Biochemical and molecular studies of Moringa (Moringa oleifera Lam): seed storage proteins and DNA fingerprinting

Seed storage proteins were fractionated and partially purified from Moringa (Moringa oleifera Lam.) seeds. The dried mature seeds of Moringa were found to contain 17.12% (w/w) total soluble proteins. Proteins were fractionated into globulin, albumin, prolamin and glutelin using the modified Osborne procedure. The low molecular weight peptides present in the first three fractions were identified to be the bioactive peptides with flocculation activity. The globulin fraction was purified by gel filtration chromatography to separate the globulins from the low molecular weight peptides. The globulin isolate was found to be a 49 kD protein. The peptide isolate had the smallest size, 20 kD. The glutelin fraction on the other hand, had two polypeptides with molecular weight of 17.5 kD and 35 kD as determined by SDS-PAGE. The 17.5 kD glutelin polypeptide was found to be glycosylated, while the 49 kD globulin was not, as determined by PAS staining method. The 20 kD peptide isolate was able to bind and precipitate higher levels of tannic acid and proanthocyanidin, as compared to the globulin and glutelin fractions using the Hagerman-Buttler assay. The 49 kD globulin isolate was completely digested by chymotrypsin after 60 minutes of reaction. The 35 kD non-glycosylated polypeptide of the glutelin fraction was readily degraded within 5 minutes of reaction. The 17.5 kD glycosylated glutelin and the 20 kD peptide fraction were fairly resistant to chymotrypsin digestion. Digestion of the total proteins showed that the higher molecular weight polypeptides were primarily degraded after 60 minutes of reaction. The protein isolates were also confirmed to have some activity against representative gram-positive and gram-negative bacteria; S. aureus and E. coli respectively. Genomic DNA was extracted from young; immature leaves of several samples from different accessions of M. oleifera according to the method by Doyle and Doyle (1990) with minor modifications. It was found that some gene-specific primers were able to amplify specific DNA sequences in M. oleifera. PCR amplicons ranging from 120 bp to 2 kb also produced some polymorphic bands among samples of different accessions. This led to DNA fingerprinting of M. oleifera varieties using lunasin gene and cereal prolamin gene primers. Phylogenetic analyses among samples of different accessions were done by scoring the presence of polymorphic bands. Based on molecular analyses, there is genetic diversity among moringa samples collected from diverse geographical areas in the Philippines and other parts of the world. BLAST search of the DNA sequences of a representative non-polymorphic and polymophic DNA markers showed that the gene-specific primers anneal to non-specific regions of the genome aside from the gene axons at lower annealing temperatures.