广西巴马小型猪内源性反转录病毒进化年代分析

doi:10.3864/j.issn.0578-1752.2015.04.15

摘要/Abstract

摘要： 【目的】确定广西巴马小型猪内源性反转录病毒（PERV-BM）的进化年代。【方法】对先前扩增、测序获得的 9个PERV-BM的LTRs，一个PERV-BM全长基因组序列，登录号为HM159246及GenBank上发表的所有背景清楚的猪内源性反转录病毒（PERV）的LTRs及env序列进行分析。首先利用DAMBE软件对上述序列比对分析，合并同源性较高的，筛选有差异代表性的核酸序列的数据集。然后利用MEGA5.2软件Neighbor-Joining方法计算PERV-BM遗传进化关系。利用DAMBE软件对上述筛选的序列集进行替换饱和度计算，分析核酸进化速率的稳定性；利用MEGA5.2软件，通过最大相似法对筛选数据制作的遗传进化树进行分子钟行为分析。最后对广西封闭群内的广西巴马小型猪3′-LTR序列进行替代饱和度及分子钟分析，对符合分子钟行为的LTRs序列，以假设的恒定进化速率计算PERV-BM的进化年代。【结果】经过DAMBE软件分析后，选出80多个GenBank上发表的有代表性的env、LTRs序列数据集。对PERV-BM (HM159246) 序列全长遗传进化分析显示，PERV-BM与中国的PERV核酸序列EF133960和GU980187，荷兰的AF356697亲缘关系较近，与韩国的HQ540595和美国的AF038600和NC_003059亲缘关系则较远。利用DAMBE软件对筛选数据集替换饱和度检测结果显示，S和V值随PERV序列之间进化分歧的增加呈线性的增加。LTRs序列数据集替换饱和曲线呈线性关系，没有替换饱和现象发生，LTRs的进化随时间持续且稳定。数据集env序列替换饱和曲线不完全呈线性关系，表明env序列集进化速率不稳定。利用MEGA5.2的最大相似法对LTRs和env数据集的进化树进行分子钟行为检测结果显示，LTRs序列集接受分子钟假说，env进化不符合分子钟行为，这和不饱和度检测的结果一致。因此可用LTRs进化的近分子钟行为进行PERV进化年代的计算。利用DAMBE软件对广西封闭群内的广西巴马小型猪3′-LTR序列进行替代饱和度及分子钟分析结果显示，3′-LTR的进化符合分子钟行为可用于进化年代计算。假设以灵长类ERV的积累突变率，每个核苷酸每年的替换率2.3×10^-9—5.0×10^-9，计算PERV-BM约进化于3.3×10⁶—7.1×10⁶年前。【结论】PERV-BM进化年代与欧洲小型猪PERV进化时间7.6×10⁶年前相近。

关键词: 广西巴马小型猪, 猪内源性反转录病毒, 进化年代

Abstract: 【Objective】The objective of this study is to determine the evolutionary time of the endogenous retrovirus (PERV-BM) in Bama mini-pigs.【Method】Analyses were performed on nine LTRs and one full-length genomic sequence of PERV-BM (accession number HM159246) amplified and sequenced by this lab and on the LTRs and env sequences of all PERV with clear backgrounds which have been published on GenBank. Firstly, through comparing the above sequences by using the DAMBE software, nucleic acid sequence data sets with high homology were combined and those that were typically different were screened. Then the phylogenetics of PERV-BM was calculated using the Neighbor-Joining method of MEGA5.2 software. DAMBE software was used to calculate the substitution saturations of the screened data sets and analyze the stability of their nucleic acid evolution rates. MEGA5.2 software was used to analyze the molecular clock of the phylogenetic tree built on the screened data according to their maximum similitude degree. Finally, substitution saturation and molecular clock analysis were done on the 3'-LTR sequences of the Bama miniature pigs in Guangxi closed group, then the sequences that were in consistence with the working of molecular clock were calculated by the hypothesized constant rate of evolutionary change in order to figure the evolutionary time of PERV-BM. 【Result】With all the analyses done with DAMBE software, the sequences of over 80 LTRs and env genes which are representatives in GenBank were selected. The phylogenetic analysis results of full-length sequence PERV-BM (HM159246) showed that it had closer genetic relationship with China PERV nucleic acid sequences EF133960 and GU980187 and Netherlands PERV nucleic acid sequences AF356697, but it was a bit distant from United States nucleic acid sequences AF038600 and NC_003059 and South Korea nucleic acid sequences HQ540595. Results of substitution saturation showed that the S and V values of the PERV sequences increased linearly with the increase of evolutionary divergence. The substitution saturation curve of LTRs sequence data sets had a linear relationship, without any substitution saturation; the evolution of LTRs was constant and stable with time. The substation saturation curve of env sequence data sets was not completely linear, indicating that the evolutionary rate of env sequence was unstable. The molecular clock test of the evolutionary tree established by the screened data sets with the maximum likelihood method by MEGA5.2 showed that LTRs sequence sets consisted with molecular clock hypothesis, while the evolutionary behavior of env did not meet the molecular clock. This was in accordance with saturation detection. With this regard, the approximate molecular clock behavior of LTRs could be used to calculate the evolutionary time of PERV-BM. Substitution saturation and molecular clock analysis on the 3'-LTR sequences of the Bama miniature pigs in Guangxi closed group showed that 3'-LTR sequence evolutionary behavior conformed to the molecular clock and could be used to calculate the evolutionary time. Supposed that the ERV mutation accumulation rate of the primate, the annual per nucleotide substitution rate 2.3×10^-9—5.0×10^-9, is used to calculate the evolutionary time, the result is that PERV-BM speciated approximately 3.3×10⁶—7.1×10⁶ years ago. 【Conclusion】The evolutionary time of PERV-BM is similar to that of the European mini-pigs which was 7.6×10⁶ years ago.

Key words: Guangxi Bama mini-pigs, PERV, evolutionary time

饶桂波，钟雅婷，欧阳康，马玲，黄红梅，吴健敏. 广西巴马小型猪内源性反转录病毒进化年代分析[J]. 中国农业科学, 2015, 48(4): 778-787.

RAO Gui-bo, ZHONG Ya-ting, OUYANG Kang, MA Ling, HUANG Hong-mei, WU Jian-min. Analysis of Evolutionary Time of Porcine Endogenous Retrovirus in Guangxi Bama Mini-pigs[J]. Scientia Agricultura Sinica, 2015, 48(4): 778-787.

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