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2025年8月27日 星期三
  2017, Vol. 25 Issue (11): 1791-1798    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
大白菜eIF(iso)4E.c基因hAT超家族活性转座子插入突变体的鉴别
刘栓桃,张志刚,王立华,李巧云,赵智中,卢金东,王淑芬,徐文玲,刘贤娴,刘辰
山东省农业科学院蔬菜花卉研究所
Identification of an Active hAT Superfamily Transposon Insertion Mutation in eIF(iso)4E.c of Chinese cabbage (Brassica rapa ssp. pekinensis)
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摘要 转座子是基因组中一段可移动的DNA序列,其有时可以创造突变,也可以使突变回复。在检测12份大白菜(Brassica rapa ssp. pekinensis)材料的真核生物翻译起始因子异构体(isoform of eukaryotic translation initiation factor 4E, eIF(iso)4E.c)的基因组序列多样性时,从两份材料即大秦白(DaQinBai)和826的eIF(iso)4E.c第3内含子中发现了1个长达3 195 bp的大插入片段。生物信息学比对发现,该大片段插入导致插入靶位点两侧有8 bp的正向重复、同时大片段两侧有17 bp的反向重复,这一结构特征与hAT超家族转座子非常吻合。在线工具预测发现,该大片段包含1个由两个外显子组成的编码框,预测的编码产物是1个含DUF659结构域的蛋白,该结构域出现在很多具有转座酶功能的蛋白中,由此推测该大片段是1个活动的转座子,由于其来自大白菜自交系大秦白和826,故将其命名为BraD8。将BraD8的全序列在大白菜基因组数据库(the Brassica database, BRAD)比对,发现在A04和A09上有很多同源性高达80%以上的片段,长度介于200~600 bp,这种组织结构非常类似于玉米(Zea mays)的Ac/Ds(activator/dissociation)转座系统。鉴于Ac/Ds转座系统中Ds以多拷贝存在于基因组中的事实,本研究分别在BraD8的5'和3'端设计特异引物,以大白菜单拷贝基因为内参,采用qRT-PCR方法,测定了12份大白菜材料中BraD8的5'和3'端的拷贝数。结果表明,不同材料中5'和3'端的拷贝数存在显著差异,在大秦白中,BraD8的5'和3'仅有少数几个拷贝,而在826中BraD8的5'和3'端则大约分别有19和45个拷贝;另外,在73和06-247中只有3'端、而322,8407,71-3-62和T03基因组中则5'和3'端都没有检测到。这些结果暗示,大白菜中的BraD8及类似结构就是属于hAT超家族的Ac/Ds转座系统,该研究将为今后BraD8及类似结构在大白菜转座子标签方面的研究提供借鉴。
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刘栓桃
张志刚
王立华
李巧云
赵智中
卢金东
王淑芬
徐文玲
刘贤娴
刘辰
关键词 大白菜hAT超家族活动转座子BraD8BraD8-类元件转座子插入突变体eIF(iso)4E.c    
Abstract:Transposon is a DNA sequence that can change its position within a genome and sometimes creat or reverse mutations. During the cloning of Chinese cabbage (Brassica rapa ssp. pekinensis) isoform of eukaryotic translation initiation factor 4E (eIF(iso)4E.c), a large DNA fragment with 3 195 bp was found to insert into its third intron of 2 accession (DaQinBai and 826) of 12 Chinese cabbage inbred lines. Bioinformatics analysis revealed that a set of typical 8 bp target site duplications and 17 bp short terminal inverted repeats were found in two side of the fragment, in which the structure was more like that of hAT (after hobo from Drosophila melanogaster, Ac from Zea mays, and Tam3 from Antirrhinum majus) superfamily transponson. The large inserted fragment contained 2 exons which consisted of an intact ORF coding for a product with DUF 659 domain and transposase-like function. The transponson was named BraD8 after Chinese cabbage lines DaQinBai and 826. Further analysis showed that BraD8-like elements were ubiquitous in B. rapa genome and mostly located on chromosome A04 and A09. Its organization in B. rapa genome was like that of activator/dissociation (Ac/Ds) in maize (Zea mays). The copy number of 5'-and 3'-termini of BraD8 varied greatly in different Chinese cabbage lines. DaQinBai contained only a few copies of both 5'-and 3'-terminus, while line 826 contained about 19 and 45 copies respectively. Both termini varied greatly in lines 94 610 and 05-46; 73 and 06-247 only contained the 3'-termini, and neither terminus was detected in lines 322, 8 407, 71-3-62 and T03. PCR amplification was performed by using 17 bp TIR sequence as single primer and genomic DNA from DaQinBai and 826 as templates. The intact BraD8 sequence was only amplified from DaQinBai, while small fragments forming three main band ranging from 1 000~2 000 bp were observed in 826. Sequencing analysis revealed that the small fragments shared higher than 80% identity with the 3'-termini of BraD8 and were named BraD8-like elements. To explore the distribution of BraD8 in B.rapa genome, BLAST search was performed in B. database with the intact BraD8 sequence as query. The search results didn’t find complete identical BraD8 sequence but a lot of fragments ranged from 200 to 600 bp. They were mostly scattered on chromosome A04, A09. The pattern of BraD8/BraD8-like elements in B. rapa genome was line Ac/Ds transponson in maize. Considering the theoretical research and practical applications of Ac/Ds in maize, the characterization of BraD8 will potentially facilitate molecular genetic studies and functional genes identification through transposon tagged protocol in Chinese cabbage .
Key wordsB. rapa L. ssp pekinensis (Chinese cabbage)    active hAT superfamily transposon    BraD8/BraD8-like elements    Transposon insertion mutation    eIF(iso)4E.c
收稿日期: 2017-03-03      出版日期: 2017-11-01
基金资助:山东省自然科学基金;山东省农业良种工程项目;山东省农业科学院农业科技创新工程-作物分子育种服务平台
通讯作者: 赵智中     E-mail: zhaozhizhong454@163.com
引用本文:   
刘栓桃 张志刚 王立华 李巧云 赵智中 卢金东 王淑芬 徐文玲 刘贤娴 刘辰. 大白菜eIF(iso)4E.c基因hAT超家族活性转座子插入突变体的鉴别[J]. , 2017, 25(11): 1791-1798.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2017/V25/I11/1791
 
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