 45℃and optimum pH between 6.0-8.0. N-terminal sequence of Aoz1 was determined by electroblotting enzyme protein to PVDF membrane as NH2-A-E-Q-T-D-S-T-W-G-L-D-R-I-S-H-E-D-Y-S-A. NCBI -BLASTP showed the terminal sequence was same with a segment closed to N-terminus of PII. Three degenerate primers (256-fold degenerate) was designed according to terminal sequence for cDNA gene encoding mature protein of Aoz1 by 3'RACE system for rapid amplification of cDNA ends. Regulatory region of Aoz1 was cloned by SMART-RACE and a full-length cDNA clone containing cap structure (Gm7) and poly(dA) tail was obtained (AF516146). BLASTN showed the identity between PII and Aoz1 was 97% and they shared a same 61-bp intron. It was concluded that Aoz1 was a homolog of PII. P83492. The alkaline protease (designated Lmz1) was purified from supernatant of Clonostachys rosea (syn. Gliocladium roseum). The purified protease showed a molecular mass of 33kDa, pI of pH 10.5, optimum temperature of 55-60℃and optimum pH between 11.0-12.0. N-terminal sequence of Lmz1 was determined by electroblotting enzyme protein to PVDF membrane as NH2-A-T-Q-S-N-A-P-?-?-?. No significant similarity of the terminus and cDNA sequence, AY207377, were found by BLASTP and BLASTN. P83454. The alkaline protease (designated Lmz) was purified from supernatant of Coprinus comartus by frozen-dry concentration, hydrophobic interaction chromatography and gel filtration. It showed a molecular mass of 31kDa, pI of pH 9.0, optimum temperature of 55-60℃and optimum pH between 10.0-11.0. N-terminus of Lmz was determined as NH2-G-L-T-T-Q-K-S-A-P-W-G-L-G-S-I-S. The terminus was same as that of alkaline protease from Aspergillus oryzae. (ii) To express successfully a fungal alkaline protease (Lmz1) in a high levelexpression system. The specific primers with EcoRI recognition site and extra bases were designed. The cDNA gene encoding mature protease (Lmz1) was amplified with high fidelity pfu DNA polymerase and specific primers against total cDNA population of Clonostachys rosea. The recombinant expression vector contain desired DNA fragment was correctly constructed and introduced into yeast host Pichia pastoris GS115 by electroporation. The recombinant alkaline protease was characterized by protease activity, PCR amplification against yeast total DNA, ELISA and western blotting. It demonstrated a molecular mass of 42kDa based on blot analysis. The expression level of Lmz1 was approximately 9mg/ml by UV spectrophotometric method. In summary, the novelty of this reseach was embodied as following. (i)Infectious extracellular serine proteases from Clonostachys rosea and Coprinus comatus and their roles during the infection to nematodes were first reports. (ii)The cDNA gene encoding a novel mature alkaline proteases was cloned successfully by 3'RACE. Lmz1, belongs to serine family as a new protease member, is a novel alkaline protease. (iii)It was prominent that an extracellular protease from a mycoparasitic fungus Clonostachys rosea was successfully expressed in a heterologous high level system. The genetic engineering strain could be used for production of larger amount alkaline protease. Thus the unique alkaline protease could be applied in more areas for some new usage. (iv)A successful screening model of key enzymes during the infection to nematodes and a typical flowchart for gene cloning & expression were established based on good experiment foundation. Genetic improvement of biocontrol strains is the major aim for biological control of parasitic nematodes. This study laid a good foundation for the research & development of highly effective biocontrol strains.