建鲤基因组中一个ty3-gypsy反转录转座子的发现与分析

摘要

转座子是动植物基因组的重要组成部分,在前期研究中发现建鲤(Cyprinus carpio var.jian)基因组中存在一个ty3-gypsy反转录转座子类型的转座子,并将其命名为JRE转座子(Jian carp Retrotransposon, JRE )。为了研究JRE反转录转座子在建鲤基因组中的功能,采用 PCR 扩增、荧光定量 PCR 和原位杂交等方法对 JRE 转座子的特性进行了研究。JRE反转录转座子全长5126 bp,具有5¢端470 bp和3¢端453 bp长末端重复片段(long terminal repeat end, LTR),中间的开放阅读框(ORF)包括核心蛋白基因(gag)和酶基因区域(pol),其长度为4203 bp。pol基因具有典型的ty3-gypsy 反转录转座子结构,基因顺序为 PR-RT-RH-IN 基因。对 pol 基因的同源分析表明,其与虾夷扇贝(Mizuh-opecten yessoensis)、栉孔扇贝(Azumapecten farreri)、大堡礁海绵(Xiphophorus maculates)和斑剑尾鱼(Xiphophorus maculates)pol基因相似性分别为40.7%、40.0%、32.8%和30.1%,因此JRE可能属于JULE反转录转座子家族。采用实时定量 PCR对 JRE 转座子在建鲤基因组内的拷贝数进行了测定,结果表明其拷贝数为124,同时对不同组织中的mRNA表达量的研究表明, JRE转座子在建鲤肝、肾、血、肌肉、性腺5种组织中均有表达,在肾和肝中表达量略高。染色体原位杂交结果表明, JRE转座子在建鲤的染色体上随机分布,没有明显的规律性。本研究表明, JRE转座子具有典型的反转录转座子结构,属于JULE转座子的分枝,在染色体上的分布不多,其转录活性并不是很高,对我们了解建鲤基因组构成和特点增加了知识储备,同时为利用转座子的活性进行转基因研究提供了一种新的途径和工具。%Transposable elements are major constituents of eukaryote genomes and have a significant effect on genome structure and stability. They also contribute to the genetic diversity and evolution of organisms. Knowledge of their distribution among several genomes is an essential condition to study their dynamics and to better understand their role in species evolution. Long terminal repeat (LTR)-retrotransposons have been reported in diverse eukaryote species, indicating a ubiquitous distribution. Among retrotransposons, the transposable element (TE) class, which are specific to eukaryotes, transpose via an RNA intermediate. Based on their structure, LTR-retrotransposons have been divided into two classes: ty3-gypsy and ty1-copia. In previous research, we found a ty3-gypsy-like retrotransposon, named JRE, in the genome of Jian carp,Cyprinus carpio var.jian. To investigate the function of JRE in the genome of Jian carp, PCR amplification, fluorescence quantitative PCR and fluorescent in situ hybridization were used to explore the structure and characteristics of JRE retrotransposon. Multiple alignments of DNA and protein sequences were constructed using MEGA 5.0 software and were manually curated using BioEdit. Pairwise distances were estimated using the option pairwise deletion of gaps in MEGA5.0. The full-length JRE retrotransposon is 5126 bp, which includes two LTRs of 470 bp at the 5¢ end and 453 bp at the 3¢ end, and two open reading frames (ORFs) between them. One ORF of 4203 bp encodes the group-specific antigen (GAG) and polyprotein (POL). The pol gene has a typical Ty3/gypsy retrotranspo-son structure, and the gene order is protease, reverse transcriptase, RNase H, and integrase (PR–RT–RH–IN). A phy-logenetic analysis of the pol gene showed that it has similarities of 40.7%, 40%, 32.8%, and 30.1% to retrotransposons of the Yesso scallop, Farrer’s scallop, a Great Barrier Reef sponge, and the spot swordtail fish, respectively. Therefore, JRE might belong to the JULE retrotransposon family. The copy number of the JRE retrotransposon in the genome of the Jian carp is 124, as determined by real-time quantitative PCR. The mRNA of the JRE retrotransposon is expressed in five Jian carp tissues: the liver, kidney, blood, muscle, and gonad, and is slightly higher in the kidney and liver than in the other tissues. Fluorescent in situ hybridization shown that JRE is randomly distributed in Jian carp chromosomes and no regular patterns were found. In conclusion, the complete structure of a retrotransposon found in a previous study was determined. Detailed analyses indicated that it has the typical structure of an LTR retrotransposon. Phylogenetic analysis showed that it is highly similar to JULE transposons. Its copy number in the Jian carp is not high, suggesting that its transcriptional activity is low. These findings extend our knowledge of the structure and features of the Jian carp genome, and might provide a new method of transgenic fish production.