中国科学院昆明动物研究所机构知识库

在药用植物研究中，现代生物学研究是历史发展的必然。药用植物现代化的含义除了药理研究外还包括对植物本身研究的现代化。研究药用植物的基础生物学、有效成分的生物合成机理，对育种和有效成分生产都有重要科学意义。目前已有很多高新技术和现代方法逐步应用于药用植物现代化研究中，其中，基因组测序和基因编辑技术，是当代生物学发展的最前沿技术。本研究选取云南最具代表性的两种药用植物——三七和铁皮石斛，利用现代最前沿的基因组测序技术和CRISPR/Cas9体系进行了基因组解析和基因编辑，对药用植物现代生物学研究具有非常重要的意义。三七（Panax notoginseng（Burk.）F. H. Chen，2n=2′ = 24），俗名田七，是五加科人参属多年生草本植物，据古代中医药典记载，三七具有散瘀止血，改善血液循环等功效。经过数十年的药理学研究，三七特有的多种次级代谢产物（人参皂苷和三七皂苷）已经被成功分离、鉴定、并已赋予药用价值。这些研究成果在制药市场得到了广泛应用，通过设计和生产各种口腔和局部用药，可治疗心血管疾病、创伤、软组织疼痛。为了进一步获得三七基因组信息，本研究成功绘制了三七参考基因组图谱，这项研究将成为三七分子育种继EST测序和RNA-seq之后的又一重要成果。本研究采用de novo 组装方法，得到了2.39Gb的基因组草图序列，其中contig N50=16kb, scaffold N50=96kb。我们通过CEGMA（core eukaryotic genes mapping approach）软件对基因组组装完整性进行评价，结果显示80%的超保守基因落在同一条组装的序列上边，96%的超保守基因能够注释出来。我们同时用了BUSCO（benchmarking universal single-copy orthologs）这个软件对三七基因组的组装和注释进行了评价，使用植物共有的保守的单拷贝基因。结果显示82.4%的植物共有的保守的基因可以在组装的基因组中找到，有3.3%的基因在组装的序列中片段存在。此外，为了更好的注释蛋白质编码基因，我们完成了三七转录组测序，并结合基因组数据，分析了12个萜烯合成酶（terpene synthases ，TPSs）基因的功能和表达量。总之，我们完成了三七高度重复的复杂基因组测序、组装和注释，探索出了一套适合三七的全基因组辅助选择的现代育种方法，鉴定了有效药用成分生物合成基因及农艺性状控制的关键基因，并且在六个组织器官转录组数据的支撑下，对IPP/DMAPP（异戊烯基二磷酸/二甲丙基二磷酸）生物合成途径和TPS基因进行了基因组学分析。不仅为三七现代生物学研究奠定了良好的基础，同时还为人参属植物的新药研发及成分鉴定提供了丰富的遗传资源。铁皮石斛（Dendrobium officinale，D. officinale）为兰科多年生附生草本植物，具有独特的观赏价值和广泛的药用功效，种子无需春化作用便可生长，是兰科植物中最具特色的物种之一，被称为“中华九大仙草”之首。铁皮石斛基因组图谱的绘制，使其成为研究兰科植物进化机制、遗传资源解读的模式物种。尽管如此，铁皮石斛现有的遗传学研究方法依然较少，本研究在农杆菌介导的转基因体系基础上，验证了几个新的高效启动子，可用于铁皮石斛基因过表达和CRISPR/Cas9基因敲除体系，也是兰科植物基因编辑体系的主要方法。在我们建立的铁皮石斛转基因体系中，pCambia-1301-35SN载体包含CaMV 35S启动子，超绿色荧光蛋白（Superfolder green fluorescence protein，SG）基因和β-glucuronidase (GUS)报告基因，通过农杆菌介导的转基因体系转入植物组织中，当受体植物出现荧光表型可以说明SG基因成果转入植物并且翻译成功能蛋白质。我们将4个新的启动子（(MtHP, CVMV, MMV, PCISV)）和CaMV 35S启动子分别与GUS基因一起整合在相同的载体骨架上，并将两者做对比，结果显示，MMV, CVMV, and PCISV与 35S 启动子同样有效，说明这4个新的启动子也可用于铁皮石斛转基因体系中。此外，我们在铁皮石斛中成功建立了CRISPR/Cas9基因敲除体系，选择5个木质素合成通路上的关键基因（C3H, C4H, 4CL, CCR 和 IRX)），结果显示，每个基因都发生了不同程度的插入、缺失、替换。以上结果表明，本研究建立的2种基因编辑技术体系（基因过表达和基因敲除）可以在铁皮石斛中得到有效应用，不仅可以培育出铁皮石斛新品种，还很好的促进了铁皮石斛现代化研究的进程。

In the study of medicine Plants, its modernization has a historical inevitability. The implication of the medicinal plants modernization is not only limited to pharmacological research, but also includes the modernization of the plant itself. Studies on the basic biology and the biosynthetic mechanism of active ingredients in medicinal plants have great scientific significance in the breeding and the production of active ingredients. Currently, there are many advanced technologies and novel methods been applied in the medicinal plants modernization research. Genome sequencing and gene editing techniques are the latest cutting edge technologies in current biological research. In this study, we chose two representative medicinal plants in Yunnan province, Panax notoginseng and Dendrobium officinale, to do the genome sequencing using the Illumina sequencing and gene editing via CRISPR/Cas9 system, which have great importance on biological research in medicinal plants.Panax notoginseng (Burk.) F. H. Chen (2n = 2′ = 24, common name sanqi or tianqi), belonging to the Araliaceae family, was a slow-growing plant documented in the ancient Chinese medicine textbooks for its ability to ameliorate blood stasis and improve blood circulation. After decades of pharmacological research, a variety of P. notoginseng specific secondary metabolites (notably ginsenosides and notoginsenosides) were isolated, identified, and implicated in conferring medicinal properties. These discoveries allowed the design and production of numerous modern oral and topical drugs to treat cardiovascular diseases, contusion, and soft tissue pain. In order to obtain the genome information, we propose the construction of a reference P. notoginseng genome. This information will be an important addition to the existing expressed sequence tag and RNA-seq data for P. notoginseng genetics. The de novo assembly process yielded