CSIR-Central Institute of Medicinal and Aromatic Plants, 226022, Lucknow, India.
Mol Biol Rep. 2022 Jun;49(6):4555-4563. doi: 10.1007/s11033-022-07300-9. Epub 2022 May 8.
The flux of isoprenoids and the total accumulation of triterpenoid saponins known as centellosides in C. asiatica are controlled by the key genes of the Mevalonate pathway (MVA). These genes were reported to have positive regulation of the pathway in providing isoprenoid moieties. Though, some information is available on the pathway and secondary metabolites. However, most of the pathway steps are not characterized functionally.
For the study, full-length pathway gene Hydroxymethyl glutaryl-CoA-synthase (CaHMGS; GenBank accession number: MZ997833), was isolated from previously annotated transcriptome data of Centella asiatica leaves. HMGS has been successfully cloned and heterologously expressed in bacteria E. coli strain DH5α. The cloned gene has been sequenced and further characterized through in silico studies by different bioinformatics tools. Also, the gene sequences have been submitted in NCBI. In silico studies of isolated gene sequence revealed the nature, characteristics of genes. The ORF of HMGS is 1449 bp encoding 482 amino acids. Predicted molecular weight (MW) of HMGS was 48.09 kDa and theoretical pI was 5.97. Blast results and Multiple sequence alignments of the gene showing the similarity with HMGS of other plants of their respective families. The Molecular Evolutionary Genetic Analysis (MEGA) version 10.1.6 was used to construct a phylogenetic tree. Differential tissue-specific expression of different plant parts was also checked. Tissue expression patterns unveiled that the highest expression level of the CaHMGS had been seen in the roots and lowest in the node of the plant. Functional complementation experiment of the CaHMGS in Saccharomyces cerevisiae wild strain YSC1021 and haploid strain YSC1021 which lack HMGS protein confirmed that the CaHMGS gene encodes functional CaHMGS that catalyzed the biosynthesis of mevalonate in yeast.
The gene was reported, cloned and characterized first time in Centella asiatica. Understanding this biosynthetic pathway gene will further help in the improvement of plants for enhanced secondary metabolites production.
在积雪草中，异戊烯类化合物的通量和被称为积雪草酸的三萜皂苷的总积累受甲羟戊酸途径（MVA）的关键基因控制。这些基因被报道在提供异戊烯部分方面对途径有正向调控作用。尽管如此，关于该途径和次生代谢物已有一些信息。然而，大多数途径步骤尚未进行功能表征。
在这项研究中，从先前注释的积雪草叶片转录组数据中分离全长途径基因羟甲基戊二酰辅酶 A 合酶（CaHMGS；GenBank 登录号：MZ997833）。HMGS 已在细菌大肠杆菌 DH5α 中成功克隆和异源表达。克隆的基因已测序，并通过不同的生物信息学工具进行了计算机分析进一步表征。此外，基因序列已提交给 NCBI。对分离基因序列的计算机分析揭示了基因的性质和特征。HMGS 的 ORF 为 1449bp，编码 482 个氨基酸。HMGS 的预测分子量（MW）为 48.09kDa，理论等电点（pI）为 5.97。基因的 Blast 结果和多重序列比对显示与其他植物科的 HMGS 具有相似性。使用 Molecular Evolutionary Genetic Analysis（MEGA）版本 10.1.6 构建了系统发育树。还检查了不同植物部位的差异组织特异性表达。组织表达模式揭示了 CaHMGS 在根中的表达水平最高，在植物节点中的表达水平最低。在缺乏 HMGS 蛋白的酿酒酵母野生型 YSC1021 和单倍体菌株 YSC1021 中对 CaHMGS 的功能互补实验证实，CaHMGS 基因编码的 CaHMGS 具有催化酵母中甲羟戊酸生物合成的功能。
本研究首次在积雪草中报道、克隆和表征了该基因。了解该生物合成途径基因将有助于进一步提高植物的次生代谢产物产量。
