Molecular Cloning Of CDNA Gene Encoding Chitinase And Structure Identification Of Antifungal Substance For Trichoderma Longibrachiatum T05

Trichoderma longibrachiatum is one species of Trichoderma genus that has biocontrol potential. There are some reports on application of Trichoderma longibrachiatum to control plant diseases, but less study in the northeast of china, and the biocontrol mechanism is still not revealed. Chitinase is one important cell wall degrading enzyme in biocontrol mechanism of Trichoderma, plays the important role in the process of hyperparasitism on mycelium of plant pathogen. Meanwhile, the active secondary metabolites produced by Trichoderma is another important part in the biocontrol mechanism, they can inhibit the growth of plant pathogen directly, and make it collapsed, also have the synergetic antagonism with cell wall degrading enzymes. Thus, the study on the two part of Trichoderma biocontrol mechanism has great significance to reveal the biocontrol mechanism of Trichoderma longibrachiatum.In this study, molecular phylogenetics was adopted to identify one strain of Trichoderma which has potential ability on biocontrol many plant pathogens in northeast of china. The chitinase cDNA gene was cloned from the Trichoderma longibrachiatum mycelia which cultured with chitinase induction medium by the methods of specific primer PCR, RT-PCR and RACE. The basic information of chitinase, function sites, secondary structure characteristics and3D characteristics were analyzed by the online tools such as NCBI and SWISS-MODEL, the molecular biological software such as BioEdit, ClustalX2.0, Chimera and Cn3D. Meanwhile, the active secondary metabolites produced by Trichoderma longibrachiatum T05were extracted, separated and its chemical structure was identified. The main results as follows:1. According to the relative specific rDNA ITS sequences of Trichoderma genus, rDNA ITS1+5.8S+ITS2sequences region and tefl sequence were amplified and sequenced, and aligned with the ITS and tefl sequences of Trichoderma in GenBank of NCBI. Meanwhile, a online Trichoderma identification tool TrichoKey2.0was used for the further identification, the test strain was identified as Trichoderma longibrachiatum. The result was completely same as the morphology identification.2. Using transparent circle method and DNS colorimetric method, the chitinase and P-1,3-glucanase activity of Trichoderma longibrachiatum T05were studied, further proved the conclusion of chitinase and P-1,3-glucanase were inducible enzyme, laid the foundation for cloning chitinase and P-1,3-glucanase complete cDNA gene, provided the optimum time that enzyme reaching the maximum expression, got the ready for extracting total RNA contained abundant mRNA.3. Firstly, the fragment sequences of chitinase gene was obtained by specific primers PCR, then the specific primer was designed for amplifying the fragment of cDNA gene, the fragment of cDNA gene of chitinase from T. longibrachiatum T05, the3'-end and5'-end of chitinase cDNA gene were amplified by RACE, the complete cDNA gene (1080bp) was obtained by joining together with software ContigExpress, named as TL-ch42.4. TL-ch42gene contained a full open reading frame (ORF) of1080bp in length. The start codon was ATG, the stop codon was TGA, endoded a putative protein of359amino acids. The length of5'and3'untranslatin region was519bp and221bp, respectively. The base composition was A22.22%, C31.76%, G24.72%, T21.30%, the G+C content of TL-ch42was56.48%, and the A+T content was43.52%. The analysis result of putative amino acid composition was as follows:Ala12.78%, Cys0.28%, Asp7.22%, Glu2.50%, Phe4.17%, Gly7.78%, His1.39%, Ile4.44%, Lys5.00%, Leu7.50%, Met2.22%, Asn6.67%, Pro3.89%, Gln3.06%, Arg2.50%, Ser8.61%, Thr5.56%, Val6.11%, Trp2.22%, Tyr5.83%, thereinto, the Ala content was highest, and Cys was lowest. The predicted molecular wight of putative protein was38.99kDa, and the pI was5.12. There were35amino acid residues with negative charge (Asp+Glu) and27amino acid residues with positive charge (Arg+Lys). The putative protein contained5400atoms with a molecular formula of C1752H2645N457O537S9. The protein was a stable protein with a instability index of28.01. The aliphatic index was77.30and the total average hydrophobicity is-0.192.5. The most probable splice site of signal peptide of TL-ch42was predicted to be located between the22nd and23rd amino acid, which was recognized as TSA-SP. The protein contained4hydrophobic regions which were located between the8th-27th amino acid,36th-56th amino acid,123rd-141st amino acid,201st-223rd amino acid. The transmembrane region was also predicted to be located in these regions. The secondary structure was composed of random coil, a helix and β strands with a percentage of61.84%,23.96%and14.21%, respectively. The result of subcellular location of TL-ch42showed that this protein was mainly located in lysosome (lumen) and in outside, microbody (peroxisome) and endoplasmic reticulum (membrane). The tertiary structure of TL-ch42putative protein was predicted using SWISS-MODEL online and the structure was exported by Chimera and Cn3D.6. Using the separation and analysis methods of TLC, column chromotography and HPLC, the antifungal secondary metabolites from the fermentation liquid of Trichoderma longibrachiatum T05. The antifungal part of secondary metabolites was determined and its chemical structure was identified with the method of biological activity tracing. The results showed that ethyl acetate was the optimum solvent to extract the antifungal secondary metabolites, petroleum ether:ethyl acetate=2:1was the optimum developer for TLC. Nine point of samples were observed under UV light and with I2. Nine components were obtained by silica column chromotography, different ratio of petroleum ether and ethyl acetate as eluent, yield rate of different component was calculated after condensing and drying solvent, and the antifungal activity was tested. The yield rate of component VI was higher, that is24.76%, and its inhibition rate on the tested plant pathogen was highest, showed that the main part of antifungal substance was in it. The232mg pure sample of component Ⅵ-1was obtained by sephadex LH20column chromotography with MeOH and water as eluent. The purity of component Ⅵ-1was above95%after HPLC analysis. After High resolution mass spectrometry determination, the deduced formula for this coumpound was C28H32O8. The mass spectrum was same as bislongiquinolide in NIST, the abundant was95%.