Cloning And Analysis Of Reverse Transcriptase Of Line-retrotransposons Of Tree Peony

Paeonia suffruticosa Andrews is ligneous plants which belong to the deciduousshrub in Paeoniaceae, Paeonia L. and Sect. Mouton DC. It has has great medicinalvalue and economic value. After thousands of years of natural selection and artificialintroduction and cultivation in China, peony has accumulated a wealth of geneticdiversity resources, but currently who negative research of retrotransposon on peonyhas not been reported. This test uses PCR cloning and analysis of reverse transcriptaseof LINE-retrotransposons of Tree Peony (Paeonia), otherwise, to evaluate thegermplasm resources. The main results were as follows:1. The PCR used degenerate primers, the factors which affects the PCR resultswere studied. The orthogonal design was used to optimize the five factors（Taq DNApolymerase, Mg2+, DNA template, dNTPs and primer concentrations) of PCR systemin tree peony. The results showed that the optimized system was as follows: a totalvolume of20μL including2.0mg/L DNA template,1.0U Taq polymerase,0.80μmol/L primer,2.0mmol/L Mg2+,87.5μmol/L dNTPs and1×PCR Buffer. Each factorhad different effects on the results. The concentration of Mg2+was the key factor ofPCR system of LINE retrotransposon RT in peony tree. The optimized PCR systemwas carried out on seven peony genus which showed that this system was stable andreliable. By annealing temperature gradient optimization we constructed the thermalcycle with the following parameters:94℃for5min, followed by35cycles of94℃for1min,53℃for1min,72℃for2min, followed by final extension step at72℃for8min. A fragment about600bp was amplified from DNA genoms of seven speciesin P. suffruticosa. All materials amplification results are consistent, and have no otherthan products of the strip. Results show that the LINE retrotransposons RT genes existin P. suffruticosa widely.2. A fragment of580bp was amplified by PCR from the genomic DNA of tree peony (Paeonia Suffruticosa Andrews.‘Luoyanghong’) using the degenerateoligoncleotide primers. The amplicons were recovered, purified and cloned. Positiveclones were identified and sequenced. Finally,32different sequences of reversetranscriptase from tree peony ‘Luoyanghong’ LINE retrotransposons wereobtained. The length of the nucleotide sequences varied from528to593bp. Thehomology ranged from25.7%to94.2%by Clustal W and the variations werecharacterized by point mutation. Four clusters were identified with high heterogeneitythrough phylogenic analysis of their nucleotide sequences. Family I and III,respectively accounted for50%and28%of the total number of clones. There is a veryconservative point in the18th amino acid sequence of glycine (Gly) in the translatedsequence, at the same time, a conservative point in138th amino acid sequence oflysine (Lys). More termination codon mutation and frameshift mutations werepresented in32sequences.3. Compared to the homogeneity of RT sequences among P. suffruticosa and otherseven plants which were downloaded from GenBank. The results showed P.suffruticosa and Allium cepa, Beta lomatogona, Vicia faba, Antirrhinum majus, Oryzasativa relationship were near with others. It showed that the P. suffruticosa anddifferent herbal, woody plants may have a common origin. And It also proved that thehorizontal transmission of retrotransposons between different species.