a draft P. notoginseng genome of 2.39 Gb, with a contig N50 size of 16 kb and scaffold N50 size of 96 kb. Evaluation of the completeness of this genome assembly by the core eukaryotic genes mapping approach (CEGMA) showed that 198 of 248 ultra-conserved genes could be fully annotated (80% completeness), and 239 of 248 ultra-conserved genes met the criterion for partial annotation (96% completeness). We also assessed the completeness of Panax notoginseng genome and annotation with common plant benchmarking universal single-copy orthologs (BUSCOs). The result showed that 1,186 out of 1,440 plant BUSCOs (82.4%) could be found in this genome assembly, and 47 plant BUSCOs (3.3%) had fragmented matches. In addition, to facilitate the protein-coding gene annotation process, we completed the RNA sequence in P. notoginseng, analysed the function and expression of 12 (terpene synthases) TPS genes by combining the genome and transcriptome. In conclusion, we have sequenced, assembled, and annotated the highly repetitive and complex P. notoginseng genome. Based on the genome analysis, we could explore the whole genome assisted breeding method, identify the key genes involved in biosynthesis and controlled the agronomic traits. With the support of RNA-seq data from six organs of P. notoginseng, we presented the genomic analysis of the IPP/DMAPP biosynthetic pathways and TPS genes. This information, together with the predicted GLYCOSYLTRANSFERASE genes, not only lays the groundwork for studying the modern biology of P. notoginseng, but also provides good genetic resources for novel drug candidate’s research and identifying active ingredients in close-related Panax species.Orchidaceae is the second largest family of flowering plants, which is highly valued for its ornamental purposes and medicinal uses. Dendrobium officinale is a special orchid species that can grow without seed vernalization. Because the whole-genome sequence of D. officinale is publicly available, this species is poised to become a convenient research model for the evolutionary, developmental, and genetic studies of Orchidaceae. Despite these advantages, the methods of genetic manipulation are poorly developed in D. officinale. In this study, based on the previously developed Agrobacterium-mediated gene transformation system, we identified several highly efficient promoters for exogenous gene expression and successfully applied the CRISPR/Cas9 system for editing endogenous genes in the genome of D. officinale. These two basic techniques contribute to the genetic manipulation toolbox of Orchidaceae. The pCambia-1301-35SN vector containing the CaMV 35S promoter and the β-glucuronidase (GUS) and Superfolder green fluorescence protein (SG) as reporter genes were introduced into the plant tissues by the Agrobacterium-mediated transformation system. Fluorescence emission from the transformed plants confirmed the successful transcription and translation of SG genes into functional proteins. We compared the GUS activity under different promoters including four commonly used promoters (MtHP, CVMV, MMV and PCISV) with CaMV 35S promoter and found that MMV, CVMV, and PCISV were as effective as the 35S promoter. Furthermore, we applied the CRISPR/Cas9-mediated genome editing system successfully in D. officinale. By selecting five target genes (C3H, C4H, 4CL, CCR and IRX) in the lignocellulose biosynthesis pathway, we showed that, for a given target, this system can generate edits (insertions, deletions, or substitutions) at a rate of 10% to 100%. These results showed that our two genetic manipulation tools can efficiently express exogenous genes and edit endogenous genes in D. officinale. These efficient research tools will not only help create novel D. officinale varieties, but also facilitate the research process of its modern biological research.