为揭示马铃薯Y病毒(Potato virus Y, PVY)P1基因的分子变异及结构特征, 并查明福建分离物P1基因的变异来源。文章设计一对简并引物从感染PVY的马铃薯病叶扩增、克隆获得福建分离物P1基因的cDNA全长序列, 分析其核苷酸序列和氨基酸序列的特征, 并采用贝叶斯法重建P1基因的系统发育树。结果显示, 12个福建分离物均成功扩增出预期大小(约915 bp)的特异性片段, P1基因的核苷酸序列与已报道的PVY不同株系的核苷酸序列一致性为73%~99%; QK44、XT02、XT08、LH05分离物的309 nt位置均检测到一个强烈的重组信号。12个PVY分离物中, P1蛋白有85个变异的氨基酸位点, 表明其高度变异; P1蛋白的第41~275位是一个高度保守的结构功能域, 含有3个保守的活性位点(H192、D201、V235); 系统发育分析显示, 福建分离物形聚为3种不同的簇, 其对应的卷曲结构(Coiled-coil domain)和空间结构也不相同, 表明不同株系型的P1基因在系统发育关系上分化较大。该研究结果表明PVY P1基因在核苷酸序列、氨基酸序列以及空间结构具有高度变异性, 但行使P1蛋白功能的活性位点所在的氨基酸(H192、D201和V235)均高度保守; 福建分离物P1基因的变异源主要来自碱基突变和基因重组。
To understand the sequence variation and the putative protein structure of P1 gene in Potato virus Y (PVY) and to identify the sources of the variation, P1 gene in PVY isolated from Fujian Province was amplified by reverse-transcription polymerase chain reaction (RT-PCR) using a pair of degenerate primers designed from the conserved regions of published sequences. Sequence variation and putative protein structure were analyzed, and phylogenetic tree was reconstructed using Bayesian inference method. Expected fragments of 915 bp in size were amplified from 12 samples collected from Fujian Province by RT-PCR. The 12 sequences shared 73%-99% nucleotide identity with the reference sequences from GenBank. A strong recombination signal was identified at position 309 in sequences of isolates QK44, XT02, XT08 and LH05. Among the 12 sequences, 85 amino acid variants were detected, indicating high sequence variation in the P1 protein. However, positions 41-275 in the protein were highly conserved, especially in three active sites (H192, D201 and V235). Phylogenetic analysis grouped the sequences into three clades, each with different Coiled-coil domains and 3D-structures, suggesting divergent phylogenetic relationship among the groups. The above results show P1 gene in PVY is highly variable but contains 3 conserved active sites (H192, D201, V235) and the high genetic variation in the gene is primarily due to mutation and recombination.
[1] 詹家绥. 植物病原物的群体遗传学. 遗传, 2012, 34(2): 157-166.
[2] MC, Karasev AV, Furutani N, Taniguchi M, Kano Y, Sato M, Natsuaki T, Maoka T. Occurrence of Potato virus Y strain PVY NTN in foundation seed potatoes in Japan, and screening for symptoms in Japanese potato cultivars. Plant Pathol , 2013, 62(5): 1157-1165.
[3] 沈建国, 史凤阳, 方治国, 谢联辉, 詹家绥. 中国马铃薯Y病毒的检测鉴定及CP基因的分子变异. 中国农业科学, 2013, 46(15): 3125-3133.
[4] AMQ, Lefkowitz E, Adams MJ, Carstens EB. Virus Taxonomy: ninth report of the international committee on taxonomy of viruses. Amsterdam: Elsevier Academic Press, 2011.
[5] BY, Miller WA, Atkins JF, Firth AE. An overlapping essential gene in the Potyviridae . Proc Natl Acad Sci USA , 2008, 105(15): 5897-5902.
[6] 沈建国, 史凤阳, 常飞, 谢联辉, 詹家绥. 马铃薯Y病毒 pipo 基因的分子变异及结构特征分析. 遗传, 2013, 35(9): 1125-1134.
[7] RP, Valkonen JPT, Gray SM, Boonham N, Jones RAC, Kerlan C, Schubert J. Discussion paper: the naming of Potato virus Y strains infecting potato. Arch Virol , 2008, 153(1): 1-13.
[8] MC, Maoka T, Natsuaki T, Natsuaki KT. PVY NTN-NW , a novel recombinant strain of Potato virus Y predominating in potato fields in Syria. Plant Pathol , 2010, 59(1): 31-41.
[9] J, Vassilakos N, Moury B. Potato virus Y : a major crop pathogen that has provided major insights into the evolution of viral pathogenicity. Mol Plant Pathol , 2013, 14(5): 439-452.
[10] MS, Akanda AK. Performance of seed potato produced from sprout cutting, stem cutting and convetional tuber against PVY and PLRV. Bangladesh J Agril Res , 2009, 34(4): 609-622.
[11] JL, Nolte P, McIntosh C, Davidson R. Effect of Potato virus Y on yield of three potato cultivars grown under different nitrogen levels. Plant Dis , 2006, 90(1): 73-76.
[12] J, Navrátil M. P1 peptidase-a mysterious protein of family Potyviridae. J Biosci , 2011, 36(1): 189-200.
[13] 陈士华, 吴祖建, 林奇英, 谢联辉. 马铃薯Y病毒P1基因的克隆与序列分析. 中国病毒学, 2003, 18(4): 376-380.
[14] X, Karasev AV, Brown CJ, Lorenzen JH. Sequence characteristics of Potato virus Y recombinants. J General Virol , 2009, 90(12): 3033-3041.
[15] B, Jia JL, Wang XQ, Wang ZY, Yang BH, Li XD, Zhu XP. Molecular characterization of two recombinant Potato virus Y isolates from China. Arch Virol , 2012, 157(2): 401-403.
[16] 代昌明, 杨军, 宋纪真, 罗朝鹏, 王燃, 林福呈. 马铃薯Y病毒贵州黔西烟草分离物P1基因序列分析. 植物病理学报, 2012, 42(1): 88-92.
[17] KS, Bollinger RC, Paranjape RS, Gadkari D, Kulkarni SS, Novak NG, Ingersoll R, Sheppard HW, Ray SC. Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J Virol , 1999, 73(1): 152-160.
[18] PSL, Posada D, Gravenor MB, Woelk CH, Frost SD. GARD: a genetic algorithm for recombination detection. Bioinformatics , 2006, 22(24): 3096-3098.
[19] K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol , 2013, 30(4): 772-780.
[20] JAA. MrModeltest v2. 3. Program distributed by the author. Evolutionary Biology Centre, Uppsala University, 2008.
[21] F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. MrBayes 3. 2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol , 2012, 61(3): 539-542.
[22] JM, Fraile A, Moreno I, Obies CI, Drake JW, García-Arenal F. The rate and character of spontaneous mutation in an RNA virus. Genetics , 2002, 162(4): 1505-1511.
[23] A, Ohshima K. Potyviruses and the digital revolution. Annu R ev P hytopathol , 2010, 48(1): 205-223.
[24] A, López-Moya JJ, García JA. Recombination and gene duplication in the evolutionary diversification of P1 proteins in the family Potyviridae . J Gen Virol , 2007, 88(Pt3): 1016-1028.
[25] AK, Mukasa SB, Kalkkinen N, Valkonen JP. Recombination and selection pressure in the ipomovirus sweet potato mild mottle virus ( Potyvirid
