Next Generation Sequencing and Transcriptome Analysis Predicts Biosynthetic Pathway of Sennosides from Senna (Cassia angustifolia Vahl.), a Non-Model Plant with Potent Laxative Properties

Senna (Cassia angustifolia Vahl.) is a world’s natural laxative medicinal plant. Laxative properties are due to sennosides (anthraquinone glycosides) natural products. However, little genetic information is available for this species, especially concerning the biosynthetic pathways of sennosides. We present here the transcriptome sequencing of young and mature leaf tissue of Cassia angustifolia using Illumina MiSeq platform that resulted in a total of 6.34 Gb of raw nucleotide sequence. The sequence assembly resulted in 42230 and 37174 transcripts with an average length of 1119 bp and 1467 bp for young and mature leaf, respectively. The transcripts were annotated using NCBI BLAST with ‘green plant database (txid 33090)’, Swiss Prot, Kyoto Encylcopedia of Genes & Genomes (KEGG), Cluster of Orthologous Gene (COG) and Gene Ontology (GO). Out of the total transcripts, 40138 (95.0%) and 36349 (97.7%) from young and mature leaf, respectively, were annotated by BLASTX against green plant database of NCBI. We used InterProscan to see protein similarity at domain level, a total of 34031 (young leaf) and 32077 (mature leaf) transcripts were annotated against the Pfam domains. All transcripts from young and mature leaf were assigned to 191 KEGG pathways. There were 166 and 159 CDS, respectively, from young and mature leaf involved in metabolism of terpenoids and polyketides. Many CDS encoding enzymes leading to biosynthesis of sennosides were identified. A total of 10,763 CDS differentially expressing in both young and mature leaf libraries of which 2,343 (21.7%) CDS were up-regulated in young compared to mature leaf. Several differentially expressed genes found functionally associated with sennoside biosynthesis. CDS encoding for many CYPs and TF families were identified having probable roles in metabolism of primary as well as secondary metabolites. We developed SSR markers for molecular breeding of senna. We have identified a set of putative genes involved in various secondary metabolite pathways, especially those related to the synthesis of sennosides which will serve as an important platform for public information about gene expression, genomics, and functional genomics in senna.狭叶番泻（Senna, Cassia angustifolia Vahl.）是全球广泛应用的天然泻下药用植物，其泻下活性成分为番泻苷（sennosides，蒽醌苷类天然产物）。然而目前该物种的遗传信息仍较为匮乏，尤其是关于番泻苷生物合成通路的相关数据极少。 本研究依托Illumina MiSeq测序平台，对狭叶番泻的幼叶与成熟叶组织开展转录组测序，共获得6.34 Gb的原始核苷酸序列。经序列组装，幼叶样本得到42230条转录本，平均长度为1119 bp；成熟叶样本得到37174条转录本，平均长度为1467 bp。 本研究采用NCBI BLAST工具，结合绿色植物数据库（txid 33090）、Swiss-Prot数据库、京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）、直系同源基因簇（Cluster of Orthologous Genes, COG）以及基因本体论（Gene Ontology, GO）对转录本进行功能注释。结果显示，幼叶样本中95.0%（40138条）的转录本可通过BLASTX比对至NCBI绿色植物数据库，成熟叶样本中该比例达97.7%（36349条）。此外，本研究利用InterProscan工具在蛋白结构域水平开展相似性分析，幼叶样本中共34031条转录本可注释至Pfam结构域，成熟叶样本中该数量为32077条。 所有转录本均可被归类到191条KEGG通路中，其中幼叶与成熟叶样本分别有166条和159条编码序列（Coding Sequence, CDS）参与萜类化合物与聚酮类化合物的代谢过程。本研究鉴定出大量参与番泻苷生物合成的酶编码CDS。 对幼叶与成熟叶的转录组文库进行差异表达分析，共得到10763个在两类组织中均存在差异表达的CDS，其中2343个（占比21.7%）CDS在幼叶中呈上调表达。研究还发现多个与番泻苷生物合成功能相关的差异表达基因。 此外，本研究鉴定出大量编码细胞色素P450（Cytochrome P450, CYPs）家族蛋白及转录因子（Transcription Factor, TF）的CDS，推测其在初生代谢与次生代谢过程中发挥潜在调控作用。本研究开发了可用于狭叶番泻分子育种的简单序列重复（Simple Sequence Repeat, SSR）分子标记。 本研究鉴定出一系列参与各类次生代谢通路的候选基因，尤其是与番泻苷合成相关的基因，可为后续狭叶番泻的基因表达、基因组学及功能基因组学研究提供重要的公共信息支撑平台。