毛头鬼伞真菌具有抗烟草花叶病毒活性的y3基因的克隆及对烟草的转化  

Cloning of y3 gene encoding a tobacco mosaic virus inhibitor from Coprinus comatus and transformation to Nicotiana tabacum

作　　者：王学仁[1,2] 何涛[1] 张改娜[1] 郝建国[1] 贾敬芬[1] 

机构地区：[1]西北大学生命科学学院,陕西省生物技术重点实验室,西安710069 [2]西安文理学院生命科学系,西安710065

出　　处：《微生物学报》2010年第2期182-190,共9页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30671087);西安市科技创新支撑计划(YF07109)~~

摘　　要：【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]The protein Y3 was a TMV inhibitor which was encoded by y3 gene. The aim of this work was to clone the full length of y3 gene from Coprinus comatus and to reveal its inhibitory function to TMV in in vivo conditions. [ Methods] We amplified the unknown 5＇- terminal eDNA sequence of y3 gene with 5 ＇- Full RACE Core Set （TaKaRa） , obtained the full length of this gene by RT-PCR, constructed the expression plasmid pCAMBIA1301-y3 via inserting gene y3 sequence, CaMV 35 S promoter, and NOS terminator at MCS and transformed it into Nicotiana tabacum via agrobacterium- mediation. [ Results] The full length of y3 gene was 534 bps including one ORF encoding 130 amino acid residues （GenBank Accession No. GQ859168; EMBL FN546262）. The eDNA sequence and its deduced amino acid sequence showed high similarity （94%） to the published fragment of y3 gene sequence. Northern blot analysis proved the transcription of y3 gene in transgenie tobacco plants. The transgenie plants inoculated with TMV expressed the inhibitory activity to TMV. [ Conclusion ] We cloned the full length of y3 gene and obtained transgenic tobacco plants. The expression of y3 gene in transgenie plants improved the inhibitory activity to TMV. The cloning and expression analysis of y3 gene might provide background information for future studying of y3 gene.

关 键 词：COPRINUS comatus y3基因 Y3蛋白 植物表达载体构建 

分 类 号：S572[农业科学—烟草工业]