Isolation and Characterisation of Genes Expressed in Zygotic Embryos and Suspension Cultures of Oil Palm (Elaeis Guineensis Jacq.)

The bottleneck of the current oil palm tissue culture technique is the low rate of embryogenesis and the development of viable embryoid lines. Therefore, it is vital to increase the efficiency of callusing, embryogenesis, germination and proliferation of embryoids so that the number of subcultures per line can be reduced without effecting the number of shoots produced in-vitro. Thus, it is necessary to elucidate and understand the molecular processes that are involved during somatic embryogenesis of oil palm particularly those involved in specifying embryogenic competence. Based on the role of cell division cycle (cdc/cdk) genes and cyclins in cell division cycle control of other eukaryotes, it is likely that cyclins are also partially involved in the regulation of somatic embryogenesis. Hence, an attempt to isolate cyclin genes from the oil palm zygotic embryo cDNA library was made using heterologous cyclin cDNA probes from Arabidopsis. 32 putative clones designated as OPZE, were isolated from screening. A preliminary characterisation was carried out on these clones in order to identify clones with sequences related to the cell division cycle. This was achieved by hybridising the peR amplified OPZE clones with amplified cDNA from suspension cultures and mature leaves separately. The OPZE clones were categorised into 3 subpopulations according to their tissue-specific expression pattern: a, b and c. Randomly selected clones from these subpopulations were sequenced partially and used for sequence homology searches using DNA sequence databases. Most clones did not have any significant homology to any known sequences in the database, thus they were designated as novel clones. Three clones OPZEJA, OPZE3A and OPZE5A that had significant homology to oleo sin, calmodulin and tumour suppressor protein respectively were selected for northern analysis. From the northern analysis studies, it was found that OPZEJA (oleosin) is zygotic embryo specific and both OPZE3A and OPZE5A are ubiquitously expressed in all evaluated tissues. In order to complement this study, a partial length homeobox gene, OPHbJ (KnottedJ -like) from oil palm was isolated and was found to be expressed specifically in meristematic tissues. However, the specific functions of these genes during oil palm embryogenesis are still unknown

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A putative proline-rich protein of B. napus

Proline-rich proteins are among the major protein components of plant cell walls. So far, two different proline-rich cell wall proteins have been described in Brassica napus. This paper reports a study on expression and sequence analysis of a novel class of a proline-rich putative protein, tentatively designated Ae4. The largest ORF of Ae4 encodes 166 amino acid residues without the start and stop codons. Ae4 is a partial length cDNA. The Ae4 gene expression was investigated and the results demonstrate that it accumulates in all vegetative tissues tested as well as in the embryogenic culture of Brassica napus. However, expression of Ae4 was undetectable in the non-embryogenic and cytokinin-treated embryogenic tissues. These results indicate that the Ae4 gene might play a role in somatic embryo formation

Molecular characterization of Dg3, a cDNA that encodes a novel lipid transfer protein in Brassica napus

In this study, we have analysed the sequence of Dg3 clone using bioinformatic tools, determined copy number of this transcript in the genome of B. napus and expression levels at various tissues/organ. The cDNA contained 307-base pair open reading frame encoding 102 amino acid residues, 60-base pair 5`-untranslated region and 127-base pair 3`-untranslated region. The predicted mature protein has a molecular weight of 9.2 kDa and is acidic, with a predicted isoelectric point (pI) of 6.2. The Dg3 sequence has all the conserved structural characteristics of plant LTPs and showed highest homology to LTPs from other plant species. The transcripts of Dg3 were detected in all tested tissues but highest expression was in siliques and in vitro embryogenic cultures. Possible roles of Dg3 during somatic embryogenesis and normal plant development are discussed

Comparisons of different RNA extraction methods on woody tissues of the tropical tree, Aquilaria malaccensis

Aquilaria malaccensis (karas) produces oleoresin in its wood (known as agarwood) when responding to wounding and microbial infection. Wood tissues are known to contain high levels of polysaccharides, polyphenolics and secondary metabolites, which make RNA extraction challenging. In this work, six different methods for extracting RNA from wood tissues of A. malaccensis were compared. RNA yield, purity, and integrity number, were used as parameters to evaluate the efficiency of each method. Conventional methods yielded RNA with good purity but the RNA integrity was poor. The commercial RNeasy Plant Mini kit protocol was modified by means of scaling-up the reaction and combining all aliquots in the same RNeasy spin column, and yielded the highest yield while maintaining the integrity of the RNA. We found that this kit with some modifications was most suitable for extracting RNA from healthy wood and agarwood. This study is essential for future molecular studies on agarwood

Isolation, cloning and characterisation of new fragrance-related floral transcripts of Vanda Mimi Palmer

