Analysis of common bean (Phaseolus vulgaris L., genotype BAT93) calmodulin cDNA using computational tools.

BACKGROUND: Common bean (Phaseolus vulgaris L.) is an important part of the human diet and serves as a source of natural products. Identification and understanding of genes in P. vulgaris is important for its improvement. Characterization of expressed sequence tags (ESTs) is one of the approaches in understanding the expressed genes. For the understanding of genes expression in P. vulgaris pod-tissue, research work of ESTs generation was initiated by constructing cDNA libraries using 5-day and 20-day old bean-pod-tissues. Altogether, 5972 cDNA clones were isolated to have ESTs. While processing ESTs, we found a transcript for calmodulin (CaM) gene. It is an important gene that encodes for a calcium-binding protein and known to express in all eukaryotic cells. Hence, this study was undertaken to analyse and annotate it.
OBJECTIVE: The objective of this study was to analyze and annotate P. vulgaris CaM (PvCaM) gene cDNA and its deduced protein (amino acids) sequence.
MATERIALS AND METHODS: Both strands of PvCaM cDNA clone were sequenced using M13 forward and reverse primer to elucidate the nucleotide sequence. The cDNA sequence and deduced protein sequence were analyzed and annotated using bioinformatics tools available online. The secondary structures and three-dimensional (3D) structure of PvCaM protein were predicted using the Phyre automatic fold recognition server.
RESULTS: Results showed that PvCaM cDNA is 818 bp in length. The cDNA analysis results showed that it contains an open reading frame that encodes for 149 amino acid residues. The deduced protein sequence analysis results showed the presence of conserved domains required for CaM function. The predicted secondary structures and 3D structure are analogous to the Solanum tuberosum CaM.
CONCLUSIONS: This study analyzed and annotated PvCaM cDNA and protein. However, in order to obtain a complete understanding of PvCaM protein, further study on its expression, structure and regulation is essential.

INTRODUCTION

The animal products such as eggs, meat and milk are sources of dietary protein. But, legumes are a well-known and widely used as a source of dietary proteins, particularly by poor people.[1] By understanding the importance of common beans (Phaseolus vulgaris L.) in food supply chain, the Phaseomics international consortium was developed to establish the necessary framework of knowledge and materials for the advancement of bean genomics, transcriptomics, and proteomics. The major goal of this Phaseomics international consortium is to help in generating new common bean varieties suitable and desired by farmers and consumers.[2] As a part of this consortium, research work was undertaken for the generation of P. vulgaris expressed sequence tags (ESTs).[3]

So far, 5972 ESTs has been generated, and while processing and analyzing generated ESTs, calmodulin (CaM) EST was identified. CaM is known to play an important regulatory role in a bimodular mechanism of calcium control in eukaryotes.[456] Therefore, to elucidate the P. vulgaris CaM (PvCaM) cDNA clone sequence, it was fully sequenced, and cDNA and deduced amino acid (protein) sequences were analyzed and annotated in this study using computational tools. The PvCaM gene cDNA sequence, deduced protein sequence, predicted secondary structures and three-dimensional (3D) structure is reported in this paper.

MATERIALS AND METHODS

Phaseolus vulgaris L. (genotype BAT93) seeds were provided by Patricia Lariguet, Laboratoire de Biologie Moléculaire des Plantes Supérieures, Department of Plant Biology, University of Geneva, Geneva, Switzerland. Seed germination and seedlings maintenance was done as stated by Bhore et al.[3]

The PvCaM cDNA clone was identified from the ESTs generated from 5-day-old (days after anthesis) bean-pod-tissue cDNA library. The cDNA library was constructed (unpublished data) using “CloneMiner cDNA library construction kit” obtained from Invitrogen Corporation.

