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虽然表达序列标签(ESTs,mRNA片段序列)已广泛用于高效基因发现和补充基因组注释的工作,最近,与实时荧光定量反转录PCR(qRT-PCR)结合,它也开始应用于种系遗传学、转录谱及其蛋白组学方面。通过对油料木本植物小桐子(J.curcas)的生殖组织基因表达水平的分析,预期可能找到一些与油脂合成相关的基因。这些研究成果在将来可能用于提高种子产油木质类植物的油产量。为此,分别构建了两个cDNA文库,一共得到9289条平均长度为603bp的EST序列。这些序列经过组装得到4502条独立基因序列(UniSeqs),分别包括1427条重叠群(包含1条以上EST序列)和3075条单一序列(包含1条EST序列)。用Gene Ontology(GO)分类软件对这些组装的序列进行了基因注释,并利用qRT-PCR对其中50条EST序列在叶子,花和种子中的相对表达水平进行了定量分析,发现在两个cDNA文库中有6条高丰度的EST序列(Contig1452,Contig1482,Contig1510,Contig1514,Contig1534和Contig1535)在一或两个小桐子组织中也是高表达。这些基因编码高表达的油脂合成相关蛋白或者转录因子。此实验为小桐子的基因组注释及其在增加公共数据库内储备序列的数量方面提供了EST序列。尤其是实时荧光定量反转录PCR分析验证的6条在cDNA文库中频繁出现的,高丰度的,具有推定功能的EST序列,特别是qRT-PCR检测出的与油脂合成或转录因子相关的EST编码基因群可能在小桐子的油脂合成中起着重要的作用。
Although expressed sequence tags (ESTs, mRNA fragment sequences) have been used extensively for efficient gene discovery and replenishment of genomic annotations, it has also recently begun to be applied to germline inheritance in combination with real-time fluorescent quantitative reverse transcription PCR (qRT-PCR) Learn, Transcription and Proteomics. By analyzing the reproductive tissue gene expression levels of the oilwood woody plant J. curcas, it is expected that some genes related to lipid synthesis may be found. These findings may in the future be used to increase the oil yield of seedy oilwood plants. For this purpose, two cDNA libraries were constructed respectively, and a total of 9289 EST sequences with an average length of 603 bp were obtained. These sequences were assembled to obtain 4502 unique gene sequences (UniSeqs), including 1427 contigs (including more than one EST sequence) and 3075 single sequences (including one EST sequence). The assembled sequences were gene annotated with Gene Ontology (GO) software and the relative expression levels of 50 ESTs in leaves, flowers and seeds were quantitatively analyzed by qRT-PCR. It was found that in the two cDNAs Six highly abundant ESTs (Contig1452, Contig1482, Contig1510, Contig1514, Contig1534 and Contig1535) were also highly expressed in one or two Jatropha curcas tissues. These genes encode highly overexpressed oil synthesis related proteins or transcription factors. This experiment provides genomic annotations for Jatropha curcas and provides EST sequences for increasing the number of stock sequences in public databases. In particular, six frequently observed, highly abundant, putative EST sequences verified by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) in cDNA libraries, especially the qRT-PCR detection related to lipid synthesis or transcription factors EST coding genes may play an important role in the lipid synthesis of Jatropha curcas.