| Transposons are genetic elements that can move, spread and generate mutations through insertions near or within genes, and that constitute an important fraction of eukaryote genomes. Transposable elements (TEs) are usually classified in two different groups according to their mode of transposition: retrotransposons transpose through an RNA intermediate, transposons transpose directly via a DNA intermediate. Retrotransposons are the most abundant and widespread class of transposable elements and generate stable mutations. The Ty1-copia-like retrotransposons are the best studied retrotransposons group in plant. Ty1-copia-like retrotransposons have been found associated with genome size, genome structure, gene function and the evolution of plant genes and genomes.Apple (Malus×domestica Borka.) is an important fruit tree planted widely in the world. Until now studies on Ty1-copia-like retrotransposons within apple genome are little. In this study, we carried out some works: existence of Ty1-copia-like retrotransposons in the genomes of different wild and cultural species of apple genus; heterogeneity and methylation level among retrotransposon; the roles of retrotransposon in genomic and chromosomal organization; transcription activity of retrotransposons in apple calli in vitro; the characterizatics of the long terminal regions (LTRs) of the retrotransposons and their contribution to genetic diversity and bud mutations using SSAP techniques based on LTRs.1 The factors that affected the PCR results were studied, using conserved reverse transcriptase region primers of Ty1-copia-like retrotransposons. The results showed that the optimized content of Mg2+ was 5mmol/L; dNTP was 200μmol/L; Primer was 0.5μmol/L; Taq polymerase was lU/50ul in the PCR system. Only one fragment of about 270bp was amplified within the genome of 12 wild species and 23 cultivars of Mallus Mill., which indicated that Ty1-copia-like retrotransposons were archaic components of apple genome. The nested insertion of near and within the reverse transcriptase gene of Ty1-copia-like retrotransposons wasn't found in apple genome.2 The reverse transcriptase conservative region of Ty1-copia-like retrotransposons were amplified from Gala apple using the optimized PCR system. The amplification product was isolated, cloned and sequenced. Forty-five clones containing reverse transcriptase(RT) conservative region were obtained (accession number: AY849580 -AY849592 and DQ105035-DQ105066). Cluster analysis of these sequences showed a great heterogeneity among RT domains isolated from the same genotype. The nucleotide acid sequences ranged from 196bp to 279bp. The homogeneity of nucleotide acid ranged from 40% to 99.2%, amino acid from 31.3% to 100%. Eleven of the sequences contained frameshifted translations, thirteen contained stop condons mutations of one, two or three, three contained recombination mutations, one contained insertion mutation, five contained deletion mutations, and twenty-two contained substitution mutations in the conserved SLYGLKQ of plant retrotransposons. According to the result of phylogenetic analysis of conceptually translated amino acid sequences, the forty-five clones were divided into ten families. The sequence number of family 1-7 was forty-two, accounting for 93% of the total number of clones. Transposable elements could exist in the seven families. The family 8-10 contained one clone respectively. Compared to the homogeneity of RT conserved region among apple and other species, the species presenting RT sequences most similar to apple RT clones were Norway spurce(gymnosperm), tomato and potato.3 Southern-blot hybridization using the total Gala RT PCR product as probes showed that multiple copies are integrated throughout the apple genome. The banding patterns among DNA extraction from control, tissue culture and genetic transformation showed no differences. Some Ty1-copia-like retrotransposons, presenting in apple, were found to be methylated, while no differences in methylation were observed among DNA extraction from the three materials. The methylation of retrotransposons maintained the stability of the host gene and genome, and promoted the genetic diversity of apple genotype and the evolution of apple genome.4 Transcriptionally activity of Ty1-copia-like retrotransposons were found in apple calli, propagated on MS solid medium supplemented with 2mg/L of 2,4-D, 0.2mg/L of BA and 0.5mg/L of NAA for a month. Calli, propagated on the medium, showed abnormal growth and lost the possibility of regeneration. RT-PCR was used for amplifying the conserved domain of reverse transcriptase gene using degenerated oligonucleotide primer. The 270bp of fragments were amplified from the mRNA of the above calli, while weren't detected from the mRNA of other materials. Therfore, 2mg/L of 2,4-D could induce the transcription activity of Ty1-copia-like retrotransposons within apple genome. The 270bp of frangments were isolated, cloned and sequenced. Twenty-one aimed sequences were obtained (accession number: AY849591-AY849596 and DQ105060-DQ105074). All 21 clones were different from each other, nucleotide acid ranged from 45.3% to 99.6%, and amino acid from 36.8% to 98.9%. Two of the sequences contained frameshifted translations, four contained stop condons mutations, three contained recombination mutations, one contained insertion mutation, one contained deletion mutations, and nine contained substitution mutations in the conserved SLYGLKQ of plant retrotransposons. A phylogenetic tree was constructed based on their predicted amino acids and twenty-one clones were divided into five families. The first, the second and the fourth family showed high homogeneity to the RT of beet, Prunus×yedoensis, Norway spruce, and A. thaliana respectively. The homogeneity of the clone prt52 in the second family and BAA02268.1 in Prunus×yedoensis was 91.6%.5 We adopted a modified version of Pearce et al.'s methodology for isolating RNase-LTR fragment of Ty1-copia-like retrotransposons in apple. The length of twenty sequences ranged from 107bp to 500bp isolated from the same genotype. Based on typical model of Ty1-copia-like retrotransposons, PPTs and IR were understood. PPTs of nine sequences consisted of purines, PPTs of eleven of continuous purines with a pyrimidine insertion. IR of nineteen sequences was the canonical "TG". One didn't contain 3'-LTRs owing to recombination mutation. Four of twenty sequences contained recombination mutations. Based on the homogeneity of amino acid sequence of RNaseH, twenty sequences were divided into six groups. Sequence length and combination, RNaseH structure and PPTs structure and starting location were different in the same group. In every group, there existed clones with more than 90% nucleotide identity, which showed that these retrotransposons could preserve transposable activity in the past. "CAAT box" and "TATA box", potential regulatory motifs from transcription, and conserved cis-acting regulator elements were discovered within LTRs in the great number of sequences using plantCARE sortware.6 The insertional polymorphism of LTR-10 element within the genome of 8 wild species and 28 cultivars of Mallus Mill. was studied by sequence-specific amplification polymorphism(SSAP). The result revealed high level of genetic diversity. The proportion of polymorphism products was 88.2%. Phylogenetic tree based on the SSAP data showed that SSAP marker was able to resolve different species lineages within apple genus. All species were divided into three groups. The first group consisted of foreign cultivar species. The second group included wild species. The third group only contained Malus asiatica var. rinid (Koidz.) Asami owing to complexity of genetic materials. The species in the second and third group origined from China. The 180bp amplification fragment was present in Wellspur Delicious, not in starking from which the former mutated, suggesting that retrotransposon activity might be involved in the bud mutation of Wellspur Delicious. |
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