Analysis of the transcription factors expressed in the mature seed embryos of Moringa oleifera Lam. using RNA-sequencing and de novo transcriptome assembly

Moringa oleifera Lam. is well known for its numerous documented properties, particularly its significant applications in nutrition, therapeutics, biocontrol, energy, and bioremediation. These properties are the consequences of the vibrant physiological processes of the plant in the context of the ever-changing biotic and abiotic factors, in which transcription factors play substantial roles. Transcription factors (TFs) are the regulators of gene expression. Transcription factors enable the activation or repression of transcription. Along with the advent of ultrahighthroughput sequencing technologies such as RNA sequencing (RNA-Seq), in combination with bioinformatics techniques, the investigation of the TFs of M. oleifera was made possible. This research aimed to identify transcripts encoding for transcription factors in the mature embryos of Moringa oleifera Lam. through RNAsequencing and de novo transcriptome assembly (SOAP and Trinity assemblies); and determine their gene expression levels. In this study, the cataloguing and functional annotation of highly expressed TFs in M. oleifera were performed. Annotations were made based on BLAST, plant TF databases, TAIR, NCBI, gene2go, KEGG and ATTED-II. Highly expressed transcripts were homologs of A. thaliana. Other putative TFs were homologous to Theobroma cacao. Highly expressed putative TFs from SOAP as well as highly expressed TFs from TriAnn showed involvement in various seed processes. Some of the TFs were associated with non-seed related functions. It is recommended that validation of the functions of these putative M. oleifera transcription factors be performed through quantitative real-time PCR which can quantify the abundance and expression of TF genes in the mature seed embryos of M. oleifera in real time. Validation of genes encoding for TFs using quantitative realtime PCR which is an efficient method for the detection and quantitation of gene expression and can shed light on the functions of the transcription factors encoded by the TF transcripts particularly in their involvement in the many attributes of the seed embryos of M. oleifera such as in the developmental process, production of antioxidants, oil biosynthesis and stress response

Discovering Genes Involved in the Synthesis of Secondary Metabolites From the Seeds of Moringa Oleifera Through Transcriptome Analysis

Moringa oleifera is a widely used crop that produces seeds with a plethora of benefits encompassing health and nutrition. Secondary metabolite compounds were determined in the seeds of Moringa oleifera that possess nutritional and pharmacological benefits. Although various phytochemical researchers reported the presence of secondary metabolites in M. oleifera seeds, there is a lack of research on the genes encoding for enzymes that catalyze the synthesis of secondary metabolites in the seeds of M. oleifera. In the present study, RNA sequencing was used to analyze the transcriptome of the mature seed embryos of M. oleifera. Biological pathway analysis revealed 416 upregulated genes encoding for 11 enzymes involved in the catalytic steps of the phenylpropanoid and flavonoid pathways, and 63 unigenes encoding for 8 enzymes involved in the catalytic steps of the alkaloid pathway. These findings however need further validation using qRT-PCR which is a reliable and robust technique in order to validate the presence and expression of genes encoding for enzymes leading to the synthesis of secondary metabolites in the mature seed embryos of M. oleifera

Identification and characterization of endophytic fungi associated with the leaves of Moringa oleifera Lam

Fungal endophytes live within host plants and are recently gaining interest as sources of biologically active secondary metabolites. In this research, fungal endophytes associated with leaves of Moringa oleifera Lam. were isolated, characterized and identified. Leaf samples from two moringa trees were collected from Barangay Pandan, which is an urban area and Barangay Sapang Bato considered as a rural area with the highest elevation of all the barangays of Angeles City. All leaf samples were rid of debris by rinsing with tap water. A flame-sterilized one-hole puncher was used to bore 54 explants from leaves collected from each tree of each site (6 mm diameter). Explants were surface-sterilized by washing them with distilled water followed by 70% ethanol for 20 s, and then 0.52% NaOCl solution (commercially available bleach) for 30 s before finally rinsing them with sterile distilled water. The surface sterilized explants were then transferred to plates containing malt extract agar (MEA) amended with streptomycin (250 mg L-1) to prevent bacterial growth. The isolated fungal endophytes were characterized and identified based on their morphocultural characters. Results showed that a total of 24 fungal morphospecies were isolated. These were identified as belonging to the genera Fusarium, Xylaria, Pestalotiopsis, Aspergillus, Nigrospora, Stachybotrys, Rhizoctonia, and Macrophomina. Majority of the fungal endophytes isolated failed to produce spores and therefore were considered to be Mycelia sterilia. Fourteen fungal endophytes were extracted from Barangay Pandan as compared to 10 from barangay Sapang Bato. Of the nine different taxa identified in the two sites, Mycelia sterilia, Pestalotiopsis sp. and Rhizoctonia sp. were found to be common fungal endophytes extracted in both sites

Multiplex SSR-PCR Analysis of Genetic Diversity and Redundancy in the Philippine Rice (Oryza sativa L.) Germplasm Collection

