MATERIALS AND METHODS

Plant materials and DNA extraction: DNA was extracted from young leaves (0.5-1.0 g) of field-grown cassava genotype TMS 4 (2) 1425 as described by Dellaporta et al. (1983). DNA was resuspended at a concentration of 500 ng μL^-1. The quality of the DNA was verified by running 2 μL of the DNA alongside a molecular weight marker λPst I on 0.8% agarose gel electrophoresis in 1 x TAE (Tris Acetate EDTA) buffer at 500 volts for 1 h.

Primer design: A cassava granule-bound starch synthase I gene-specific primer pair was designed from cDNA sequences (accession X14760) earlier deposited in GenBank database from nucleotide +99 to +530. The downloaded sequences were used as template to design primer specific for the genes using Lasergene sequence analysis software (DNASTAR Inc, Madison, USA). The composition of the primer pairs designed and used for the study were as follows: Forward 5’-TGGACCCAAACTATCACTC -3’ and Reverse 5’-AGTTTTGCCCCATACCTTC -3.’Synthesis of the primers was done by Integrated DNA Technologies Incorporation (Iowa, USA).

PCR amplification, cloning and DNA sequencing: Amplification was carried out in 50 μL reaction volume, which composed of 1 μL of 10x buffer, 0.5 μL of MgCl₂ (25 mM), 1 μL each of primer F and R (1 μM), 0.5 μL of dNTPs (2.5 mM), 1 μL template DNA (500 ng), 4.8 μL of H₂O and 0.2 U of Taq DNA polymerase (Bioline, USA). The PCR amplification profile consisted of initial denaturation at 94°C for one min and 30 cycles of amplification (94°C for 30 sec, 50°C for 30 sec, 72°C for 45 sec) with a final cycle of 5 min at 72°C. The PCR amplification was carried out in a Peltier thermal cycler (PTC 2000, MJ Research, India).The PCR fragments were purified and cloned into pDRIVE vector (QIAGEN, California, USA). The presence of the insert in the recombinant plasmid was confirmed by restriction digestion. Both strands of the DNA inserts were sequenced and any sequence ambiguities were resolved by re-sequencing. The three independent PCR clones were sequenced from each PCR reaction. The sequences were manually edited and vector sequences removed. DNA sequencing was performed by Iowa State University, USA.

Sequence analyses: The genomic sequence of GBSS I has been submitted to GenBank under the accession numbers HM038439. Putative introns were identified in silico by (1) alignments between genomic and cDNA sequences using CLC DNA WORKBENCH software version 6.1 and (2) and confirmed by gene prediction method using GENSCAN web server (Burge and Karlin ,1998). The genomic sequence of GBSS I obtained in this study was aligned with available cDNA sequence (accession X14760) of the gene in the GenBank to identify the locations of the intron.

Phylogenetic analysis: Sequence searches of the non-redundant and unfinished genome databases at NCBI were conducted with the isolated GBSS I ORFs as query sequence. The ClustalX package was used to create an alignment of the sequences that was then submitted to a neighbour-joining analysis to generate a branching pattern. The phylogenetic tree was displayed using the CLC TREEVIEW program. Nineteen GBSS I sequences of higher plants (and their accession numbers) included in the study were: Frangula anus (AF500430), Ipomoea batatas (AB524726), Ipomoea trifida (EU192912), Nelumbo nicifera (FJ602702), Solanum tuberosum (X58453) Musa acuminata (HQ646360), Gossypium hirsutum (FJ713477), Oryza sativa (AB066094), Hordeum vulgare (FN179380), Saccharum officinarum (AF446084), Secale cereale (FJ491377), Glycine max (EU586117), Perilla frutescens (AF210699), Vigna unguiculata (EF472252), Phaseolus vulgaris (AB029546), Pisum sativum (AJ345045), Zea mays (EU970982), Manihot esculenta (HM038439) and Triticum aestivum (AF286320).

RESULTS

Amplification, isolation and characterization of cassava GBSS I clones: Out of the three annealing temperatures (45, 50 and 55°C) and two template DNA concentrations (100 and 500 ng μL^-1) tested for amplification of GBSS I, the band was distinct at annealing temperature of 50°C using DNA template concentration of 500 ng μL^-1. These conditions were used for amplification of GBSS I in 50 μL reaction volume for fragment purification and cloning purposes (Fig. 1). The observed product size was 731 bp against the expected of 323 bp. Three independent clones obtained with the primer pair designed to amplify +99 to +530 portion of Manihot esculenta GBSS I ( accession X14760) gene were sequenced and their nucleotide sequences were all identical. Therefore, only clone pOYE303-1 was subjected to further analysis. The insert in clone pOYE303-1 (accession No. HM038439) was 731 nucleotides long and encoded a partial protein of 124 amino acids with predicted molecular weight of 13.65 kDa and calculated 10.01 isoelectric point (Fig. 2).

