现代月季Ty1-copia类逆转座子逆转录酶序列的克隆及分析

Cloning and Analysis of Ty1-copia-like Reverse Transcriptase in Mordern Rose

本研究以8个现代月季品种为试验材料,根据Ty1-copia类逆转座子逆转录酶的保守区设计简并引物,通过PCR扩增,从现代月季基因组中扩增出260 bp左右的目标条带。将扩增产物纯化后克隆于p MD18-T质粒载体,选择阳性克隆,再经菌落PCR鉴定、测序及序列分析,获得19条来源于现代月季的Ty1-copia类逆转座子逆转录酶序列。研究发现这些序列的核苷酸长度集中于258~268 bp,存在较高的异质性,同源性范围3.4%~81.6%,主要表现在缺失突变,通过核苷酸聚类分析分为四类家族。翻译成氨基酸后,发现存在移框突变和终止密码子突变的现象,将这19条来源于现代月季的逆转录酶序列与登录在NCBI上来自于其它植物的Ty1-copia类逆转座子逆转录酶的氨基酸序列聚类分析,结果表明现代月季与其它物种间具有较高的同源性,表明Ty1-copia类逆转座子逆转录酶序列在不同物种之间可能存在横向传递。

In this study, eight morden rose cultivars were employed as experimental materials and used degene- rate oligoncleotide primers corresponding to conserved domains of the Tyl-copia-like retrotransposons reverse transcriptase to amplify a fragment of 260 bp by PCR from modem roses. The amplicons were cloned into pMD 18- T vectors after purification positive clones were selected and identified by colony PCR, then sequenced and analyzed. Nineteen different sequences of reverse transcriptase with length varied from 258-268 bp from Modem roses were obtained. These sequences showed high heterogeneity mainly characterized by deletion mutation. The homology ranged from 3.4% to 81.6%. Four groups were distinguished after cluster and alignment analyses of their nucleotide sequences. The deduction of amino acids showed that flame shift mutation and stop codon mutation. The amino cluster and alignment analyses of the nineteen sequences and other published sequences of the reverse transcriptase from other plants in NCBI showed that it might be involved in high homology. It also showed that the horizontal transmission of Tyl-copia-like retrotransposons reverse transcriptase between different species.