Rice germplasm conservation is vital to ensuring the availability of a rich gene pool for future varietal improvement programs. However, with limited resources for gene banking, there is a need to identify and prioritize unique accessions in the PhilRice gene bank for maximum resource utilization. A robust and unequivocal way to identify duplicates is through multiplex SSR-PCR DNA fingerprinting. The present study established the optimal concentrations and reaction conditions for the successful amplification of PCR products using a multiplex panel composed of rice simple sequence repeat (SSR) markers, namely RM312, RM316, RM514 and RM171. The panel was then used to analyze the genetic diversity and identify duplicates among the 427 rice germplasm accessions with similar or identical variety names from the PhilRice genebank. A total of 15 alleles were detected at the 4 SSR loci. The polymorphism information content (PIC) values of the SSR markers were moderately high ranging from 0.459 to 0.643. A dendrogram was constructed using the Dice similarity coefficient and the UPGMA algorithm. The multiplex SSR-PCR panel produced unique profiles of 31 accessions that, being genetically distinct, should be maintained as part of the main collection of the genebank. There were 17 accessions identified as possible redundants having a bootstrap value greater than 95%. Additional SSR and morphological markers will be required to further strengthen the evidence for redundancy, and hence justify removal of unnecessary duplicates from the collection

Analysis of the oil biosynthesis transcripts of the Moringa oleifera Lam. mature seed embryos using RNA sequencing

Moringa oleifera seeds are capable of producing 40% edible oils that are gaining significance due to its nutritional advantages. Several studies have examined M. oleifera seed oil, nevertheless, these studies focused on the extraction of oil and methods of biodiesel production. There is a paucity of information on transcriptome level studies to determine the unigenes involved in oil biosynthesis metabolic pathways. The main objective of this study is to explore the transcriptome of the mature embryo of M. oleifera Lam. particularly the key genes related to oil biosynthesis. The transcriptome reflects the set of genes that are actively expressed at any given time produced in one or a population of cells in a given organism. Total RNA was extracted from 30 mature seed embryos obtained from 10 trees in Muñoz, Nueva Ecija, Philippines. RNA were pooled for cDNA library construction. Then, RNAsequencing was done followed by de novo sequence assembly to provide a costeffective and comprehensive means of transcriptome level information for M. oleifera. A total of 182,588 transcripts were generated in this study. Out of these transcripts, 3,556 unigenes are involved in oil biosynthesis. The most numerous group of unigenes are those involved in fatty acid biosynthesis with 1,009 unigenes, fatty acid catabolism with 982 unigenes and triacylglycerol catabolism with 608 unigenes. There are 33 unigenes encoding for transcription factors involved in regulating oil biosynthesis gene expression. This is the first transcriptome resource ever reported for M. oleifera mature seed embryo. These unigenes are unmatched in protein databases for M. oleifera. Hence, the transcriptome resource for the M. oleifera Lam. mature seed embryo generated in this study will be useful for the mapping of oil biosynthesis related genes and the understanding of metabolic pathways which could possibly be used to improve seed yield and oil content of M. oleifera

Molecular cloning and expression of chitin deacetylase 1 gene from the gills of Penaeus monodon (black tiger shrimp)

Chitin deacetylases have been identified and studied in several fungi and insects but not in crustaceans. These glycoproteins function in catalyzing the conversion of chitin to chitosan by the hydrolysis of N-acetamido bonds of chitin. Here, for the first time, the full length cDNA of chitin deacetylase (CDA) gene from crustaceans was fully cloned using a partial fragment obtained from a transcriptome database of the gills of black tiger shrimp Penaeus monodon that survived White Spot Syndrome Virus (WSSV) infection employing Rapid Amplification of cDNA Ends (RACE) PCR. The shrimp CDA, named PmCDA1, was further characterized by in silico analysis, and its constitutive expression determined in apparently healthy shrimp through reverse transcription PCR (RT-PCR). Results revealed that the P. monodon chitin deacetylase (PmCDA1) is 2176 bp-long gene with an open reading frame (ORF) of 1596 bp encoding for 532 amino acids. Phylogenetic analysis revealed that PmCDA1 belongs to Group I CDAs together with CDA1 and CDA2 proteins found in insects. Moreover, PmCDA1 is composed of a conserved chitin-binding peritrophin-A domain (CBD), a low-density lipoprotein receptor class A domain (LDL-A) and a catalytic domain that is part of CE4 superfamily, all found in group I CDAs, which are known to serve critical immune function against WSSV. Finally, high expression of PmCDA1 gene in the gills of apparently healthy P. monodon was observed suggesting important basal function of the gene in this tissue. Taken together, this is a first report of the full chitin deacetylase 1 (CDA1) gene in crustaceans particularly in shrimp that exhibits putative immune function against WSSV and is distinctly highly expressed in the gills of shrimp