关键酶编码基因的可变剪接对丹参酮合成调控机制研究

Alternative splicing (AS) can yield multiple gene isoforms from an individual protein-coding locus that is associated with plant development, signal pathways, diseases, or the responses to biotic or abiotic stress. Although the increased throughput and decreased cost of next-generation sequencing (NGS) has enabled NGS to be the strongest tool for extensively analyzing transcriptome, most gene isoforms cannot be precisely annotated due to the limitation of NGS short reads. SMRT sequencing by Pacific Biosciences (PacBio) RS is a third-generation sequencing (TGS) platform that is widely used in genome sequencing because of its long reads (average 4~8 kb). Furthermore, a previous study has addressed the problem of its higher error rate (up to 15%) by the correction of NGS reads and self-correction of the circular-consensus (CCS) reads. Thus, using SMRT sequencing to analyze transcriptome may more comprehensively evaluate full-length transcripts, alternatively spliced isoforms. ..Salvia miltiorrhiza Bunge is considered as a potential model medicinal plant for traditional Chinese medicine (TCM) research because of its significant medicinal value, relatively small genome (~538 M), short life cycle, efficient transgenic system and uncomplicated tissue culture requirements. The main active constituents of S. miltiorrhiza are lipophilic diterpenoid components and hydrophilic phenolic acids. Elucidating the biosynthetic pathways and regulatory mechanisms of the active constituents will provide the theoretical basis for cultivating this TCM and producing innovative medicine. Recently, an abundance of RNA-seq data on S. miltiorrhiza has been published with the aim of extensively studying the biosynthetic pathway and metabolic regulation. However, the short-reads from NGS made the assembling of the full-length transcripts and accurately identifying the different gene isoforms very challenging and complicated. ..Here, we combined SMRT sequencing and NGS sequencing to execute a single-molecule long-read survey of the S. miltiorrhiza transcriptome to discover the full-length transcripts, alternatively spliced isoforms. Our experiments focused on various root tissues revealed the complexity of the AS event, and characterized 40% of the detected gene loci expressing alternatively spliced isoforms which included key enzyme-coding genes involved in tanshinone biosynthesis. Furthermore, we will examine the AS event in different organs (root, stem, leaf and flower) and in detail investigate differential expression for AS of key enzyme-coding genes. In addition, the functions of AS of key enzyme-coding genes should be characterized and identified by RNA interference and overexpression technologies. Therefore, our study will provide a model for analyzing the full-length transcriptome and biosynthesis of active constituents in other medicinal plants.

蛋白编码基因的可变剪接在植物发育、响应环境胁迫等方面发挥调控作用，该调控方式对中药材活性成分合成的作用鲜有报道。本项目以中药丹参为例，研究丹参酮合成是否受关键酶编码基因的可变剪接调控。课题组已获得丹参基因组图谱，发现丹参酮生物合成相关的17个关键酶编码基因，6个存在可变剪接。据此，本项目利用二代测序精度高和三代测序片段长的优势，获得丹参根的周皮、韧皮部和木质部等不同组织、根茎叶花等不同器官的全长转录组，通过比较丹参基因组序列，研究解析关键酶编码基因的可变剪接种类和数量，并从转录组水平阐明这些可变剪接的组织和器官表达特异性，证实可变剪接对丹参酮合成具有调控作用。再通过RNAi和过量表达技术研究揭示关键酶编码基因的可变剪接对丹参酮合成的调控机制。本项目对丹参道地药材形成的调控机制研究、丹参优良品种选育、丹参酮相关药物资源开发具有指导意义，为研究其它中药材活性成分生物合成的调控机制提供新思路。

蛋白编码基因的可变剪接在植物发育、响应环境胁迫等方面发挥调控作用，该调控方式对中药材活性成分合成的作用鲜有报道。本项目以中药丹参为例，研究丹参酮合成是否受关键酶编码基因的可变剪接调控。利用课题组获得的丹参基因组图谱，发现丹参酮生物合成相关的17个关键酶编码基因，6个存在可变剪接。据此，本项目首次结合PacBio第三代测序平台及Illumina第二代测序平台，在植物领域解析全长转录本，从组学水平分析丹参基因可变剪接现象，并揭示脂溶性丹参酮生物合成途径及其调控相关基因的差异表达及可变剪接变化规律。第三代测序平台具有读长优势，但存在测序质量较低的缺陷。利用二代高质量的短reads对单分子测序低质量的长reads进行校正，然后将二代短reads比对丹参基因组分析丹参转录本的剪接位点，根据剪接位点、校正后高质量的长reads以及丹参基因组数据分析丹参全长转录本及可变剪接信息。综合解剖学和化学分析结果证实，丹参酮生物合成具有组织特异性，其合成与积累的主要部位是丹参根的周皮组织。根据丹参酮的含量差异及根三个组织部位的基因表达差异，阐明丹参酮生物合成途径上游萜类合酶的表达变化规律，并预测15个CYP450s、5个SDRs和1个2OGD基因参与丹参酮下游的生物合成。同时发现丹参根转录本中约40%的基因存在可变剪接，可变剪接类型多样，且不同可变剪接体可能与萜类化合物合成有关。基于丹参基因组筛选2OGD超家族编码基因；通过系统进化分析、差异表达及共表达分析筛选与丹参酮生物合成相关的候选2OGD编码基因，发现13个2OGD编码基因在根周皮、韧皮部和木质部中表达与丹参酮的积累相一致，且2OGD5在丹参根中显著高表达；采用RNAi转基因及代谢组学技术揭示2OGD5抑制的转基因毛状根中丹参新酮、隐丹参酮、丹参酮IIA的含量显著降低。本项目对丹参道地药材形成的调控机制研究、丹参优良品种选育、丹参酮相关药物资源开发具有指导意义，为研究其它中药材活性成分生物合成的调控机制提供新思路。.发表论文10篇，2篇被选为封面文章，其中SCI收录论文8篇，影响因子大于5的论文3篇。2019年，项目负责人入选国家百千万人才工程，被授予“有突出贡献中青年专家”称号。