Screening, Identification And Cellulase Gene Cloning Of High Effective Straw Cellulose-Degrading Fungus Strains

The annual output of crop straws is about 650 million tons in China, containning more than 200 million tons cellulose, which is the most abundant natural renewable resources. Because of their complex structural components, crop residues are hard to degrade in soil that would affect soil moisture and future management practices. Therefore, most of crop residues were abandoned and burned in many rural areas, and causing environmental pollution and serious waste of straw cellulose resources. For the purpose of constructing biodegradation technique of straw cellulose, systemetic studies were developed in this paper and the obtained reults are as follows .360 straws cellulose-degrading strains were isolated from 300 soil samples from Jilin, Zhejiang, Sichuan, Hebei and Beijing areas. According to the size of degradation halo, five high efficient fungus strains were selected and named as T1, HD5, HD6, HD7, HD24, respectively. These five fungus could grow strongly on medium with straw cellulose as a sole carbon and energy sources. Based on the results of morphology, 18 S rDNA and ITS sequence analysis , strains T1, HD5, HD6, HD7, HD24 were identified as Hypocrea lixii (Trichoderma harizianum), Aspergillus flavus, Aspergillus fumigatus, Trichoderma koningiopsis and Penicillium oxalicum, respectively.Strains Hypocrea lixiiT1,Aspergillus flavus HD5,Aspergillus fumigatus HD6,Trichoderma koningiopsisHD7, Penicillium oxalicum HD24 had better effects on straws cellulose degradation in agar plates and broth condition. In the experiment of agar plates, the clear halo for each strain could expand to 20-25mm after seven days incubated at 30℃. In broth medium, the whole-activity (FPA) of strains T1, HD5, HD6, HD7, HD24 had reached the highest level in the first 3 days, which were 25.4 U, 34.36U, 28.85U, 31.19U and 32.05U, respectively, and significantly exceeded Trichoderma viride AS3.3711 to the extent of 21.88%,64.87%,38.43%,49.66% and 53.79%, respectively. The optimum growth temperature was 50℃for all the strains, whereas optimum pH value was varying from 5 to 6.Different strains showed specific requirement for various types and quantities of nitrogen, the optimum N rate as NaNO₃ is 1.5% for strain Hypocrea lixiiT1, the optimum N rate as (NH₄)₂SO₄ is 2%for strain Aspergillus flavus HD5, the optimum N rate as (NH₄)₂SO₄ is 0.5% for Aspergillus fumigatusHD6, the optimum N rate as (NH4) 2SO4 is 1.5% for Trichoderma koningiopsisHD7, the optimum N rate as (NH₂)₂CO is 1% for Penicillium oxalicum HD24.The addiontion of other kinds of carbon resouces would affect FPA for all of the strains greatly. Addition of sucrose compressed FPA for all of the fungus, addition of disaccharide exhibited a inhibitory effect on strains Hypocrea lixiiT1, Aspergillus flavus HD5, Aspergillus fumigatusHD6 and Trichoderma koningiopsis HD7, but it could stimulate FPA of Penicillium oxalicum HD24; application of Tween 20 had no obvious stimulative effect on strain Hypocrea lixiiT1and Aspergillus fumigatusHD6, but significantly increased the degradation efficiency of strains Aspergillus flavus HD5 and Penicillium oxalicum HD24.Three cDNA libraries were constructed using the vectorλTriplE and E. coli LE392 as host strain. Over 100,000λmacrophages were obtained from the Amp selective medium, all the libraries capacities were above 10⁹ pfu/mL. The positive recombinants were screened from agar plates by cellulose induction, three novel gene sequences coding exo-glucanase and endo-glucanase were obtained by using PCR amplification.The difference of proteins expression for Trichoderma viride AS3.3711, Aspergillus fumigatus HD6,Trichoderma koningiopsisHD7 and Penicillium oxalicum HD24 in the same culture conditions was studied in this paper. The results showed significant difference about proteins expression among the four strains by using two-dimensional gel electrophoresis (2 DE). The discrepancy of protein points were probably related to the different types or activities of cellulase.