Fig. 1:	Amplification of GBSS I by PCR. MW: DNA marker, 1 = 500 ng of DNA at 55°C, Molecular weight markers are at the left in kb

Fig. 2:	Phylogenetic relationship of GBSS I sequences of higher plants. The tree was constructed on the basis of available GBSS I sequences in the GenBank and from this study (cassava) using neighbour-joining method from bootstrapped data sets. The number of bootstrap replicates is indicated next to each branch. The scale indicates the average

Fig. 3:	Alignment of cassava GBSS I genomic and cDNA sequences. Ca GBSS I -cassava GBSS I cDNA (X14760) sequence, POYE303-1- cassava GBSS I genomic (HM038439) sequence of clone POYE303-1

HM038439 was rich in glycine (11.29%), valine (10.48%) and lysine (9.85). The BLAST search showed that GBSS I cDNA of cassava (accession No. X74160) was the most significant hit (100% similarity) with 1e-120 E-value, as expected. The clone contained the consensus BOX 1 of starch synthase gene. Box I (16 amino acids) was a glycine-rich region containing KTGGL, the substrate-binding site for ADP-glucose (Furukawa et al., 1990). The identity of HM038439 to GBSS I in sweet potato and potato is 66 and 67%, respectively. The predicted secondary structure of HM038439 consist of 9.68% alpha helix, 33.06% extended strand and 57.26% random coil.

Phylogenetic analysis: The GBSS I proteins in higher plants diverged into two groups represented by monocots and dicots (Fig. 2). Cassava GBSS I protein belongs to the dicot group; dicot GBSS I protein further divided into three subgroups. Cassava GBSS I protein is found in the subgroup of potato and sweet potato. However, it is closely related to buckthorn GBSS I than potato and sweet potato GBSS I.

In silico identification of introns: The locations of three putative introns identified by sequence alignment between the GBSS I isolated genomic sequence (accession No. HM038439) and GBSS I cDNA sequence (accession X14760) deposited in the GenBank are in Fig. 3. The putative intron locations were further confirmed by gene prediction method using GENSCAN web server. The first intron was 91 bp long and located between nucleotide 82 and 172. The first intron was 12.5% of the entire genomic sequence and made up of 30.0%T, 25.3%A, 16.5%C and 27.5%G. The second intron was 96 bp long and about 13.1% of HM038439. The second intron was located between 366 and 461 and made up of 19.8%A, 21.9%G, 43.8%T and 12.5%C. The third intron was 121 bp long and about 16.6% of HM038439. It was located between 595 and 716 nucleotide and 32.2%A, 18.2%G, 35.6%T and 14.9%C rich. When the intron sequences were removed, the nucleotide sequences of HM038439 and GBSS I cDNA were identical. All the introns of GBSS I sequence contain the splice site sequences consistent with the consensus 5’-GT–AG-3’ (Hanley and Schuler, 1988).

DISCUSSION

The successful PCR amplification of GBSS I gene indicated that the composition of oligonucleotide primer pairs used was adequate and specific for the gene. Three partial genomic clones of GBSS I was isolated and sequenced in this study. They were 100% identical at both nucleotide and amino acid levels, suggesting that the GBSS I protein in cassava has no isoform, in agreement with previous work reported in cassava (Salehuzzaman et al., 1993) and sweet potato (Wang et al., 1999). GBSSI protein is known to contain three conserved domains known as BOX I, BOX II and BOX III. One of the conserved domains, BOX I, was identified in the insert of each of the three partial gbss I genomic clones. The BOX I contains 16 amino acids among which is KTGGL motif that is highly conserved among plants and bacteria starch synthases. The motif is the ADP-glucose substrate-binding site for GBSS I. The KTGGL motif has been reported in sweet potato, maize, sorghum, pea and potato (Wang et al., 1999; Denyer et al., 2001; Edwards et al., 2002).

The phylogenetic study of higher plant GBSS I previously reported (Salehuzzaman et al., 1993) was expanded by including newly identified sequences from higher plants. The divergence of GBSS I into monocot and dicot groups have been documented. However the newly released GBSS I sequence of buckthorn is closely related to cassava GBSS I than potato and sweet potato GBSS I. The reason for these is not understood. Cassava belongs to family Euphorbiaceae and produce tubers while buckthorn is in Rhamnaceae and not producing tubers.

The observed amplification product size (731 bp) was larger than the expected amplification product size (323 bp) of the starch gene studied. Since genomic DNA was used as template DNA for PCR amplification, the difference in predicted and observed PCR product size could be attributable to the presence of introns in genomic DNA. An intron is any nucleotide sequence within a gene that is removed by RNA splicing to generate the final mature RNA product of a gene (Rose, 2008). A total of three putative introns were identified in the genomic sequence of GBSS I cloned. This is the first identification of intron sequence in cassava GBSS I gene, which is expected to have wide applications on gene expression studies and starch modification. The inclusion of introns in plant transformation constructs increased transformation efficiency (Gonzalez et al., 1998; Hankoua et al., 2006). The intron sequences can be used in the design of plant gene constructs to silence or over express starch genes in cassava or other plant genes for enhanced starch functionality (Chen et al., 2003). The introns can also be used for expression studies of other genes in other plants as the significance of introns in gene expression has recently been established (Rose, 2008).

CONCLUSION

The study produced and described, for the first time, partial genomic organization of GBSS I in cassava. The study revealed that cassava genome contains only one isoform of GBSS I. The study also identified three introns in the genomic arrangement of GBSS I. The introns will find applications in genetic modification of cassava for enhanced starch quality and in genetic manipulation of other crops.

ACKNOWLEDGMENT

The Visiting Research Fellowship opportunity given to the first author by International Institute of Tropical Agriculture (IITA) is appreciated. The financial support of Obafemi Awolowo University Research Committee is acknowledged.