Kalita Ratna, Patar Lochana, Shasany Ajit Kumar, Modi Mahendra K, Sen Priyabrata
Department of Agricultural Biotechnology, Assam Agricultural University, Jorhat, 785013, India.
Mol Biol Rep. 2015 Sep;42(9):1431-9. doi: 10.1007/s11033-015-3922-6. Epub 2015 Aug 27.
3-Hydroxy-3-methylglutaryl-CoA reductases (HMGR) plays an important role in catalyzing the first committed step of isoprenoid biosynthesis in the mevelonic (MVA) pathway (catalyzes the conversion of HMG-CoA to MVA) in plants. The present manuscript reports the full length cDNA cloning of HMGR (CaHMGR, GenBank accession number: KJ939450.2) and its characterization from Centella asiatica. Sequence analysis indicated that the cDNA was of 1965 bp, which had an open reading frame of 1617 bp and encoded a protein containing 539 amino-acids with a mol wt of 57.9 kDa. A BLASTp search against non-redundant (nr) protein sequence showed that C. asiatica HMGR (CaHMGR) has 65-81% identity with HMGRs from different plant species and multi-alignment comparison analysis showed the presence of two motif each corresponding to HMG-CoA-binding and NADP(H)-binding. The Conserved Domain Database analysis predicted that CaHMGR belongs to Class I hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase. Three-dimensional modeling confirmed the novelty of CaHMGR with a spatial structure similar to Homo sapiens (PDB id: 1IDQ8_A). Tissue Expression analysis indicates that CaHMGR is ubiquitous albeit differentially expressed among different tissues analysed, Strong expression was recorded in the nodes and leaves and low in the roots. The present investigation confirmed that nodes are vital to terpenoid synthesis in C. asiatica. Thus, the cloning of full length CDS, characterization and structure-function analysis of HMGR gene in Centella facilitate to understand the HMGR's functions and regulatory mechanisms involved in mevalonate pathway in C. asiatica at genetic level.
3-羟基-3-甲基戊二酰辅酶A还原酶（HMGR）在催化植物甲羟戊酸（MVA）途径中类异戊二烯生物合成的首个关键步骤（催化HMG-CoA转化为MVA）中发挥着重要作用。本论文报道了来自积雪草的HMGR（CaHMGR，GenBank登录号：KJ939450.2）全长cDNA克隆及其特性。序列分析表明，该cDNA长度为1965 bp，具有1617 bp的开放阅读框，编码一个含539个氨基酸、分子量为57.9 kDa的蛋白质。对非冗余（nr）蛋白质序列进行的BLASTp搜索显示，积雪草HMGR（CaHMGR）与来自不同植物物种的HMGR具有65%-81%的同一性，多序列比对分析表明存在两个基序，分别对应HMG-CoA结合和NADP(H)结合。保守结构域数据库分析预测CaHMGR属于I类羟甲基戊二酰辅酶A（HMG-CoA）还原酶。三维建模证实了CaHMGR的新颖性，其空间结构与智人相似（PDB编号：1IDQ8_A）。组织表达分析表明，CaHMGR在不同组织中虽有差异表达但普遍存在，在节和叶中表达强烈，在根中表达较低。本研究证实节对积雪草中的萜类合成至关重要。因此，积雪草中HMGR基因全长CDS的克隆、特性分析及结构功能分析有助于从基因水平了解HMGR在积雪草甲羟戊酸途径中的功能及调控机制。
