Isolation And Characterization Of Ty1-copia-like Retrotran Sposons In Paeonia Suffruticosa Andrews

Paeonia suffruticosa Andrews. is the deciduous shrub in Paeoniaceae, Paeonia L.and Sect. Mouton DC. It is one of the most important spectacular and preciousresource of plants. And it has over2000years cultivation and application history in ourcountry, after a long term it is forming rich genetic variations. Until now studies onreverse transcriptase (RT) in P. suffruticosa genome have not seen. In our study, weisolated Ty1-copia-like retrotransposons RT sequences from P. suffruticosa genomefirstly. Then the phylogenetic analysis was made.1. The PCR used degenerate primers, the factors which affects the PCR resultswere studied. Reaction system after optimization were:1×buffer,2mmol/L Mg2+,0.4mmol/L dNTPs,0.8μmol/L degenerate primers (RTS and RTF),1U Taq polymerase,DNA templates50ng/μL，double-steamed water up to25μL PCR were preformed ina thermal cycle with the following parameters:94°C for5min, followed by35cyclesof94°C for50s,43°C for50s,72°C for1min, followed by final extension step at72°C for7min, then4°C end. A fragment of300bp was amplified from DNA genomsof10species in P. suffruticosa. All materials amplification results are consistent, andhave no other than products of the strip. Results showed that the Ty1-copia-likeretrotransposons RT genes exist in P. suffruticosa widely.2. The amplicons were recovered and cloned, then sequenced and analyzed.59unique RT sequences of Ty1-copia-like retrotransposons were obtained from P.suffruticosa cv. Luoyanghong genome. Analysis of these sequences by CLUSTAL、MEGA and DNAstar softwares, the result showed that a great heterogeneity. Thelength of base sequences changes for267bp~585bp，the homogeneity scope rangedfrom13.8.%~92.6%.Translated the59sequences for amino acid sequences andanalysised, the result showed that there are1~9any number of termination anticodonmutations in54sequences. The same length base sequences are different between theamino acid sequences. So the bases insert and alternative mutations are existed in P. suffruticosa genome widely.3. These59Ty1-copia retransposon RT sequences were designated as9Family(Family1~9) by using the phylogenetic. Every Family contains different sequencenumber. Family1including30sequences, covering the whole sequences of about50%.Their existence history was more remote, the ability of transcription turned to begreater activity. Family4, Family8and Family9each contains only1sequence, eachother between them and with other Family relationship goes faraway, explained thehistory of its existence is relatively short.4. Compared to the homogeneity of RT sequences among P. suffruticosa and other28plants which were downloaded from GenBank.The results showed P. suffruticosaand Petunia hybrid, Calibrachoa parviflora, Lycopersicon esculentum, Cicerarietinum, Orobanche tunetana, Solanum tuberosum, Phelipanche bungeana, Lyciumbarbarum, Lycium chinense, Camellia sinensis, Fragaria viridis, Prunus mume,Helianthus annuus, Ephedra distachya in relationship were near with others. It showedthat the P. suffruticosa and different herbal, woody plants may have a common origin.And It also proved that the horizontal transmission of retrotransposons betweendifferent species.