Saw Hoon Lim

Random amplified polymorphic DNA analysis of the moth orchids, Phalaenopsis (Epidendroideae: Orchidaceae)

AbstractThe genetic distance and relationships of 149 accessions representing 46 species in the genus Phalaenopsis and four species in Paraphalaenopsis were studied using random amplified polymorphic DNA (RAPD) markers. The genus Paraphalaenopsis was used as an outgroup. A total of 20 random primers were screened and out of these, six random primers provided 123 polymorphic bands and zero monomorphic bands. Pairwise genetic distances between accessions were estimated according to Nei and Li (1979). Cluster analysis of data using the UPGMA algorithm placed the species in seven groups that are mostly congruent with those based on morphological characters erected by previous workers. As observed from the banding patterns, Ph. doweryensis, which is suspected to be a hybrid of Ph. gigantea and Ph. kunstleri or Ph. cochlearis, is not. RAPD markers can thus be successfully applied in this economically important group of orchids for the study of relationships and to distinguish taxa up to the specific level.

The isolation, molecular characterization and expression of dihydroflavonol 4-reductase cDNA in the orchid, Bromheadia finlaysoniana

Abstract The cDNA, ODFR, encoding dihydroflavonol 4-reductase (DFR) was isolated from a cDNA library constructed from Bromheadia finlaysoniana flowers using a homologous probe generated via PCR. Southern blot analysis suggests that DFR is represented by a single gene in this orchid while Northern blot and reverse transcriptase-PCR analysis showed that DFR is expressed in all purple colored tissues. A phylogenetic tree constructed based on multiple alignments among DFR sequences showed that the DFR genes isolated from barley, maize and B. finlaysoniana, which are all monocotyledons, are more similar to each other than to DFR sequences from dicotyledonous plants.

Porphobilinogen deaminase is encoded by a single gene in Arabidopsis thaliana and is targeted to the chloroplasts

Porphobilinogen deaminase (PBG deaminase) is an early enzyme of the pathway for chlorophyll and heme synthesis. Using degenerate oligonucleotide primers, based on amino acid sequence data for purified PBG deaminase from pea, a fragment was amplified from Arabidopsis genomic DNA by PCR, and then used to isolate both a cDNA and a genomic clone for PBG deaminase from Arabidopsis. The cDNA, shown to be full-length by primer extension, encodes a precursor protein of 382 residues, which can be imported into isolated chloroplasts and processed to the mature size. The genomic clone encodes an identical sequence to the cDNA, except for the presence of four introns within the coding region of the mature protein, and 1.7 kb of upstream sequence. There is no obvious TATA box within 50 bp of the transcription start. Southern blot analysis suggests that PBG deaminase is encoded by a single gene in the Arabidopsis genome, and RNase protection experiments demonstrated that this gene is expressed in both leaves and roots. These results support the conclusion that there is only one form of PBG deaminase in all plant cells, which is located in the plastid.

Cloning and characterization of full-length cDNA clones encoding chalcone synthase from the orchid Bromheadia finlaysoniana

Abstract cDNA clones encoding chalcone synthase (CHS) (EC 2.3.1.74), a key enzyme involved in flavonoid and anthocyanin biosynthesis were isolated from a cDNA library constructed from flowers of the orchid, Bromheadia finlaysoniana (Lindl.) Rchb.f using a homologous probe generated via PCR. The complete nucleotide sequences of the 3 clones, OCHS3 , OCHS4 and OCHS8 were determined. The lengths of OCHS3 , OCHS4 and OCHS8 are 1445, 1 382 and 1 439 bp, respectively. All the cDNAs contained a single open reading frame of 1 185 bp, encoding a polypeptide of 394 amino acids with a calculated molecular mass of 42.9 kDa. The nucleotide sequences of OCHS3 , OCHS4 and OCHS8 showed that they were not identical but exhibited a high degree of similarity (> 97 %). The deduced amino acid sequence showed 76–82 % homology, whereas the nucleotide sequence showed 59–68 % homology to CHS of other plants. Southern blot analysis indicated that CHS is possibly encoded by a small multigene family in the genome of B. finlaysoniana . Northern blot analysis revealed that CHS was expressed at very high levels in young leaves which are flushed with anthocyanin, whereas it was expressed at much lower levels in flowers which are faintly coloured and completely undetectable in other organs. Within different floral organs, CHS was found to be expressed at the highest levels in sepals, followed by stalks and columns whereas it was hardly detectable in lips and petals. However, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, a significantly more sensitive method compared to northern hybridization, demonstrated that CHS transcripts could be amplified from all parts of the plant and all floral organs examined.

Characterization, expression and phylogenetic study of R2R3-MYB genes in orchid

AbstractcDNA fragments representing 21 R2R3-MYB genes were isolated by RT-PCR from the Dendrobiumorchid hybrid Woo Leng. Six full-length cDNA clones were obtained from a flower cDNA library, four of which, DwMYB1, DwMYB2, DwMYB8 and DwMYB10, represent typical plant R2R3-MYB genes. The conceptual DwMYB4 protein is truncated at the C-terminal region and contains the R2 repeat and the N-terminal half of the R3 repeat (R2R3′). DwMYB4 expression is restricted to flowers. DwMYB9 contains an 8 amino acid N-terminal deletion in the R2 repeat (R2′R3) and is expressed at high levels in mature flower and inflorescence, but at very low levels in young flower buds. DwMYB8 and DwMYB10 show similar expression patterns and share very high sequence similarity in the N-terminal part of the MYB domain. Analysis of amino acid substitution indicated that the pattern and type of substitution between Arabidopsis and maize are quite different. Maize may have more conserved substitution in the MYBBRH domain than Arabidopsis.

