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【目的】蛋白质Y3具有抗烟草花叶病毒(TMV)活性并由y3基因编码。本文的目的是从真菌毛头鬼伞(Coprinus comatus)中克隆y3基因全长并在植物体中展现其对TMV的抑制活性。【方法】我们利用试剂盒5′-Full RACE Core Set(TaKaRa)扩增了y3基因cDNA5′-端未知序列,通过RT-PCR获得了全长序列,并把该全长序列与CaMV 35 S启动子和NOS终止子一起插入多克隆位点(MCS)构建了植物表达载体pCAMBIA1301-y3,用于农杆菌介导的烟草转化。【结果】y3基因全长534碱基对,包含1个开放阅读框(ORF),编码一条含130个氨基酸残基的肽链(GenBank检索号:GQ859168;EMBL:FN546262)。其cDNA序列和由它推到的氨基酸序列均与已发表的y3基因部分片段有高度相似性(94%)。Northern杂交分析证实了y3基因在转基因烟草中得到表达。接种TMV的转基因植株表现出抗TMV的活性。【结论】我们克隆了y3基因全长并得到了转基因植株。在转基因植株中,由于y3基因的表达改善了植株的抗病毒活性。y3基因的克隆和表达无疑为该基因的进一步研究奠定了基础。
【Objective】 Protein Y3 has anti-tobacco mosaic virus (TMV) activity and is encoded by y3 gene. The purpose of this paper was to clone the full-length y3 gene from Coprinus comatus and demonstrate its inhibitory activity against TMV in plants. 【Method】 The full-length sequence of 5’-end cDNA of y3 gene was amplified by RT-PCR using the kit 5’-Full RACE Core Set (TaKaRa). The full-length sequence was then ligated with CaMV 35 S A plant expression vector, pCAMBIA1301-y3, was constructed for insertion into the multiple cloning site (MCS) with the NOS terminator for Agrobacterium-mediated tobacco transformation. 【Result】 The y3 gene was 534 bp in length and contained an open reading frame (ORF) encoding a peptide chain of 130 amino acid residues (GenBank accession number: GQ859168; EMBL: FN546262). Both its cDNA sequence and the deduced amino acid sequence are highly similar (94%) to the published partial fragment of y3 gene. Northern blot analysis confirmed that the y3 gene was expressed in transgenic tobacco. The transgenic plants inoculated with TMV showed anti-TMV activity. 【Conclusion】 We cloned y3 gene and obtained transgenic plants. In transgenic plants, the antiviral activity of plants is improved due to the expression of the y3 gene. The cloning and expression of y3 gene undoubtedly lay the foundation for the further study of this gene.