Yang Mengquan, You Wenjing, Wu Shiwen, Fan Zhen, Xu Baofu, Zhu Mulan, Li Xuan, Xiao Youli
CAS Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China.
University of Chinese Academy of Sciences, Beijing, 100039, China.
BMC Genomics. 2017 Mar 22;18(1):245. doi: 10.1186/s12864-017-3615-8.
Huperzia serrata (H. serrata) is an economically important traditional Chinese herb with the notably medicinal value. As a representative member of the Lycopodiaceae family, the H. serrata produces various types of effectively bioactive lycopodium alkaloids, especially the huperzine A (HupA) which is a promising drug for Alzheimer's disease. Despite their medicinal importance, the public genomic and transcriptomic resources are very limited and the biosynthesis of HupA is largely unknown. Previous studies on comparison of 454-ESTs from H. serrata and Phlegmariurus carinatus predicted putative genes involved in lycopodium alkaloid biosynthesis, such as lysine decarboxylase like (LDC-like) protein and some CYP450s. However, these gene annotations were not carried out with further biochemical characterizations. To understand the biosynthesis of HupA and its regulation in H. serrata, a global transcriptome analysis on H. Serrata tissues was performed.
In this study, we used the Illumina Highseq4000 platform to generate a substantial RNA sequencing dataset of H. serrata. A total of 40.1 Gb clean data was generated from four different tissues: root, stem, leaf, and sporangia and assembled into 181,141 unigenes. The total length, average length, N50 and GC content of unigenes were 219,520,611 bp, 1,211 bp, 2,488 bp and 42.51%, respectively. Among them, 105,516 unigenes (58.25%) were annotated by seven public databases (NR, NT, Swiss-Prot, KEGG, COG, Interpro, GO), and 54 GO terms and 3,391 transcription factors (TFs) were functionally classified, respectively. KEGG pathway analysis revealed that 72,230 unigenes were classified into 21 functional pathways. Three types of candidate enzymes, LDC, CAO and PKS, responsible for the biosynthesis of precursors of HupA were all identified in the transcripts. Four hundred and fifty-seven CYP450 genes in H. serrata were also analyzed and compared with tissue-specific gene expression. Moreover, two key classes of CYP450 genes BBE and SLS, with 23 members in total, for modification of the lycopodium alkaloid scaffold in the late two stages of biosynthesis of HupA were further evaluated.
This study is the first report of global transcriptome analysis on all tissues of H. serrata, and critical genes involved in the biosynthesis of precursors and scaffold modifications of HupA were discovered and predicted. The transcriptome data from this work not only could provide an important resource for further investigating on metabolic pathways in H. serrata, but also shed light on synthetic biology study of HupA.
蛇足石杉是一种具有重要经济价值的传统中草药，具有显著的药用价值。作为石松科的代表性成员，蛇足石杉产生多种具有生物活性的石松生物碱，尤其是石杉碱甲，它是一种治疗阿尔茨海默病的有前景的药物。尽管它们具有药用重要性，但公开的基因组和转录组资源非常有限，石杉碱甲的生物合成在很大程度上尚不清楚。先前对蛇足石杉和龙骨石松454-ESTs的比较研究预测了参与石松生物碱生物合成的假定基因，如赖氨酸脱羧酶样（LDC-like）蛋白和一些细胞色素P450（CYP450）。然而，这些基因注释并未进行进一步的生化表征。为了了解蛇足石杉中石杉碱甲的生物合成及其调控，对蛇足石杉组织进行了全转录组分析。
在本研究中，我们使用Illumina Highseq4000平台生成了大量蛇足石杉的RNA测序数据集。从根、茎、叶和孢子囊这四种不同组织中总共生成了40.1 Gb的 clean数据，并组装成181,141个单基因。单基因的总长度、平均长度、N50和GC含量分别为219,520,611 bp、1,211 bp、2,488 bp和42.51%。其中，105,516个单基因（58.25%）通过七个公共数据库（NR、NT、Swiss-Prot、KEGG、COG、Interpro、GO）进行了注释，分别对54个GO术语和3,391个转录因子（TFs）进行了功能分类。KEGG通路分析表明，72,230个单基因被分类到21个功能通路中。在转录本中鉴定出了负责石杉碱甲前体生物合成的三种候选酶，即LDC、CAO和PKS。还对蛇足石杉中的457个CYP450基因进行了分析，并与组织特异性基因表达进行了比较。此外，还进一步评估了在石杉碱甲生物合成后期两个阶段修饰石松生物碱骨架的两类关键CYP450基因BBE和SLS，它们共有23个成员。
本研究是对蛇足石杉所有组织进行全转录组分析的首次报道，发现并预测了参与石杉碱甲前体生物合成和骨架修饰的关键基因。这项工作的转录组数据不仅可以为进一步研究蛇足石杉的代谢途径提供重要资源，还为石杉碱甲的合成生物学研究提供了线索。