Species determination of Malaysian Bactrocera pests using PCR-RFLP analyses (Diptera: Tephritidae).

BACKGROUND Identification of Bactrocera carambolae Drew and Hancock, B. papayae Drew and Hancock, B. tau Walker, B. latifrons Hendel, B. cucurbitae Coquillett, B. umbrosa Fabricius and B. caudata Fabricius would pose a problem if only a body part or an immature stage were available. Analysis of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of cytochrome oxidase I (COI) gene using primers COIR, COIF, UEA7 and UEA10 and restriction enzymes (MseI, RsaI and Alu1) was carried out. The banding profiles in the electrophoresis gel were analysed. RESULTS The COI gene in six Bactrocera spp. was successfully amplified by COIR and COIF, as well as UEA7 and UEA10, while B. caudata was amplified successfully only by UEA primers. Using COI amplified PCR products and restriction enzymes, distinct banding profiles for B. tau, B. latifrons, B. cucurbitae and B. umbrosa were observed, but not for B. carambolae and B. papayae. However, using UEA7, UEA10 and RsaI, B. caudata could be identified, while B. carambolae and B. papayae might possibly be separated from one another. It was also shown that adult body parts or immature life stages of B. carambolae, B. papayae, B. latifrons and B. cucurbitae produced the same banding profiles as the adults. CONCLUSION PCR-RFLP analyses are able to identify positively five Bactrocera species, while B. papayae and B. carambolae might possibly be separated from one another, even if immature life stages or adult body parts are used.

Amplified fragment length polymorphism analysis of vandaceous orchids

Abstract We investigated the application of the PCR-based fingerprinting technique, amplified fragment length polymorphism (AFLP), in orchids. The optimal AFLP patterns have been determined using primer combinations of EcoRI +4 and MseI +3 selective nucleotides. The same reproducible AFLP patterns were demonstrated in genomic DNAs isolated both from: (1) different orchid tissues, e.g. leaves and flowers; and (2) orchid flowers collected at different times. Genomic variations among different cultivars of vandaceous orchid hybrids were successfully determined by AFLP analysis. More than 10% of the AFLP bands were polymorphic DNA when siblings, derived from the same original crosses (two cultivars of Aranda Christine, five cultivars of Mokara Willie How), were used. Only 0.3–0.7% of the AFLP patterns were shown to be polymorphic when different cultivars, originating from somatic mutations during meristem culture for massive propagation, were used (two cultivars of Ar. Christine, four cultivars of M. Chark Kuan).

Proteomic analysis of the molecular response of Raji cells to maslinic acid treatment.

Maslinic acid, a natural pentacyclic triterpene has been shown to inhibit growth and induce apoptosis in some tumour cell lines. We studied the molecular response of Raji cells towards maslinic acid treatment. A proteomics approach was employed to identify the target proteins. Seventeen differentially expressed proteins including those involved in DNA replication, microtubule filament assembly, nucleo-cytoplasmic trafficking, cell signaling, energy metabolism and cytoskeletal organization were identified by MALDI TOF-TOF MS. The down-regulation of stathmin, Ran GTPase activating protein-1 (RanBP1), and microtubule associated protein RP/EB family member 1 (EB1) were confirmed by Western blotting. The study of the effect of maslinic acid on Raji cell cycle regulation showed that it induced a G1 cell cycle arrest. The differential proteomic changes in maslinic acid-treated Raji cells demonstrated that it also inhibited expression of dUTPase and stathmin which are known to induce early S and G2 cell cycle arrests. The mechanism of maslinic acid-induced cell cycle arrest may be mediated by inhibiting cyclin D1 expression and enhancing the levels of cell cycle-dependent kinase (CDK) inhibitor p21 protein. Maslinic acid suppressed nuclear factor-kappa B (NF-κB) activity which is known to stimulate expression of anti-apoptotic and cell cycle regulatory gene products. These results suggest that maslinic acid affects multiple signaling molecules and inhibits fundamental pathways regulating cell growth and survival in Raji cells.

Primer design and optimization for RAPD analysis of Nepenthes.

Primers with higher G+C content produced better random amplified polymorphic DNA (RAPD) profiles in Nepenthes. The occurrence of clustered Gs and Cs in the center of the primer seemed also to influence the banding patterns. It was also observed that for certain polymerases, the use of different buffers other than that recommended by the manufacturer provided a better amplification profile for Nepenthes.

A simple and efficient method of DNA isolation from orchid species and hybrids

A simple and reliable method for extracting DNA has been developed for orchid species and hybrids. The high quality of DNA obtained is suitable for amplification via the polymerase chain reaction (PCR) for producing random amplified polymorphic DNA (RAPD) markers.