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研究了苹果基因组中Ty1-copia类逆转座子的转录活性、多样性及其甲基化水平。在培养基MS+2,4-D2mg/L+BA0.2mg/L+NAA0.5mg/L上诱导了1个月的愈伤组织中用RT-PCR方法检测到了270bp的目的条带,而对照、继代组培苗和不含2,4-D的愈伤中均没有目的条带的扩增,说明一定浓度的2,4-D可以诱导苹果中Ty1-copia类逆转座子的表达活性。将目的条带回收、克隆和测序后进行分析:所克隆的21条序列虽都为Ty1-copia类逆转座子逆转录酶保守序列,但存在很大的异质性,21个克隆的核甘酸序列变化范围为196-290bp;翻译成氨基酸后存在缺失、插入、移框和终止密码子突变,7个克隆在氨基酸保守序列‘SLYGLKQ’处有1-2氨基酸位点的突变,说明这些序列对应的逆转录酶多数可能已失去了其原有功能。将21条序列进行聚类分析,除ART20外,其它克隆可聚为3类,并分别与GenBank上某些物种的相应序列同源性很高,表明在不同物种间可能存在逆转座子的横向传递作用。甲基化试验表明苹果基因组内Ty1-copia类逆转座子甲基化水平高。这些都表明Ty1-copia类逆转座子对苹果基因型的多样性及基因组的进化具有重要意义。
The transcriptional activity, diversity and methylation level of Ty1-copia retrotransposons in the apple genome were studied. A 270 bp band of interest was detected by RT-PCR in callus induced for 1 month on medium MS + 2,4-D2 mg / L + BA 0.2 mg / L + NAA 0.5 mg / L whereas control , There was no amplification of the target bands in the secondary callus and 2,4-D-free callus, indicating that certain concentrations of 2,4-D can induce Ty1-copia retrotransposon expression activity in apple . The target bands were recovered, cloned and sequenced for analysis. Although the 21 cloned sequences were both Ty1-copia reverse transcriptase conserved sequence, but there is a great heterogeneity, 21 cloned nucleotides The sequence ranged from196-290bp. There were deletions, insertions, frameshift and stop codon mutations after translated into amino acids. The seven clones had 1-2 amino acid mutations at the amino acid conserved sequence ’SLYGLKQ’, indicating that these sequences correspond to The reverse transcriptase majority may have lost its original function. The 21 sequences were clustered, and except for ART20, the other clones could be clustered into three groups, which were highly homologous to the corresponding sequences of some species in GenBank, indicating that transposon transversions may exist between different species Transmission effect. Methylation tests showed Ty1-copia retrotransposon methylation levels were high in the apple genome. All of these show that the Ty1-copia retrotransposons are of great importance to the diversity of genotypes and genome evolution in apple.