李属坏死环斑病毒辽宁桃树分离物基因组分析

Analysis of the Complete Genome of Prunus necrotic ringspot virus Isolate Liaoning-peach

采用RT-PCR和RACE技术,克隆了李属坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV)辽宁桃树分离物全长基因组。用Vector NTI Advance 11软件进行基因组结构注释。用SDTv.1.0软件分析该分离物与Gen Bank中公布的其他PNRSV分离物间的核酸一致性,并用MEGA5.0软件和RDP3软件对各PNRSV分离物进行系统发育分析和重组分析。结果显示:PNRSV辽宁桃树分离物基因组由3条正义单链RNA组成。RNA1由3 332个核苷酸构成,具有一个开放阅读框(nt 30~3 167),编码一个约117.0k D的复制酶相关蛋白P1。RNA2由2 591个核苷酸构成,具有一个开放阅读框(nt 27~2 426),编码一个约99.0 k D的复制酶相关蛋白P2。RNA3由1 943个核苷酸构成,具有两个互不重叠的开放阅读框分别编码一个运动蛋白(nt 175~1 026)和一个外壳蛋白(nt 1 100~1 774),这两个开放阅读框的间隔区为73个核苷酸。PNRSV辽宁桃树分离物的RNA1、RNA2和RNA3与其他已经公布的PNRSV分离物间,核酸序列一致性分别为91.8%~98.8%,93.3%~99.2%和87.7%~99.0%。基于RNA1、RNA2和RNA3全长序列构建的系统发育进化树,分别将已报道的PNRSV分离物划分为3个、2个和3个组,PNRSV辽宁桃树分离物分别属于RNA1Ⅰ组、RNA2Ⅰ组和PV96组。基于RNA1、RNA2和RNA3全长序列进行的重组分析表明各PNRSV分离物基因组间不存在重组。PNRSV辽宁桃树分离物基因组序列是世界首个来源于桃树的PV96组基因组序列。

The complete genomic c DNAs of Prunus necrotic ringspot virus（PNRSV）strain Liaoningpeach were amplified using RT-PCR and RACE techniques. The obtained sequences were analyzed using Vector NTI Advance 11 for genome organization. Using software SDTv.1.0,the sequences were aligned with the other PNRSV sequences published in Gen Bank for nucleotide identity. Using software MEGA5.0 and RDP3,phylogenetic analysis and recombinational analysis were performed for collected PNRSV isolates. The PNRSV Liaoning-peach has a segmented genome,consists of three positive single-stranded RNAs. The RNA1 has 3 332 nucleotides（nts）,containing an open reading frame（ORF）,encoding a replicationassociated polymerase P1（ca. 117.0 k D）（nt 30–3 167）. The RNA2 has 2 591 nts,containing an ORF,encoding a replication-associated polymerase P2（ca. 99.0 k D）（nt 27–2 426）. The RNA3 has 1 943 nts,containing two non-overlapping ORFs. The two ORFs are separated by an intergenic spacer of 73 nts,and encode a movement protein（nt 175–1 026） and a coat protein（nt 1 100–1 774） respectively. The identities of nucleotide sequences of RNA1,RNA2 and RNA3 of PNRSV Liaoning-peach with other published PNRSV isolates were 91.8%–98.8%,93.3%–99.2%,87.7%–99.0%,respectively. Based on the phylogenetic trees constructed from complete sequences of RNA1,RNA2 and RNA3,PNRSV isolates were classified into three,two and three groups,respectively. The PNRSV Liaoning-peach was classified into group RNA1 group I,RNA2 group I and group PV96,respectively. Recombination analysis of complete sequences of RNA1,RNA2 and RNA3 suggested that no recombinational cases occurred among the collected PNRSV isolates. This was the first report of complete genome sequences of PNRSV infecting peach worldwide.