Orchidaceae belongs to the largest family of flowering plants, with approximately 880 genera worldwide. Among these, more than 120 genera are found naturally in Peninsular Malaysia [
Volatile compounds emitted by fully open flower of
Peak | Relative retention time (min) | Main spectrum fragments (m/z) | Compound name |
---|---|---|---|
Monoterpene | |||
1 | 8.636 | 36,41,53,67,79,93,105,121 | Ocimene |
2 | 9.147 | 41,43,59,81,93,112 | Linalool oxide |
3 | 9.592 | 41,43,69,71,93,107,121,136 | Linalool |
| |||
Sesquiterpene | |||
10 | 16.492 | 41,43,69,71,93,107,123,136,162 | Nerolidol |
| |||
Benzenoid | |||
4 | 9.702 | 51,77,105,136 | Methylbenzoate |
5 | 10.030 | 39,51,65,78,91,105,122 | Benzyl acetate |
| |||
Phenylpropanoid | |||
6 | 10.783 | 39,43,65,79,91,108,150 | Phenylethanol |
8 | 11.926 | 39,43,65,78,91,104 | Phenylethyl acetate |
| |||
Indole | |||
9 | 13.084 | 39,50,63,74,90,117 | Indole |
| |||
Formanilide | |||
7 | 12.260 | 39,52,65,76,93,161 | Formanilide |
Plant volatiles are synthesized from three major biosynthesis pathways including phenylpropanoids, fatty acids derivatives and terpenoids pathways. Mevalonic acid (MVA) and methyl-
Acetyl-CoA-C-acetyltransferase (ACA), classified as Thiolase II, is involved in catalysing the condensation of two units of acetyl-CoA (2C) to acetoacetyl-CoA (4C) in the mevalonate pathway. ACA is a crucial molecule involved in terpene biosynthesis, production of volatile benzenoids, and the biosyntheses of hormones and cholesterols [
Over the years, the numbers of fragrance-related cDNAs that have been isolated and characterized particularly in vandaceous orchids are limited to the reports from our research group, working on this highly fragrant vandaceous orchid,
We reported here a novel finding of an ACA-like gene from our previously constructed
The standard CTAB protocols of Yu and Goh [
From the
The PCR products were gel-purified using QIAquick Gel Extraction Kit (Qiagen, CA, USA) and subsequently cloned into the yT&A vector using the protocol provided in the yT&A Cloning kit (Yeastern Biotech, Taipei, Taiwan). The ligation mixture consisted of 1X ligation buffer A, 1X ligation buffer B, yT&A cloning vector: PCR product (1 : 3), 0.2 unit of YEA T4 DNA ligase and topped up with distilled water to a final volume of 10
In order to get the full length
First strand cDNA of
Endogenous controls and the primers sets used in real-time RT-PCR. Actin, tubulin, and cyclophilin were used as endogenous controls in the real-time RT-PCR analysis.
Primers identity/amplicon size | GenBank accession | Primers sequences |
---|---|---|
Actin (236 bp) | AF246716 | Forward: 5′-CAGTGTTTGGATTGGAGGTTC-3′ |
Reverse: 5′- CCAGCAGCAGTCAGGAAAA-3′ | ||
| ||
Tubulin (227 bp) | GW393564 | Forward: 5′- CTCCCGCATTGACCATAAAT-3′ |
Reverse: 5′-GGAACCACACCCAAACTCTC-3′ | ||
| ||
Cyclophilin (200 bp) | GW393531 | Forward: 5′-TTGGATGTCGTGAAGGCAAT-3′ |
Reverse: 5′-CAACACAAGAAGATAGCACAGCA-3′ |
Full length
Protein sequence comparison using ClustalW and Blastx indicates that VMPACA has 81% protein sequence identity to the ACAs encoded by
Amino acid sequence alignment of VMPACA (putative acetyl-CoA-C-acetyltransferase) and other closely related protein sequences from
There are two types of thiolase based on their catalytic activities: Thiolase I (acetyl-CoA C-acyltransferase) and Thiolase II (acetyl-CoA C-acetyltransferase). Thiolase I is also known as a degradative thiolase while Thiolase II is known as a synthetic thiolase [
A phylogenetic tree was constructed using MEGA 4.0 software to estimate the relationships between the putative VMPACA and other plant ACAs. The phylogenetic tree analysis suggested that VMPACA is highly similar to ACA from
Phylogenetic tree of VMPACA with homologues proteins by using MEGA 4.0. The bootstrap neighbor-joining method was used to construct the tree. The phylogenetic tree analysis of ACA has included
So far, no work corresponding to acetyl-CoA-C-acetyltransferase of vandaceous orchids has been reported. To characterize
Expression profile of putative
Expression of genes encoding scent-biosynthesis enzymes was previously reported to be spatially regulated during flower development as different fragrance-related transcripts were shown to have different tissue-specific expression patterns whereby some were upregulated solely in the reproductive parts and some in the vegetative parts [
Analysis of the expression level of putative
A full length
The authors would like to thank the Ministry of Higher Education Malaysia and UPM for financial support through the Fundamental Research Grant Scheme (02-12-10-1002FR) and Research University Grant Scheme (05-04-08–0551RU), respectively.