Characterization Of A Novel DNase And Sequence Analysis Of A CDNA Encoding MT From Ganoderma Lucidum

Ganoderma lucidum, a kind of Chinese medecinal fungis was collected into the《Pharmacopeia of the Peoples' Republic of China-2000》. It contains various kinds ofactivities which are effective to human diseases. As far papers reported involved in Glucidum studied mostly focused on polysaccharide and small molecules activities and itstreatment effect. But studies concerning its functional proteins were seldom been found.As for my research concentrated on revealing some new active proteins and gene from G lucidum. In this paper there were two parts were involved:â… The purification and characterization of a novel deoxyriboendonuclease;â…¡Gene cloning and sequence analysis of a cDNA encoding metallothionein from Ganoderma lucidum.1．Purification and characterization of a novel deoxyriboendonuclease fromGanoderma lucidumA novel deoxyribonuclease (DNase) from the fruiting bodies of Ganoderma lucidum had been isolated, purified and designated as GLDNase.The GLDNase was purified by ammonium sulphate precipitation, Blue Sepharose 6 Fast Flow and SP Sepharose Fast Flow. It was partially characterizesd. It was shown that the GLDNase was a single-chained, and molecular mass of 14．7 kDa as analyzed on SDS-PAGE. But it showed a major mass peak of 13807 Da while was analyzed in an electrospray-mass analyzer. An isoelectric point of pH6．6 was estimated by isoelectric focusing. Concentration of carbohydrate was 2．7% detected by Phenol-sulfuric acid method. Amino acid analysis of the N-terminus was PLDTGRYHIYTW/T/CDGG., which was distinctly different from the N-terminal of other protein in GeneBank analysised by BLAST program.Digested by trypsin , two big fragment of GLDNase were sequenced as QVETWDRLDELPIR and ESPGLQGVSSVPLR on matrix-assisted laser desorption-ionization time of flight mass spectrometry. The first peptides had high similarity with one peptide fraction large subunit of Methanopyrus kandleri DNA polymeraseâ…¡.The second had high similarity with one peptide fraction of dihydrolipoamide dehydrogenase.GLDNase acted on both double-stranded and single-stranded DNA, but preferentially on ssDNA. The optimum pH of GLDNase was pH 8．4 and its optimum temperature was 40℃.It was an endonuclease but not a restriction endonuclease. A divalent cation Mg²⁺ is required for the endonuclase activity as a co-factor. The Mg²⁺ ions could be replaced by Ca²⁺, Zn²⁺, Mn²⁺, but the activity decreased. The activity of the purified enzyme was completely inhibited by 10 mM EDTA. The oligonucleotides produced had a phosphate group at the 5'-termini and a hydroxyl group at the 3'-termini.These results indicated that the nucleolytic properties of GLDNase were essentially the same as those of other well characterized DNasesI. So GLDNase was a member of DNasel family.Antiviral activity of GLDNase against TMV was tested by means of local lesion and leaf discs assay. The results indicated that GLDNase exhibited certain antiviral activity of inhibiting virus infection, and no significantly inhibited the replication of virus. The inhibition of TMV was 85．2% when the concentration of GLDNase was 100ug·mL^-1 .2．Cloning and Sequence analysis of a cDNA encoding metallothionein from Ganoderma lucidumMetallothioneins are low molecular weight, cysteine-rich and metal binding proteins whose synthesis is usually induced by metal ions. MT plays important biological functions such as protective role against excess reactive heavy metal ions, free radical scanengers and reservoir of essential metals that can be donated to other metalloproteins. To our know, none of G lucidum MT gene had been reported to date.A partial sequence of G lucidum MT gene was identified by RT-PCR and RACE PCR method. The full-length cNDA of G lucidum MT was of 389bp by including 5' untranslated region of 121bp, 3' UTR of 122bp and an open reading frame (ORF) of 102bp encoding a polypeptide of 33 amino ancids. The predicated protein from G lucidum MT gene sequence contained 7 cysteine residues organized in cys-x-cys, cys-x-x-cys, cys-x-x-x-x-cys motifs as classically described for MTs. The contens of cysteine in the G lucidum MT amino acids sequence was 21．2%, and glycine and alanine were 12．1% respectively. Sequence analysis revealed that the cDNA of MT from G lucidum was 80% homologous to Paxillus involutus and 61% homologous to Agaricus bisporus. The result of sequence analysis indicated that MT from Ganoderma lucidum was the member of metallothionmerin family.