CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, Uttar Pradesh, India.
PLoS One. 2013;8(4):e60375. doi: 10.1371/journal.pone.0060375. Epub 2013 Apr 3.
Artemisia annua is the source of antimalarial phytomolecule, artemisinin. It is mainly produced and stored in the glandular secretory trichomes present in the leaves of the plant. Since, the artemisinin biosynthesis steps are yet to be worked out, in this investigation a microarray chip was strategized for the first time to shortlist the differentially expressing genes at a stage of plant producing highest artemisinin compared to the stage with no artemisinin. As the target of this study was to analyze differential gene expression associated with contrasting artemisinin content in planta and a genotype having zero/negligible artemisinin content was unavailable, it was decided to compare different stages of the same genotype with contrasting artemisinin content (seedling--negligible artemisinin, mature leaf--high artemisinin). The SCAR-marked artemisinin-rich (~1.2%) Indian variety 'CIM-Arogya' was used in the present study to determine optimal plant stage and leaf ontogenic level for artemisinin content. A representative EST dataset from leaf trichome at the stage of maximal artemisinin biosynthesis was established. The high utility small scale custom microarray chip of A. annua containing all the significant artemisinin biosynthesis-related genes, the established EST dataset, gene sequences isolated in-house and strategically selected candidates from the A. annua Unigene database (NCBI) was employed to compare the gene expression profiles of two stages. The expression data was validated through semiquantitative and quantitative RT-PCR followed by putative annotations through bioinformatics-based approaches. Many candidates having probable role in artemisinin metabolism were identified and described with scope for further functional characterization.
黄花蒿是抗疟植物青蒿素的来源。它主要产生和储存在植物叶片的腺毛分泌性毛中。由于青蒿素生物合成步骤尚未被阐明，在这项研究中，我们首次设计了一个微阵列芯片，以筛选在植物产生最高青蒿素的阶段与没有青蒿素的阶段之间差异表达的基因。由于本研究的目的是分析与植物内差异青蒿素含量相关的差异基因表达，而没有零/可忽略不计青蒿素含量的基因型可用，因此决定比较具有不同青蒿素含量的同一基因型的不同阶段（幼苗-无青蒿素，成熟叶片-高青蒿素）。本研究使用了 SCAR 标记的青蒿素丰富（~1.2%）的印度品种“CIM-Arogya”来确定青蒿素含量的最佳植物阶段和叶片发生水平。建立了一个来自最大青蒿素生物合成阶段叶片毛状体的代表性 EST 数据集。使用包含所有重要青蒿素生物合成相关基因的高效小型定制黄花蒿微阵列芯片、建立的 EST 数据集、内部分离的基因序列以及从黄花蒿 Unigene 数据库（NCBI）有策略地选择的候选基因，比较了两个阶段的基因表达谱。通过半定量和定量 RT-PCR 验证了表达数据，然后通过基于生物信息学的方法进行可能的注释。鉴定并描述了许多可能在青蒿素代谢中起作用的候选基因，并为进一步的功能表征提供了依据。
