Isolation Of Anti-TMV Y3 Gene From Coprinus Comatus And Its Characterization Expression

A variety of fungus proteins with biological activity possess potentially applicable value. They include ribosome inactivating proteins, antifungal proteins, ribonucleases, ubiquitin-like proteins, lectins, cellulases, laccases, invertases etc. Among them, there is an anti-TMV protein, designated as Y3 protein and encoded by y3 gene. Y3 protein was isolated from the fungus shaggy mane (Coprinus comatus (Mull.:Fr.) S. F. Gray), and showed inhibition on multiplication of tobacco mosaic virus (TMV). Because y3 gene might be a potential gene resource for antiviral genetic engineering, further studies, including full-length cDNA sequencing and identification of antiviral activity, should be completed.In this work gene y3 was isolated from fungus C. comatus. The sequencing of this gene was carried out. Plant expression vector harboring gene Y3 was constructed and transformed into tobacco. The summary is as follows:(1) Using Y-shaped adapter dependant extension (YADE), y3 gene of full-length DNA sequence was isolated from C. comatus mycelia and characterized. The putative full DNA sequence of y3 gene consisted of 1289 bps (GenBank Accession No. HM204931), which included an encoding region and two flanking regions at the 5'-and 3'-terminal.(2) Cloning and characterization of the full-length cDNA y3 from of C. comatus mycelia was carried out with 5'-RACE. The putative full sequence of cDNA of y3 gene was composed of 534 bps with an ORF. The ORF encoded a polypeptide of 130 amino acid residues. Accession No. in GenBank is GQ859168和Accession No. in EMBL is FN546262。Comparison of the polypeptide sequence encoded by full-length y3 gene with the partial amino acid sequence reported before showed that there were 101 amino acids shared with the published Y3 sequence, and there were additional 29 amino acids newly derived from the full-length cDNA sequence.(3) Through alignment of the DNA sequence of y3 gene with its cDNA, it was revealed that y3 gene was composed of 5 exons and 4 introns. Utilizing the software of promoter prediction on line and based on our experimental data, the transcription start site of y3 gene was predicted at A (No.321 bp) that followed TaTa box in DNA sequence.(4) The full length of y3 gene we obtained exhibited high similarity (up to 94%) to partial known fragment of gene y3 sequence. According to the full-length cDNA sequence, gene y3 had been cloned and used to construct plant expression plasmid.(5) The plasmid was constructed via inserting gene y3 sequence, at the MCS of pCAMBIA1301 along with CaMV 35S promoter and NOS terminator. The expression plasmids were transferred into Nicotiana tabacumvia via Agrobacterium mediated method, resulting in transgenic plantlets. The transcription of y3 gene was characterized by Northern blot analysis. Inoculation tests on transgenic plantlets with TMV demonstrated inhibitory activity against TMV. This illustrated that y3 gene had been transcripted and expressed in transgenic tobacco plants and y3 gene was proved to have certain inhibitory function against TMV in vivo.(6) In order to extract DNA or RNA simply and effectively, a published protocol for extracting DNA and RNA was improved and applied to isolate DNA and RNA of fungi and tobacco. Compared with commonly used method and commercial kits, the simplified protocol could be used to extract DNA and RNA not only from fungi but also from plants. The isolated DNA and RNA were stable and their activities could be kept when stored under low temperatures.(7) A new 28S rRNA sequence with 906 bps was isolated from mycelia of C. comatus (GenBank Accession No. GU568178). The sequence was obtained from an untargeted band when we cloned antiviral protein gene y3. This sequence was used to search homologous sequences using BLAST algorithm in NCBI, and to perform cluster with its homologues using the Clustal w and MEGA methods. The results confirmed that this sequence was a new 28S rRNA, and the phylogenic relationship of C. comatus was found to be dispersed from each other. Moreover, two homologous counterparts between the sequences of new 28S rRNA and the y3 gene were the possible cause of formation of the untargeted band in PCR amplification of y3 gene. Within these two homologous counterparts, one counterpart had the sequence similar to a PCR primer for y3 gene, and the other one was a primer-like fragment. In this research, obtaining of the new sequence of 28S rRNA was a new example of the formation of untargeted bands during PCR amplification. This finding and clustering analysis results of 28S rRNA would be helpful for researches in Fungus Genomics and the establishment of their molecular classification system.