Rather Gulzar A, Sharma Arti, Pandith Shahzad A, Kaul Veenu, Nandi Utpal, Misra Prashant, Lattoo Surrinder K
Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi, 180001, India.
Department of Botany, University of Jammu, Jammu Tawi, 180006, India.
Plant Mol Biol. 2018 Jan;96(1-2):197-215. doi: 10.1007/s11103-017-0690-9. Epub 2017 Dec 21.
Comprehensive transcriptome analysis of leaf and root tissues of Nothapodytes nimmoniana unravels several putative pathway genes, transcription factors and CYPs related to camptothecin (CPT) biosynthesis. Additionally, post-transcriptional suppression by artificial microRNA (aMIR) of NnCYP76B6 (geraniol 10-hydroxylase) suggests its role in CPT biosynthesis. Tissue-specific LC-MS/MS analysis revealed the presence of secologanin as the central intermediate of MIA pathway in N. nimmoniana. Nothapodytes nimmoniana is a rich source of potent anticancer drug camptothecin (CPT) whose biosynthetic pathway is unresolved due to the lack of genomic and transcriptomic information. Present investigation entails deep transcriptome analysis of N. nimmoniana which led to identification of putative pathway genes and regulatory components involved in CPT biosynthesis. Using Illumina HiSeq 2500 sequencing platform a total of 31,172,889 (6.23 Gb) and 31,218,626 (6.24 Gb) raw reads were generated from leaf and root wood, respectively. These were assembled de novo into 138,183 unique contigs. Additionally, 16 cytochrome P450 transcripts related to secondary metabolism were also identified. Further, transcriptome data pool presented 1683 putative transcription factors of which transcripts corresponding to WRKY TFs were the most abundant (14.14%). A total of 2741 transcripts were differentially expressed out of which 478 contigs showed downregulation in root wood and 2263 contigs were up-regulated. Further, comparative analyses of 17 genes involved in CPT biosynthetic pathway were validated by qRT-PCR. On basis of intermediates, two distinct seco-iridoid pathways are involved in the biosynthesis of monoterpene indole alkaloids either through multiple isomers of strictosidinic acid or strictosidine. Tissue-specific LC-MS/MS analysis revealed the presence of secologanin as the central intermediate of MIA pathway in N. nimmoniana. Geraniol-10 hydroxylase (NnCYP76B6) an important enzyme in CPT biosynthesis which specifically shunts geraniol into the secologanin pathway was also cloned from the trancriptome resource. In planta transient expression of NnCYP76B6 showed a significant enhancement in mRNA transcript levels coincident with enhanced CPT accumulation. Further, artificial microRNA (aMIR) mediated downregulation of NnCYP76B6 resulted in reduction of mRNA transcript levels as well as CPT content in comparison to control. These empirical results suggest a plausible regulatory role for NnCYP76B6 in CPT biosynthesis and also establish a valuable repository for deciphering various structural, rate limiting and regulatory genes of CPT biosynthetic pathway.
对印度蛇根木叶片和根部组织进行的全面转录组分析，揭示了几个与喜树碱（CPT）生物合成相关的假定途径基因、转录因子和细胞色素P450。此外，通过人工microRNA（aMIR）对NnCYP76B6（香叶醇10-羟化酶）进行转录后抑制，表明其在CPT生物合成中的作用。组织特异性LC-MS/MS分析表明，裂环马钱子苷是印度蛇根木中MIA途径的核心中间体。印度蛇根木是强效抗癌药物喜树碱（CPT）的丰富来源，由于缺乏基因组和转录组信息，其生物合成途径尚未明确。目前的研究对印度蛇根木进行了深度转录组分析，从而鉴定出参与CPT生物合成的假定途径基因和调控成分。使用Illumina HiSeq 2500测序平台，分别从叶片和根部木材中产生了总共31,172,889（6.23 Gb）和31,218,626（6.24 Gb）条原始读数。这些读数被从头组装成138,183个独特的重叠群。此外，还鉴定出16个与次生代谢相关的细胞色素P450转录本。此外，转录组数据库显示有1683个假定的转录因子，其中对应于WRKY转录因子的转录本最为丰富（14.14%）。共有2741个转录本差异表达，其中478个重叠群在根部木材中显示下调，2263个重叠群上调。此外，通过qRT-PCR对参与CPT生物合成途径的17个基因进行了比较分析验证。基于中间体，单萜吲哚生物碱的生物合成涉及两条不同的断环马钱子苷途径，分别通过异胡豆苷酸或异胡豆苷的多种异构体。组织特异性LC-MS/MS分析表明，裂环马钱子苷是印度蛇根木中MIA途径的核心中间体。香叶醇-10羟化酶（NnCYP76B6）是CPT生物合成中的一种重要酶，它特异性地将香叶醇分流到裂环马钱子苷途径，该酶也从转录组资源中克隆得到。NnCYP76B6在植物中的瞬时表达显示mRNA转录水平显著提高，同时CPT积累增加。此外，与对照相比，人工microRNA（aMIR）介导的NnCYP76B6下调导致mRNA转录水平以及CPT含量降低。这些实验结果表明NnCYP76B6在CPT生物合成中可能具有调控作用，也为破译CPT生物合成途径的各种结构、限速和调控基因建立了一个有价值的资源库。
