目的 bHLH轉(zhuǎn)錄因子家族在植物生長發(fā)育、環(huán)境脅迫以及次生代謝調(diào)控等過程中發(fā)揮重要作用,初步篩選金錢草Lysimachia christinae與黃酮類物質(zhì)合成相關(guān)的bHLH基因,為后續(xù)深入研究金錢草黃酮類物質(zhì)合成代謝提供基礎(chǔ)。方法 基于PacBio平臺開展金錢草全長轉(zhuǎn)錄組測序分析,系統(tǒng)篩選鑒定金錢草LcbHLHs基因家族成員,對其蛋白序列特征、跨膜結(jié)構(gòu)域、信號肽、亞細(xì)胞定位、系統(tǒng)進(jìn)化等進(jìn)行預(yù)測分析,并檢測部分LcbHLHs基因在不同器官和不同透光率下的表達(dá)模式,利用煙草瞬時表達(dá)系統(tǒng)驗證LcbHLH26和LcbHLH27的功能。結(jié)果 金錢草全長轉(zhuǎn)錄組測序共獲得37.29 Gb數(shù)據(jù)量,去冗余后得到15 045條轉(zhuǎn)錄本序列,平均長度為2 288 bp,N₅₀長度為2 463 bp。基于金錢草全長轉(zhuǎn)錄組數(shù)據(jù)共篩選得到45個LcbHLHs基因家族成員,氨基酸數(shù)目為214～777 aa,相對分子質(zhì)量為22 634.57～85 044.11,等電點4.58～9.36,均為親水蛋白,均定位在細(xì)胞核上。與擬南芥AtbHLHs蛋白序列進(jìn)行聚類分析發(fā)現(xiàn),金錢草LcbHLHs家族成員聚在14個亞族上。基于金錢草不同器官及不同透光率處理的RNA-Seq轉(zhuǎn)錄組數(shù)據(jù),分別檢測到23和4個差異表達(dá)的LcbHLHs基因,qRT-PCR驗證部分LcbHLHs基因的差異表達(dá)與轉(zhuǎn)錄組數(shù)據(jù)結(jié)果基本一致。過表達(dá)金錢草LcbHLH26、LcbHLH27可以顯著提高煙草葉片總黃酮含量。結(jié)論 獲得金錢草全長轉(zhuǎn)錄組信息,鑒定分析了金錢草LcbHLHs基因家族,為深入研究bHLH基因在金錢草黃酮類物質(zhì)合成調(diào)控中的功能奠定基礎(chǔ)。

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

國家重點研發(fā)計劃項目（2021YFD1601000）；重慶市基本科研業(yè)務(wù)經(jīng)費（2023jbky-018）；重慶市教委科學(xué)技術(shù)研究計劃項目（KJQN202215114，KJQN202315114）；重慶英才計劃創(chuàng)新創(chuàng)業(yè)團隊（CQYC202203091179）