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Differential Gene Expression of the Enzymes of Galactomannan Degradation in the Developing Endosperms of Normal and "Makapuno" Coconut (Cocos nucifera Linn.)
Dissertation Abstract:
The differential gene expression of the enzymes of galoctamannan degradation in the developing endosperms of normal and makapuno coconut was determined by cloning, sequencing, and characterization of the genes encoding a-galoctosidase, b-mannanase, and b-mannosidase.
RNA samples were extracted from different developmental stages of the normal and makapuno endosperms using a modified method of Burgos et al. (1995)*. RNA yields for the 6-7 months normal and makapuno nuts were 268 and 196 μg/5-8g FW, respectively. The RNA yield and purity decreased as the nut matured and the makapuno relatively contained lesser amount of RNA compared to normal endosperms in all ages sampled. The normal and makapuno RNA were reverse-transcribed. The forward and reverse primers for each of the gene were designed, synthesized and used in PCR amplification. The PCR products were cloned in TOPO TA plasmid, sequenced, and analyzed.
For a-galoctosidase, a 348 bp sequence from normal nut cDNA (CnAgln1) was sound to be identical to 348 bp sequence from makapuno (CnAglm1). The BLASTN 2.1.3 and BLASTP 2.2.2 searches revealed homology to the 5’ and 3’ ends of the sequences used in the primer design. The Vector NTI pairwise alignment showed that the putative genes were relatively most similar to plant mammalian and some fungal a-galoctosidases. Northern blot analysis using DIG-labeled CNAgln1 revealed a high level of mRNA of the putative a-galoctosidase in the normal six- to seven-month-old endosperm and very low level in makapuno.
The b-mannanase clones from the normal nuts were CnMan-n1 (505 bp), CnMan-n2 (627 bp) for the normal clones and CnMan-m1 (505 bp), and CnMan-m2 (629 bp) and CnMan-m3 (665 bp) for the makapuno clones. The deduced amino acid MAN-N1 (167) was almost identical to MAN-M1 (167). MAN-N2 (209) and MAN-M2 were more similar to MAN-N1 and M1, whereas MAN-M3 was the least similar to the others. MAN-N1 and MAN-M1 were relatively most similar to the coffee and tomato b-mannanases, even showing a short 4 amino acid long conserved region. MAN-N2 was relatively most alike to Piromyces sp., MAN-M2 to Caldocellum saccharolyticum b-mannanase, and MAN-M3 to Streptomyces lividans b-mannanase.
For b-mannosidase, four primers were designed − one from the 5’ end (manos1), another from the 3’ (manos2), and two other internal primers (manos3 and manos4). It was only from the manos1-manos4 combination where products were obtained. Two clones from the makapuno cDNA, CnMndm1 and CnMndm2 both showed 391 bp and their deduced amino acid sequences MNDM1 and MNDM2 had 130 amino acids. These sequences were 99 percent homologous and the VNTI pairwise alignment exhibited a considerable degree of similarity with the L. esculentum b-mannosidase.
The results of the previous biochemical studies pertaining to the makapuno phenotype in comparison to the current molecular studies were further discussed.
*Burgos, R.C., V.L. Chiang, X.H. Zhang, E.R. Campbell, G.K. Padilla, and W.H.
Campbell. 1995. “RNA isolation for plant tissues recalcitrant to extraction to guanidine.”
Biotechniques 19:734-37.