刺梨全长转录组测序分析及黄酮类化合物生物合成相关基因挖掘

Full-length transcriptome sequencing analysis and identification of putative genes for biosynthesis of flavonoid in Rosa roxburghii

关键词:  刺梨, 单分子实时测序技术, 全长转录组, 生物信息学分析

Key words:  Rosa roxburghii, single-molecule real-time sequencing technology, full-length transcriptome, bioinformatics analysis

摘要:

刺梨是一种药食两用植物,在炎症和癌症研究方面显示出巨大的潜力。为进一步推动刺梨的研究和应用,建立一个完整的刺梨全长转录组数据库非常必要,这有助于揭示其复杂的分子机制和生物活性物质的合成途径。为深入探究刺梨功能基因信息,选择三代建库方法,采用PacBio Seque Ⅲ测序平台,利用单分子实时测序技术(single molecule real-time, SMRT)对刺梨6种组织混合样品进行全长转录组测序,使用生物信息学手段对其进行分析。结果表明:(1)共获得25 003条去冗余后的isoforms序列,平均长度为2 471 bp。成功预测到24 357个CDS序列,平均长度为1 727 bp,其中大部分CDS长度为300～3 000 bp。(2)使用GO、KEGG等7个数据库进行比对,共注释24 859个基因,占比达99.42%; 挖掘出黄酮类化合物生物合成相关转录本99个。(3)鉴定出1 930个基因分别属于82种转录因子家族,并且有55个转录因子可能参与调控刺梨黄酮类化合物的生物合成。(4)共95个LncRNA,SSR位点12 588个,进一步利用Primer 3软件进行刺梨SSR的引物设计,共筛得10 545对SSR引物。该研究结果丰富了刺梨基因数据库,为下一步分子标记开发、生长发育、抗逆、次级代谢产物的生物合成及遗传改良和育种提供了理论基础。

Abstract:

Rosa roxburghii, a medicinal and edible plant native to Southwest China, is renowned for its rich bioactive compounds, including flavonoids, vitamin C, and polysaccharides, which exhibit significant anti-inflammatory, anticancer, and antioxidant properties. To advance the genetic research and application of R. roxburghii, this study aimed to construct a comprehensive full-length transcriptome database and identify key genes involved in flavonoid biosynthesis. Using PacBio Sequel II single-molecule real-time(SMRT)sequencing, mixed samples from six tissues(flower, leaf, stem, young bark, mature bark, and fruit)were analyzed. Bioinformatics tools were employed for transcriptome assembly, functional annotation, and structural characterization. The results were as follows:(1)A total of 25 003 non-redundant isoforms were obtained, with an average length of 2 471 bp. Among these, 24 357 coding sequences(CDS)were predicted, averaging 1 727 bp, with the majority ranging between 300 - 3 000 bp.(2)Functional annotation using seven databases(GO, KEGG, Nr, Swiss-Prot, TrEMBL, KOG, and Pfam)revealed that 24 859 isoforms(99.42%)were annotated. Notably, 99 transcripts were linked to flavonoid biosynthesis pathways, including phenylalanine ammonia-lyase(PAL), chalcone synthase(CHS), and flavonol synthase(FLS).(3)A total of 1 930 transcription factors(TFs)from 82 families were identified, with 55 TFs(e.g., WRKY, MYB, and bHLH)potentially regulating flavonoid biosynthesis.(4)Structural analysis predicted 95 long non-coding RNAs(LncRNAs)and 12 588 simple sequence repeats(SSRs), from which 10 545 SSR primer pairs were designed. The research findings enrich the genetic database of R. roxburghii, providing a theoretical foundation for the next steps in molecular marker development, growth and development, stress resistance, biosynthesis of secondary metabolites, as well as genetic improvement and breeding.