Polymerase Chain Reaction (PCR)-Based Cloning of Partial cDNAs of Selected Genes in Normal and Mutant ‘Makapuno’ Endosperms of Coconut (Cocos nucifera L.)

Reggie Y. de la Cruz, Rita P. Laude, Ma. Genaleen Q. Diaz, Antonio C. Laurena, Merlyn S. Mendioro, Evelyn Mae T. Mendoza

Abstract


Makapuno, a mutant coconut (Cocos nucifera L.) with over-proliferating endosperm, is commonly grown in the Philippines and the Dutch East Indies. The science behind the makapuno phenomenon has not been completely understood. To determine the molecular basis of this phenomenon, we designed primers and cloned genes involved in glycolysis, galactomannan degradation, alcoholic fermentation, fatty acid biosynthesis, cytokinin biosynthesis, polyamine synthesis and cell cycle regulation. The total ribonucleic acid (RNA) from normal and makapuno endosperms of coconut was isolated and reverse transcribed. The complementary deoxyribonucleic acids (cDNAs) were used as template for polymerase chain reaction (PCR). The PCR products were then cloned in PCR cloning vector, pGEM-T Easy. Thirteen (13) partial cDNA sequences were obtained. Basic Local Alignment System Tool (BLAST) and InterProScan analyses revealed that the cDNAs harbored conserved domains and were highly homologous (68–98%) to equivalent sequences from other plant species. Pairwise alignment of the 13 partial cDNAs and their predicted protein sequences in normal and mutant makapuno coconut revealed absence of sequence differences.

Key Words: cDNA, cloning, coconut endosperm, endosperm overgrowth, makapuno

Abbreviations: BLAST – Basic Local Alignment System Tool, BLASTN – Basic Local Alignment System tool for nucleotide, BLASTP – Basic Local Alignment System Tool for protein, cDNA – complementary deoxyribonucleic acid, GAPD – glyceraldehyde-3-phosphate dehydrogenase, IPT – isopentenyl transferase, PABP – polyA-binding protein, PK – pyruvate kinase, RRM –RNA recognition motif, RT-PCR – reverse transcription-polymerase chain reaction


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