Isolation of Tyl-copia-like Retrotransposon Sequences from the Apple Genome by Chromosome Walking Based on Modified SiteFinding-polymerase Chain Reaction  

作　　者：Guiling ZHAO Zhihong ZHANG Haiyue SUN He LI Hongyan DAI 

机构地区：[1]College of Horticulture, Shenyang Agricultural University, Shenyang 110161, China [2]College of Forestry, Shenyang Agricultural University, Shenyang 110161, China

出　　处：《Acta Biochimica et Biophysica Sinica》2007年第9期675-683,共9页生物化学与生物物理学报（英文版）

基　　金：This work was supported by a grant from the National Natural Science Foundation of China （No. 30500348）

摘　　要：Long terminal repeat （LTR） retrotransposons are powerful tools for studying genetic biodiversity, genome evolution, gene mutation, gene cloning and gene expression. The scarcity of retrotransposon sequence information restricts the development of these studies in higher plants. In the present study, 31 reverse transcriptase （RT） genes of Tyl-copia-like retrotransposons were identified from the apple genome by amplifying the RT coding region using degenerate primers. Nineteen RT genes showed extreme heterogeneity in terms of fragment size, base pair composition and open reading frame integrality. Originating from one 266 bp cloned RT gene, a 1966 bp Tyl-copia-like retrotransposon （named Tcrml）, including RT-ribonuclease H-LTR domain sequences, was achieved by chromosome walking based on modified SiteFinding-polymerase chain reaction. The comparison between Tcrml and other LTR retrotransposons in gene structure and sequence homology shows that Tcrml is the first Tyl-copia-like retrotransposon including an LTR domain in the apple genome. Dot blot analysis revealed that Tcrml copy number in the apple was approximately 1× 10^3 copies per haploid genome.Long terminal repeat （LTR） retrotransposons are powerful tools for studying genetic biodiversity, genome evolution, gene mutation, gene cloning and gene expression. The scarcity of retrotransposon sequence information restricts the development of these studies in higher plants. In the present study, 31 reverse transcriptase （RT） genes of Tyl-copia-like retrotransposons were identified from the apple genome by amplifying the RT coding region using degenerate primers. Nineteen RT genes showed extreme heterogeneity in terms of fragment size, base pair composition and open reading frame integrality. Originating from one 266 bp cloned RT gene, a 1966 bp Tyl-copia-like retrotransposon （named Tcrml）, including RT-ribonuclease H-LTR domain sequences, was achieved by chromosome walking based on modified SiteFinding-polymerase chain reaction. The comparison between Tcrml and other LTR retrotransposons in gene structure and sequence homology shows that Tcrml is the first Tyl-copia-like retrotransposon including an LTR domain in the apple genome. Dot blot analysis revealed that Tcrml copy number in the apple was approximately 1× 10^3 copies per haploid genome.

关 键 词：Malus domestica Tyl-copia-like retrotransposon long terminal repeat sequence modified SiteFinding-polymerase chain reaction 

分 类 号：Q5[生物学—生物化学]