基于转录组分析的腾冲嗜热厌氧杆菌thiD的热适应机制研究

RNA-seq reveals the role of thiD in the thermal adaptation of Thermoanaerobacter tengcongensis

摘要:

ThiD在腾冲嗜热厌氧杆菌中由thiD编码，是硫铵素合成途径的关键酶。thiD的结构和功能已在真菌、酵母菌和植物中得到阐明。然而，thiD在嗜热生物中的功能仍不清楚。【目的】探讨腾冲嗜热厌氧杆菌的嗜热机制并揭示thiD在不同温度下的功能及在热适应调控中的作用。【方法】利用同源重组技术构建腾冲嗜热厌氧杆菌的ΔthiD株，观察并比较野生株和ΔthiD株在50、60、75和80 ℃下的生长趋势。通过转录组测序分析ΔthiD株与野生株在75 ℃的差异表达基因。通过实时荧光定量PCR分析比较野生株和ΔthiD株中13个基因和3个sRNAs在50、60、75、80 ℃下的转录水平。【结果】成功构建了ΔthiD株，生长曲线结果显示其在50 ℃时，ΔthiD株生长速率与野生株无显著差异。然而，在60 ℃和75 ℃时，ΔthiD株生长速度明显慢于野生株。另外，ΔthiD株在80 ℃时几乎不生长。转录组结果显示，与野生株相比，ΔthiD株有503个差异表达基因，其中包括278个上调差异表达基因和213个下调差异表达基因。KEGG分析表明，以下途径与热适应有关，包括硫胺素代谢、嘧啶代谢和嘌呤代谢、肽聚糖生物合成、脂肪酸代谢途径、氨基酸代谢途径、双组分系统、DNA复制、同源重组、错配修复、磷酸转移酶系统。通过实时荧光定量PCR分析发现，在野生株和ΔthiD株中与嗜热机制相关的13个基因和3个sRNAs在特定温度下的转录水平发生了变化。【结论】thiD在腾冲嗜热厌氧杆菌热适应过程中发挥重要作用。本研究为揭示thiD在不同温度下的功能和在热适应过程中的调控机制提供实验数据和理论依据。

Abstract:

ThiD encoded by thiD in Thermoanaerobacter tengcongensis is a key enzyme in the biosynthesis of thiamine. The structure and functions of thiD have been elucidated in fungi, yeasts, and plants, while the role of thiD in thermophiles remains unclear. [Objective] This study aims to explore the thermal adaptation mechanism of T. tengcongensis and reveal the role of thiD in the thermal adaptation of T. tengcongensis at different temperatures. [Methods] The thiD-deleted mutant (ΔthiD) of T. tengcongensis was constructed by homologous recombination. The growth trends of the wild type (WT) and ΔthiD at 50 ℃, 60 ℃, 75 ℃, and 80 ℃ were observed and compared. The differentially expressed genes (DEGs) between ΔthiD and WT cultured at 75 ℃ were determined by RNA-seq. The transcript levels of 13 genes and 3 sRNAs in WT and ΔthiD at 50 ℃, 60 ℃, 75 ℃, and 80 ℃ were compared and analyzed by real-time PCR. [Results] ΔthiD was successfully constructed, with the growth rate not significantly different from WT at 50 ℃. However, ΔthiD show cased slower growth than WT at 60 ℃ and 75 ℃ and did not grow at 80 ℃. The transcriptome results revealed 503 DEGs in ΔthiD compared with WT, including 278 DEGs with up regulated expression and 213 DEGs with down regulated expression. The Kyoto encyclopedia of genes and genomes (KEGG) analysis indicated the following pathways associated with thermophilic adaptation, involving thiamine metabolism, pyrimidine metabolism and purine metabolism, peptidoglycan biosynthesis, fatty acid metabolism, amino acid metabolism, two-component system, DNA replication, homologous recombination, mismatch repair, and phosphotransferase system. The transcript levels of 13 genes and 3 sRNAs related to thermal adaptation in WT and ΔthiD changed at specific temperatures. [Conclusion] thiD plays an important role in the thermal adaptation of T. tengcongensis. This study provides experimental data and a theoretical basis for revealing the role of thiD in the thermal adaptation of thermophiles at different temperatures.