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| 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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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 .
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Received: 03 March 2017
Published: 01 November 2017
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| Fund:;the Agricultural Fine Seed Project in Shandong Province;Agricultural scientific and technological innovation project of Shandong Academy of Agricultural Sciences |
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