大白菜eIF(iso)4Ec基因hAT超家族活性转座子插入突变体的鉴别（英文）.doc

大白菜elF (iso) 4E.c基因hAT超家族 活性转座子插入突变体的鉴别(英文)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山东省农业科学院蔬菜花卉研宄所/山东省设施蔬菜生物学重点实验室/国家蔬菜改良中心山东分中心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)材料的真核土 物翻译起始因子异构体(isoform of eukaryotic translation initiation factor 4E, elE (iso) 4E. c)的基因组序列多样性时，从W份材料即大秦白 (DaQinBai)和826的elF (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h AT超家 族转座子非常吻合工具预测发现，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2 00600 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，木研宄分别在BraD8的 5’和3’端设计特异引物，以大白菜单拷W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昇，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h AT 超家族的Ac/Ds转座系统，该研宄将为今后BraD8及类似结构在人白菜转座子标 签方面的研宄提供借鉴关键词：大白菜;活动hAT超家族转座; BraD8/BraD8-类元件;转座了插入突变;真核 土物翻译起始因子异构体(elF (iso) 4E. c);赵智中，zhaozhizhong454@163.基金：山东省重点研发计划(No. 2017GNC10124)Identification of an Active hAT Superfamily Transposon Insertion Mutation in elF (iso) 4E. c of Chinese cabbage (Brassica rapa ssp. pekinensis)LIU Shuan-Tao ZHANG Zhi-Gang WANG Li-Hua LI Qiao-Yun ZHAO Zhi-Zhong LU Jin-Dong WANG Shu-Fen XU Wen-Ling LIU Xian-Xian LIU ChenVegetable and Flower Institute of ShandongAcademy of Agricultural Sciences， ShandongBranch of National Center for Vegetables Improvement， Shandong Key Laboratory for Biologyof Greenhouse Vegetables;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 (e TF (iso) 4E. c)，a large DNA fragment with 3 195 bp was found to insert into its third intron of 2 accession (Da Qin Bai 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 h AT (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 Da Qin Bai and 826. Further analysis showed that BraD8~like elements were ubiquitous in B.rapa genome and mostly located on chromosome 八04 and A09. Tts 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. Da Qin Bai contained only a few copies of both 5’-and3’-terminus, while line 826 contained about 19 and 45 copies respectively. Both termini varied greatly in lines94 610 and 05~46;73 and 06-247 only contained the 3’ -termini, and neither terminus was detected in lincs322, 8 407，71-3-62 and T03.PCR amplification was performed by using 17 bp TIR sequence as single primer and genomic DNA from Da Qin Bai and 826 as templates. The intact BraD8 sequence was only amplified from Da Qin Bai, while small fragments forming three main band ranging from 1 000J 000 bp were observed in826. Sequencing analysis revealed that the small fragments shared higher than 80%identity with the 3’-termini of BraD8 and were named BraD8-likc 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, thecharacterization of BraD8 will potentially facilitate molecular genetic studies and functional genes identification through transposon tagged protocol in Chinese cabbage.Keyword：Chinese cabbage; Active hAT superfamily transposon; BraD8/BraD8_like elements; Transposon insertion mutation; Isoform of eukaryotic translation initiation factor 4E (elF (iso) 4E.c);Transposons are classified either as RNA transposons (class I ) that transpose through an RNA intermediate or as DNA transposons (class II) that transpose via a DNA intermediate through’cut and paste’excision and genomic reintegration (Finnegan, 1990) . DNA transposons can be further categorized eis autonomous and non-eiutonomous transposons (llua-Van et al., 2005) . Autonomous elemen。