单细胞拉曼光谱揭示聚球藻PCC7942对无机碳源的利用偏好性

doi:10.13560/j.cnki.biotech.bull.1985.2025-1469

摘要/Abstract

摘要：

目的无机碳（Ci）供应形式是调控蓝细菌光合效率的关键环境因子，目前在CO₂与 $H C O 3 -$ 共存条件下，蓝细菌在单细胞尺度的无机碳利用偏好性及其分子调控机制尚不明确。以模式聚球藻PCC7942为对象，旨在单细胞水平揭示其对不同无机碳源的利用偏好。方法整合¹³C稳定同位素探针、单细胞拉曼光谱（Raman-SIP）与转录组测序技术，设置¹²C-NaHCO₃、¹³C-NaHCO₃、空气、¹²C-CO₂、¹²C-CO₂＋¹³C-NaHCO₃和¹³C-CO₂＋¹²C-NaHCO₃共6组碳源处理，以BG11为培养基，在固定光照与温度条件下培养；通过单细胞拉曼光谱检测类胡萝卜素v1峰位移，定量表征¹³C同化与碳源利用特征；选取单一碳源（¹²C-NaHCO₃、¹²C-CO₂）样本开展转录组测序，经数据质控、基因组比对、差异表达基因筛选及GO/KEGG富集分析，解析碳代谢通路调控机制。结果在CO₂与 $H C O 3 -$ 共存体系中，该菌株显著优先利用CO₂；此偏好性由拉曼光谱中类胡萝卜素v1峰的动态红移定量证实。同时，RNA-seq结果显示，CO₂处理组中共有2 750个注释差异表达基因（DEGs），其中1 083个为显著差异表达基因。卡尔文‒本森循环（Calvin-Benson cycle）多数编码酶基因的转录水平下降，而rbcLS基因表达情况则相反。结论结合单细胞拉曼与转录组技术，证实聚球藻PCC7942优先利用CO₂，并阐明了其转录调控机制，在单细胞水平构建了“碳源选择-光谱表征-转录调控”的系统关联。

关键词: 聚球藻PCC7942, 单细胞拉曼光谱, 无机碳利用偏好性, 同位素探针, 转录组测序, 卡尔文?本森循环, 类胡萝卜素, 红移

Abstract:

Objective The supply form of inorganic carbon (Ci) is a critical environmental factor regulating photosynthetic efficiency in cyanobacteria. However, the inorganic carbon utilization preference of cyanobacteria at the single-cell level and the corresponding molecular regulatory mechanism remain unclear under the coexistence of CO₂ and $H C O 3 -$ . This study used the model strain Synechococcus sp. PCC7942 to reveal its utilization preference for different inorganic carbon sources at the single-cell level. Method This study integrated ¹³C stable isotope probing, single-cell Raman spectroscopy (Raman-SIP), and transcriptome sequencing. Six carbon source treatments were set, including ambient ¹²C-NaHCO₃, ¹³C-NaHCO₃, air, ¹²C-CO₂, ¹²C-CO₂ + ¹³C-NaHCO₃, and ¹³C-CO₂ + ¹²C-NaHCO₃. Cells were cultured in BG11 medium under controlled light and temperature. The ¹³C assimilation and carbon source utilization characteristics were quantitatively characterized by detecting the shift of the carotenoid v1 peak using single-cell Raman spectroscopy. Transcriptome sequencing was performed on samples grown on single carbon sources (¹²C-NaHCO₃ and ¹²C-CO₂). The regulatory mechanism of carbon metabolism pathways was dissected through data quality control, genome alignment, screening of differentially expressed genes, and GO/KEGG enrichment analysis. Result In the CO₂/ $H C O 3 -$ coexistence system, the strain predominantly utilizes CO₂—an observation quantitatively confirmed by dynamic redshifts in the Raman carotenoid v1 peak. RNA-seq analysis revealed 2 750 differentially expressed genes (DEGs) with annotations in the CO₂-treated group, among which 1 083 were significantly differentially expressed. Most genes encoding enzymes of the Calvin-Benson cycle showed downregulated transcription, whereas rbcLS expression was upregulated. Conclusion This study integrates single-cell Raman spectroscopy and transcriptomics to demonstrate that Synechococcus sp. PCC7942 preferentially utilizes CO₂ and clarify its transcriptional regulatory mechanism. This work establishes a systematic linkage among carbon source preference, Raman spectral characterization, and transcriptional regulation at the single-cell level.

