利用 454 焦磷酸测序 GS FLX 钛平台对麻疯树（Jatropha curcas L.）五个主要组织进行从头组装和转录组分析。
De novo assembly and transcriptome analysis of five major tissues of Jatropha curcas L. using GS FLX titanium platform of 454 pyrosequencing.
机构信息
Genomics Laboratory, Department of Genetic Engineering, SRM University, Chennai, Tamil Nadu 603 203, India.
出版信息
BMC Genomics. 2011 Apr 15;12:191. doi: 10.1186/1471-2164-12-191.
BACKGROUND
Jatropha curcas L. is an important non-edible oilseed crop with promising future in biodiesel production. However, factors like oil yield, oil composition, toxic compounds in oil cake, pests and diseases limit its commercial potential. Well established genetic engineering methods using cloned genes could be used to address these limitations. Earlier, 10,983 unigenes from Sanger sequencing of ESTs, and 3,484 unique assembled transcripts from 454 pyrosequencing of uncloned cDNAs were reported. In order to expedite the process of gene discovery, we have undertaken 454 pyrosequencing of normalized cDNAs prepared from roots, mature leaves, flowers, developing seeds, and embryos of J. curcas.
RESULTS
From 383,918 raw reads, we obtained 381,957 quality-filtered and trimmed reads that are suitable for the assembly of transcript sequences. De novo contig assembly of these reads generated 17,457 assembled transcripts (contigs) and 54,002 singletons. Average length of the assembled transcripts was 916 bp. About 30% of the transcripts were longer than 1000 bases, and the size of the longest transcript was 7,173 bases. BLASTX analysis revealed that 2,589 of these transcripts are full-length. The assembled transcripts were validated by RT-PCR analysis of 28 transcripts. The results showed that the transcripts were correctly assembled and represent actively expressed genes. KEGG pathway mapping showed that 2,320 transcripts are related to major biochemical pathways including the oil biosynthesis pathway. Overall, the current study reports 14,327 new assembled transcripts which included 2589 full-length transcripts and 27 transcripts that are directly involved in oil biosynthesis.
CONCLUSION
The large number of transcripts reported in the current study together with existing ESTs and transcript sequences will serve as an invaluable genetic resource for crop improvement in jatropha. Sequence information of those genes that are involved in oil biosynthesis could be used for metabolic engineering of jatropha to increase oil content, and to modify oil composition.
背景
麻疯树是一种重要的非食用油料作物，在生物柴油生产方面具有广阔的前景。然而，油产量、油成分、油饼中的有毒化合物、害虫和疾病等因素限制了其商业潜力。利用克隆基因建立的成熟遗传工程方法可以用来解决这些限制。早些时候，从麻疯树 ESTs 的 Sanger 测序中获得了 10983 条单基因，从未克隆的 cDNA 的 454 焦磷酸测序中获得了 3484 个独特的组装转录本。为了加快基因发现的进程，我们对麻疯树根、成熟叶、花、发育种子和胚胎的标准化 cDNA 进行了 454 焦磷酸测序。
结果
从 383918 条原始reads 中，我们获得了 381957 条适合转录序列组装的质量过滤和修剪reads。对这些reads 的 de novo 拼接生成了 17457 条组装的转录本（拼接体）和 54002 条单基因。组装转录本的平均长度为 916 bp。大约 30%的转录本长度大于 1000 个碱基，最长的转录本长度为 7173 个碱基。BLASTX 分析表明，其中 2589 个转录本是全长转录本。通过对 28 个转录本的 RT-PCR 分析验证了这些组装转录本。结果表明，这些转录本是正确组装的，代表着活跃表达的基因。KEGG 途径映射显示，2320 个转录本与主要生化途径有关，包括油脂生物合成途径。总的来说，本研究共报告了 14327 个新的组装转录本，其中包括 2589 个全长转录本和 27 个直接参与油脂生物合成的转录本。
结论
本研究报告的大量转录本，加上现有的 ESTs 和转录本序列，将成为麻疯树作物改良的宝贵遗传资源。与油脂生物合成有关的基因的序列信息可用于麻疯树的代谢工程，以提高油脂含量和改变油脂成分。
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