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基于双端测序的荠苨转录组从头组装与特征分析。

De novo assembly and characterization of Camelina sativa transcriptome by paired-end sequencing.

出版信息

BMC Genomics. 2013 Mar 5;14:146. doi: 10.1186/1471-2164-14-146.

Abstract

BACKGROUND

Biofuels extracted from the seeds of Camelina sativa have recently been used successfully as environmentally friendly jet-fuel to reduce greenhouse gas emissions. Camelina sativa is genetically very close to Arabidopsis thaliana, and both are members of the Brassicaceae. Although public databases are currently available for some members of the Brassicaceae, such as A. thaliana, A. lyrata, Brassica napus, B. juncea and B. rapa, there are no public Expressed Sequence Tags (EST) or genomic data for Camelina sativa. In this study, a high-throughput, large-scale RNA sequencing (RNA-seq) of the Camelina sativa transcriptome was carried out to generate a database that will be useful for further functional analyses.

RESULTS

Approximately 27 million clean "reads" filtered from raw reads by removal of adaptors, ambiguous reads and low-quality reads (2.42 gigabase pairs) were generated by Illumina paired-end RNA-seq technology. All of these clean reads were assembled de novo into 83,493 unigenes and 103,196 transcripts using SOAPdenovo and Trinity, respectively. The average length of the transcripts generated by Trinity was 697 bp (N50 = 976), which was longer than the average length of unigenes (319 bp, N50 = 346 bp). Nonetheless, the assembly generated by SOAPdenovo produced similar number of non-redundant hits (22,435) with that of Trinity (22,433) in BLASTN searches of the Arabidopsis thaliana CDS sequence database (TAIR). Four public databases, the Kyoto Encyclopedia of Genes and Genomes (KEGG), Swiss-prot, NCBI non-redundant protein (NR), and the Cluster of Orthologous Groups (COG), were used for unigene annotation; 67,791 of 83,493 unigenes (81.2%) were finally annotated with gene descriptions or conserved protein domains that were mapped to 25,329 non-redundant protein sequences. We mapped 27,042 of 83,493 unigenes (32.4%) to 119 KEGG metabolic pathways.

CONCLUSIONS

This is the first report of a transcriptome database for Camelina sativa, an environmentally important member of the Brassicaceae. We showed that C. savita is closely related to Arabidopsis spp. and more distantly related to Brassica spp. Although the majority of annotated genes had high sequence identity to those of A. thaliana, a substantial proportion of disease-resistance genes (NBS-encoding LRR genes) were instead more closely similar to the genes of other Brassicaceae; these genes included BrCN, BrCNL, BrNL, BrTN, BrTNL in B. rapa. As plant genomes are under long-term selection pressure from environmental stressors, conservation of these disease-resistance genes in C. sativa and B. rapa genomes implies that they are exposed to the threats from closely-related pathogens in their natural habitats.

摘要

背景

从荠蓝种子中提取的生物燃料最近已成功用作环保型喷气燃料,以减少温室气体排放。荠蓝在遗传上与拟南芥非常接近,两者均属于十字花科。尽管目前有一些十字花科成员的公共数据库,如拟南芥、山柳菊、油菜、芥菜和白菜,但荠蓝没有公共的表达序列标签(EST)或基因组数据。在这项研究中,我们进行了荠蓝转录组的高通量、大规模 RNA 测序(RNA-seq),生成了一个数据库,将有助于进一步的功能分析。

结果

通过 Illumina 配对末端 RNA-seq 技术,从原始读取中去除接头、模糊读取和低质量读取(2.42 吉字节对)后,共生成约 2700 万个清洁“读取”。使用 SOAPdenovo 和 Trinity 将所有这些清洁读取分别从头组装成 83493 个 unigenes 和 103196 个转录本。Trinity 生成的转录本的平均长度为 697bp(N50=976),长于 unigenes 的平均长度(319bp,N50=346bp)。尽管如此,SOAPdenovo 生成的组装在 BLASTN 搜索拟南芥 CDS 序列数据库(TAIR)时产生了与 Trinity(22433)相似数量的非冗余命中(22435)。四个公共数据库,京都基因与基因组百科全书(KEGG)、Swiss-prot、NCBI 非冗余蛋白(NR)和直系同源群(COG),用于 unigene 注释;在 83493 个 unigenes中,最终有 67791 个(81.2%)被注释了基因描述或保守蛋白结构域,这些描述或结构域映射到 25329 个非冗余蛋白序列。我们将 83493 个 unigenes中的 27042 个(32.4%)映射到 119 个 KEGG 代谢途径。

结论

这是荠蓝转录组数据库的第一个报告,荠蓝是十字花科中一个重要的环境成员。我们表明,荠蓝与拟南芥属密切相关,与芸薹属关系较远。尽管大多数注释基因与拟南芥属的基因具有很高的序列同一性,但相当一部分抗病基因(NBS 编码的 LRR 基因)与其他十字花科植物的基因更相似;这些基因包括白菜中的 BrCN、BrCNL、BrNL、BrTN 和 BrTNL。由于植物基因组长期受到环境胁迫的选择压力,这些抗病基因在荠蓝和白菜基因组中的保守意味着它们在其自然栖息地中受到了与其密切相关的病原体的威胁。

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