Key words: Synechococcus elongatus PCC7942, single-cell Raman spectroscopy, inorganic carbon utilization preference, isotope probing, transcriptome, Calvin-Benson cycle, carotenoid, Raman shift

刘佳, 孟宇, 荆晓艳. 单细胞拉曼光谱揭示聚球藻PCC7942对无机碳源的利用偏好性[J]. 生物技术通报, 2026, 42(8): 331-340.

LIU Jia, MENG Yu, JING Xiao-yan. Single-cell Raman Spectroscopy Reveals the Inorganic Carbon Source Utilization Preference of Synechocystis sp. PCC7942[J]. Biotechnology Bulletin, 2026, 42(8): 331-340.

图/表 6

表1 培养基BG11的组分

Table 1 Composition of BG11 Medium

序号 Number	BG11培养基 BG11 medium			A₅（微量金属溶液）A₅ trace metal solution
序号 Number	成分 Component	含量 Content （mL/L）	母液质量浓度 Concentration （g/L）	成分 Component	母液质量浓度 Concentration （g/L）
1	NaNO₃	10	150.0	H₃BO₃	2.86
2	K₂HPO₄	10	4.0	MnCl₂·4H₂O	1.86
3	MgSO₄·7H₂O	10	7.5	ZnSO₄·7H₂O	0.22
4	CaCl₂·2H₂O	10	3.6	Na₂MoO₄·2H₂O	0.39
5	柠檬酸	10	0.6	CuSO₄·5H₂O	0.08
6	柠檬酸铁铵	10	0.6	Co（NO₃）₂·6H₂O	0.05
7	EDTANa₂	10	0.1
8	Na₂CO₃	10	2.0
9	A₅	1

图1 聚球藻PCC7942在添加不同碳源的液体BG11培养基中的生长曲线A：不含CO2碳源培养装置；B：含CO2碳源培养装置；C：不同碳源下菌株生长曲线

Fig. 1 Growth curve of Synechococcus elongatus PCC7942 in liquid BG11 media supplied with different carbon sourcesA: Incubation device without CO2 as carbon source. B: Incubation device with CO2 as carbon source. C: Growth curve of Synechococcus elongatus PCC7942 in liquid BG11 media supplied with different carbon sources

图2 聚球藻PCC7942在不同碳源条件下的类胡萝卜素拉曼峰动态红移（平均拉曼光谱）A-E：聚球藻PCC7942分别以13C-NaHCO3（A）、13C-CO2+5 mmol/L 12C-NaHCO3（B）、12C-CO2（C）、12C-NaHCO3（D）、12C-CO2+5 mmol/L 13C-NaHCO3（E）为碳源的类胡萝卜素拉曼峰动态红移；F-G：聚球藻PCC7942分别以13C-NaHCO3（F）、13C-CO2+5 mmol/L 12C-NaHCO3（G）为碳源的类胡萝卜素v1峰位的动态红移

Fig. 2 Dynamic red shift of S. elongatus PCC7942 (mean Raman spectra) supplied with different carbon sourcesA-E: Dynamic red shift of S. elongatus PCC7942 (mean Raman spectra) supplied with 13C-NaHCO3 (A)、13C-CO2 plus 5 mmol/L 12C-NaHCO3 (B)、12C-CO2 (C)、12C-NaHCO3 (D)、12C-CO2 plus 5 mmol/L 13C-NaHCO3 (E) as the carbon source, respectively. F-G: Dynamic red shift of S. elongatus PCC7942 (v1) supplied with 13C-NaHCO3 (F) and 13C-CO2 plus 5 mmol/L 12C-NaHCO3 (G) as the carbon source, respectively

图3 蓝细菌CO2浓缩机制（CCM）通用模型及其光合作用途径中蛋白质动态变化

Fig. 3 Generalized model for the cyanobacterial CO2-concentrating mechanism (CCM) and protein dynamic changes in its photosynthesis pathway

图4 KEGG功能富集分析

Fig. 4 KEGG functional enrichment analysis

图5 卡尔文循环途径、糖酵解途径和柠檬酸循环途径中蛋白质的动态变化A：卡尔文循环途径中蛋白质的动态变化；B：糖酵解途径和柠檬酸循环途径中蛋白质的动态变化

Fig. 5 Protein dynamics in the Calvin cycle pathway, glycolysis pathway and citrate cycle pathwayA: Protein dynamics in the Calvin cycle pathway. B: Protein dynamics in the glycolysis pathway and the citrate cycle pathway

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