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从头测序和组装积雪草叶片转录组,用于映射结构、功能和调控基因,特别是与次生代谢相关的基因。

De novo sequencing and assembly of Centella asiatica leaf transcriptome for mapping of structural, functional and regulatory genes with special reference to secondary metabolism.

机构信息

Metabolic and Structural Biology Department, CSIR-Central Institute of Medicinal and Aromatic Plants (CIMAP), P.O. CIMAP, Lucknow-226015, India.

出版信息

Gene. 2013 Aug 1;525(1):58-76. doi: 10.1016/j.gene.2013.04.057. Epub 2013 May 1.

Abstract

Centella asiatica (L.) Urban is an important medicinal plant and has been used since ancient times in traditional systems of medicine. C. asiatica mainly contains ursane skeleton based triterpenoid sapogenins and saponins predominantly in its leaves. This investigation employed Illumina next generation sequencing (NGS) strategy on a pool of three cDNAs from expanding leaf of C. asiatica and developed an assembled transcriptome sequence resource of the plant. The short transcript reads (STRs) generated and assembled into contigs and singletons, representing majority of the genes expressed in C. asiatica, were termed as 'tentative unique transcripts' (TUTs). The TUT dataset was analyzed with the objectives of (i) development of a transcriptome assembly of C. asiatica, and (ii) classification/characterization of the genes into categories like structural, functional, regulatory etc. based on their function. Overall, 68.49% of the 46,171,131 reads generated in the NGS process could be assembled into a total of 79,041 contigs. Gene ontology and functional annotation of sequences resulted into the identification of genes related to different sets of cellular functions including identification of genes related to primary and secondary metabolism. The wet lab validation of seventeen assembled gene sequences identified to be involved in secondary metabolic pathways and control of reactive oxygen species (ROS) was established by semi-quantitative and real time PCR (qRT-PCR). The validation also included sequencing/size matching of a set of semi-quantitative PCR amplicons with their in silico assembled contig/gene. This confirmed the appropriateness of assembling the reads and contigs. Thus, the present study constitutes the largest report to date on C. asiatica transcriptome based gene resource that may contribute substantially to the understanding of the basal biological functions and biochemical pathways of secondary metabolites as well as the transcriptional regulatory elements and genetic markers. This work sets the stage for multi-faceted future improvement of the plant, through discovery of new genes, marker-assisted breeding or genetic engineering, on this species as well as for other species of Apiaceae and triterpene producing medicinal plants.

摘要

积雪草(L.)城市是一种重要的药用植物,自古以来就在传统医学系统中使用。积雪草主要含有五环三萜骨架的三萜皂苷元和皂苷,主要存在于其叶片中。本研究采用 Illumina 下一代测序(NGS)策略对积雪草展开叶 cDNA 池进行了研究,并开发了该植物的组装转录组序列资源。生成的短转录本(STR)组装成连续和单核苷酸,代表了积雪草中大多数表达的基因,被称为“暂定独特转录本”(TUT)。对 TUT 数据集进行了分析,目的是(i)构建积雪草的转录组组装,(ii)根据基因的功能将基因分类/特征化为结构、功能、调控等类别。总体而言,在 NGS 过程中产生的 46,171,131 个读长中,有 68.49%可以组装成总共 79,041 个连续。序列的基因本体和功能注释导致鉴定出与不同细胞功能组相关的基因,包括与初级和次级代谢相关的基因。通过半定量和实时 PCR(qRT-PCR)验证了十七个参与次级代谢途径和活性氧(ROS)控制的组装基因序列,建立了次级代谢途径和活性氧(ROS)控制的基因。验证还包括对一组半定量 PCR 扩增子与其在计算机组装的连续/基因的测序/大小匹配。这证实了组装读长和连续的合理性。因此,本研究构成了迄今为止积雪草转录组基因资源的最大报告,可能对理解次生代谢物的基本生物学功能和生化途径以及转录调控元件和遗传标记有很大贡献。这项工作为该物种以及伞形科和三萜类药用植物的其他物种的未来多方面改进奠定了基础,包括发现新基因、标记辅助育种或遗传工程。

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