A subtracted cDNA library of open flower was constructed using the suppression subtractive hybridization (SSH) technique, to identify fragrance-related transcripts of Vanda Mimi Palmer. In total, 107 transcripts up-regulated during blooming were identified and sequenced. Only 33 clones (4 singletons and 29 contigs) showed similarities to known sequences in the public database. Of these, thirty-two clones were transcripts encoding fragrance-related enzymes including sesquiterpene synthase, (±)-germacrene D synthase, tyrosine decarboxylase and putative acyltransferase. Two fragrance-related transcripts, VMPAAT encoding a putative alcohol acyltransferase and VMPSTS encoding a sesquiterpene synthase, were subjected to full-length cDNA isolation and characterization. The full length cDNA of VMPAAT has a 1343bp open reading frame (ORF) of 448 amino acid residues whereas VMPSTS is predicted to encode a polypeptide of 561 amino acid residues with 1682bp ORF. VMPAAT and VMPSTS show high homologies with plant alcohol acyltransferase and terpene synthase, respectively. Real time RT-PCR indicated that both transcripts were expressed preferentially in floral tissues, with high levels in blooming and full bloom flowers. VMPAAT and VMPSTS transcripts were expressed in a rhythmic pattern. The results presented in this study will be potentially useful in providing additional insights into the fragrance-related pathways of Orchidaceae members, which until today is still limited

Extracellular localization of napin in the embryogenic tissues of Brassica napus spp. oleifera

Napin, a storage protein, has been reported to be transcribed abundantly during the pre-embryogenic stage and associated with the induction of Brassica napus secondary embryogenesis. In this study, we studied the distribution pattern of napin in the winter oilseed rape embryogenic tissue in comparison to that of the non-embryogenic tissue using the indirect immunofluorescence localisation coupled with the ultrastructural immunogold labelling techniques. Immunolocalisation studies revealed that the extracellular matrix layer outside the outer epidermal cell wall of B. napus embryogenic tissues contained napin. This is the first study to report the extracellular localisation of napin. In addition, we have also further characterised the expression pattern of Eg1 that encodes for napin in the B. napus embryogenic tissue

HnRNP-like proteins as post-transcriptional regulators

Plant cells contain a diverse repertoire of RNA-binding proteins (RBPs) that coordinate a network of post-transcriptional regulation. RBPs govern diverse developmental processes by modulating the gene expression of specific transcripts. Recent gene annotation and RNA sequencing clearly showed that heterogeneous nuclear ribonucleoprotein (hnRNP)-like proteins which form a family of RBPs, are also expressed in higher plants and serve specific plant functions. In addition to their involvement in post-transcriptional regulation from mRNA capping to translation, they are also involved in telomere regulation, gene silencing and regulation in chloroplast. Here, we review the involvement of plant hnRNP-like proteins in post-transcription regulation of RNA processes and their functional roles in control of plant developmental processes especially plant-specific functions including flowering, chloroplastic-specific mRNA regulation, long-distance phloem transportation and plant responses to environmental stresses

Distribution of arabinogalactan protein (AGP) epitopes on the anther derived embryoid cultures of Brassica napus

The anther-derived embryoid cultures of Brassica napus is stably embryogenic and has an extracellular matrix (ECM) layer covering the surface of the developing embryoids. In this study, the distribution of arabinogalactan protein (AGP) epitopes in the ECM layer and the embryogenic tissue of winter oilseed rape were investigated by immuno-labelling with anti-AGP monoclonal antibodies (mAb JIM4, JIM8, and JIM 13). There was no labelling by the JIM4 and JIM8 mAbs in the ECM layer, unlike what was reported in other plant species. JIM 13 epitope is developmentally regulated because it was only present in the ECM layer of the mature embryogenic tissue. These observations indicate a possible variability in the AGP epitopes present in the ECM layer among the different plant species. JIM8 and JIM 13 epitopes were found in some epidermal cells of embryogenic tissue, but not in the non-embryogenic tissue, implying that AGPs might have a specific role in embryogenic competency or determining the cell fate of the B. napus embryogenic cell

Molecular characterization of a new 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) transcript from Vanda Mimi Palmer.

A 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) transcript was successfully isolated from the floral cDNA library of Vanda Mimi Palmer (VMPDXR). The full-length cDNA of clone VMPDXR was predicted to encode a polypeptide of 473 amino acid residues with 15 bp of 5′ UTR and 230 bp of 3′ UTR including a poly-A tail. VMPDXR was predicted to have a molecular mass of 51.4 kD and a pI value of 6.04. It has two conserved domains, an N-terminal NADPH binding site (GSTGSIG) and an N-terminal proline-rich region(PPPPAWPGR). It also contains two highly homologous regions, a 78–207 amino acids stretch at the N-terminal and a 221–304 amino acids stretch at the C-terminal domain. The putative plastid transit peptide is not found in VMPDXR and it is clustered into the plant DXRs in the phylogenetic tree. VMPDXR was differentially expressed in roots, leaves, sepals, petals and column. The VMPDXR transcript levels were preferentially high in blooming and fully bloomed flowers compared to the bud. The expression of VMPDXR at different times did not appear in a rhythmic manner and no drastic fluctuation was observed at night except at 2 pm during the day