基于转录组测序与生物信息学综合分析短葶山麦冬中调控甾体皂苷积累的分子机制

Uncovering the molecular mechanisms behind steroidal saponin accumulation in Liriope muscari (Decne.) Baily through transcriptome sequencing and bioinformatics analysis

摘要:

本研究旨在以短葶山麦冬的叶与根为研究对象进行高通量Illumina转录组测序，并结合生物信息学分析参与调控短葶山麦冬主要活性成分甾体皂苷类化合物积累的相关酶基因及关键转录因子，为研究短葶山麦冬甾体皂苷积累的分子机制提供理论基础。通过转录组测序，鉴定到AACT、CAS、DXS与DXR等31个参与甾体皂苷类化合物合成的代谢酶，其中萜类化合物骨架合成阶段有16个，倍半萜和三萜生物合成阶段有3个，甾体化合物合成阶段有12个。基因差异表达显示，在叶与根之间具有34条差异表达基因(differentially expressed genes, DEGs)，分别编码甾体皂苷合成有关的15个代谢酶，通过基因共表达模式进一步分析，编码其中14个代谢酶的31条DEGs呈现共表达模式。此外，还通过共表达分析及PlantTFDB的转录因子分析工具预测到8个转录因子GAI、PIF4、PIL6、ERF8、SVP、LHCA4、NF-YB3和DOF2.4可能参与调节6个代谢酶DXS、DXR、HMGR、DHCR7、DHCR24和CAS。通过启动子分析预测这些转录因子表明，其主要调控机制涉及脱落酸响应、干旱诱导胁迫响应及光响应等过程，特别是脱落酸响应元件(abscisic acid responsive elements, ABRE)与干旱诱导胁迫响应元件(MYB binding site involved in drought-inducibility, MBS)响应途径；并通过qRT-PCR分析这8个关键转录因子和代谢酶基因，结果表明它们在叶与根中差异表达与测序结果一致。

Abstract:

The leaves and roots of Liriope muscari (Decne.) Baily were subjected to high-throughput Illumina transcriptome sequencing. Bioinformatics analysis was used to investigate the enzyme genes and key transcription factors involved in regulating the accumulation of steroidal saponins, which are the main active ingredient in L. muscari. These analyses aimed to reveal the molecular mechanism behind steroidal saponin accumulation. The sequencing results of L. muscari revealed 31 enzymes, including AACT, CAS, DXS and DXR, that are involved in the synthesis of steroidal saponins. Among these enzymes, 16 were in the synthesis of terpenoid skeleton, 3 were involved in the synthesis of sesquiterpene and triterpene, and 12 were involved in the synthesis of steroidal compound. Differential gene expression identified 15 metabolic enzymes coded by 34 differentially expressed genes (DEGs) in the leaves and roots, which were associated with steroidal saponin synthesis. Further analysis using gene co-expression patterns showed that 14 metabolic enzymes coded by 31 DEGs were co-expressed. In addition, analysis using gene co-expression analysis and PlantTFDB’s transcription factor analysis tool predicted the involvement of 8 transcription factors, including GAI, PIF4, PIL6, ERF8, SVP, LHCA4, NF-YB3 and DOF2.4, in regulating 6 metabolic enzymes such as DXS, DXR, HMGR, DHCR7, DHCR24, and CAS. These eight transcription factors were predicted to play important roles in regulating steroidal saponin accumulation in L. muscari. Promoter analysis of these transcription factors indicated that their main regulatory mechanisms involve processes such as abscisic acid response, drought-induction stress response and light response, especially abscisic acid responsive elements (ABRE) response and MYB binding site involved in drought-inducibility (MBS) response pathway. Furthermore, qRT-PCR analysis of these eight key transcription factors demonstrated their specific differences in the leaves and roots.