目的 bHLH转录因子家族在植物生长发育、环境胁迫以及次生代谢调控等过程中发挥重要作用,初步筛选金钱草Lysimachia christinae与黄酮类物质合成相关的bHLH基因,为后续深入研究金钱草黄酮类物质合成代谢提供基础。方法 基于PacBio平台开展金钱草全长转录组测序分析,系统筛选鉴定金钱草LcbHLHs基因家族成员,对其蛋白序列特征、跨膜结构域、信号肽、亚细胞定位、系统进化等进行预测分析,并检测部分LcbHLHs基因在不同器官和不同透光率下的表达模式,利用烟草瞬时表达系统验证LcbHLH26和LcbHLH27的功能。结果 金钱草全长转录组测序共获得37.29 Gb数据量,去冗余后得到15 045条转录本序列,平均长度为2 288 bp,N₅₀长度为2 463 bp。基于金钱草全长转录组数据共筛选得到45个LcbHLHs基因家族成员,氨基酸数目为214～777 aa,相对分子质量为22 634.57～85 044.11,等电点4.58～9.36,均为亲水蛋白,均定位在细胞核上。与拟南芥AtbHLHs蛋白序列进行聚类分析发现,金钱草LcbHLHs家族成员聚在14个亚族上。基于金钱草不同器官及不同透光率处理的RNA-Seq转录组数据,分别检测到23和4个差异表达的LcbHLHs基因,qRT-PCR验证部分LcbHLHs基因的差异表达与转录组数据结果基本一致。过表达金钱草LcbHLH26、LcbHLH27可以显著提高烟草叶片总黄酮含量。结论 获得金钱草全长转录组信息,鉴定分析了金钱草LcbHLHs基因家族,为深入研究bHLH基因在金钱草黄酮类物质合成调控中的功能奠定基础。

Objective The bHLH transcription factor family plays a crucial role in plant growth and development, environmental stress responses, and secondary metabolite regulation. The research preliminarily screens LcbHLHs genes related to flavonoid synthesis establishes a foundation for further exploration into the metabolic synthesis of flavonoids in Lysimachia christinae. Methods In this study, full-length transcriptome sequencing was performed based on the PacBio platform. Bioinformatic tools were employed to identify and analyze the protein sequence characteristics, physicochemical properties, transmembrane domains, signal peptides, subcellular localization and phylogenetic relationship of the L. christinae LcbHLHs gene family members. The expression patterns of certain LcbHLHs genes in different tissues and under different light transmittance were examined. Additionally, overexpression vectors of LcbHLH26 and LcbHLH27 were constructed for transient expression in tobacco to assess their functionality. Results A total of 37.29 Gb data were obtained from the full-length transcriptome sequencing of L. christinae. After removing redundancy, 15 045 transcript sequences were obtained, with an average length of 2 288 bp and an N₅₀ length of 2 463 bp. Based on the full-length transcriptome sequencing data, 45 members of the LcbHLHs transcription factor gene family were identified, with the protein size of 214 to 777 aa, the relative molecular weights of 22 634.57 to 85 044.11, and isoelectric points of 4.58 to 9.36. All proteins belonged to hydrophilic proteins and located in the nucleus. Cluster analysis with the Arabidopsis AtbHLH proteins revealed that the L. christinae LcbHLHs members grouped into 14 subfamilies. RNA-Seq transcriptome data from samples of different tissues, and samples under different light transmittance identified 23 and four differentially expressed LcbHLH genes, respectively. qRT-PCR validation confirmed that for most LcbHLH genes, the change in expression was consistent with that of RNA-seq data. Transient overexpression of LcbHLH26 and LcbHLH27 significantly increased the total flavonoid content in tobacco leaves. Conclusion The systematic identification and analysis of the bHLH family provide a foundation for further exploring functions and possible regulatory mechanisms of LcbHLH members in flavonoid synthesis in L. christinae.

R286.12

国家重点研发计划项目（2021YFD1601000）；重庆市基本科研业务经费（2023jbky-018）；重庆市教委科学技术研究计划项目（KJQN202215114，KJQN202315114）；重庆英才计划创新创业团队（CQYC202203091179）