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