Escherichia coli cells harboring recombinant plasmid with PvCaM cDNA were cultivated in 10 ml Luria-Bertani (LB) medium supplemented with 40 μg/ml Kanamycin. Plasmid DNA was isolated and purified using Wizard® Plus SV Minipreps DNA purification system (Promega). Plus and minus strand of PvCaM cDNA clone was sequenced using M13 (forward) and M13 (reverse) primer.[3]

The basic alignment analysis of cDNA sequence was carried out using blast (bl2seq) program available online at National Center for Biotechnology Information and the finalized cDNA sequence was analyzed using online bioinformatics tools. The similarity search was performed using blast programs (blastN and blastP). Bioinformatics tools available at JustBio (http://www.justbio.com/) were used to deduce the protein sequence, and to find out the general features of PvCaM cDNA and deduced protein sequence.

The deduced PvCaM protein sequence was used as a blast (blastP) input to find the most analogous protein sequence and or structure in protein data bank.[7] However, for the prediction of secondary structures and the 3D structure of PvCaM, Phyre2, a free web-based service for protein structure prediction was used.[8]

RESULTS

Full-length PvCaM cDNA clone was isolated from 5-day old bean-pod-tissue cDNA library. Plus (+) and minus (−) strands sequence was aligned and after elimination of the adaptor sequence, cDNA sequence was finalized. Analysis of the results showed that PvCaM cDNA is 818 bp in length. Identity of cDNA was confirmed by analyzing nucleotide (cDNA) and deduced amino acid sequences. Annotated nucleotide and deduced protein sequence of PvCaM is deposited in GenBank/DDBJ/EMBL under the accession number JX869966. The basic annotated features of cDNA nucleotide and deduced protein sequence are summarized in Table 1, and cDNA nucleotide sequence along with its deduced amino acid sequence is shown in Figure 1.

Table 1

The basic features of PvCaM cDNA and its deduced protein sequence

Figure 1

Nucleotide and deduced amino acid sequence of Phaseolus vulgaris L. calmodulin cDNA (GenBank Accession No: JX869966). An open reading frame (ORF) and noncoding regions are shown in capital and small letters, respectively. The deduced amino acid sequence is given below the nucleotide sequence, which is numbered at both ends of each sequence line. The ORF encodes for a protein containing 149 amino acid residues. Initiation and termination codons are shown in green and red colour, respectively; *represents stop codon

The amino acid sequence analysis results showed that PvCaM protein is rich in glutamic acid (13.4%) and aspartic acid (12.1%). Results also showed that PvCaM does contain less (<1%) cysteine, histidine and tyrosine. Interestingly, there was not a single residue of the tryptophan in PvCaM protein. BlastP (domain enhanced lookup time accelerated basic local alignment search tool) results showed the presence of putative conserved domains in PvCaM protein.

The topology of PvCaM protein to show secondary structures is depicted in Figure 2. The predicted 3D structure produced for PvCaM protein by homology modeling using Phyre2 is shown in Figure 3.

Figure 2

The predicted secondary structures of Phaseolus vulgaris L. calmodulin (PvCaM) protein

Figure 3

The predicted three-dimensional (3D) structure of Phaseolus vulgaris L. calmodulin (PvCaM) protein; (a) protein ribbon 3D structure model; (b) molecular surface 3D structure of model shown in (a)

DISCUSSION

The full-length gene or its cDNA is required for the over-expression of gene in order to increase either the production of a desired important protein or natural products.[9] To understand the secondary and tertiary structural features of the proteins, molecular modeling is commonly used.[101112] The main goal of this brief-study was to annotate PvCaM gene cDNA and its deduced protein (amino acid) sequence. The PvCaM cDNA clone was isolated from 5-day-old-pod tissue cDNA library; hence, it reflects that it is expressed in bean's 5-day-old developing-pod-tissue. However, its level of expression and its pattern of expression are not clear at this moment as we have not characterised its expression.

The guanine-cytosine (GC) content in PvCaM cDNA is 43%. It is close to, but significantly higher than the GC content (39.4%) reported in nuclear DNA of broad bean.[13] BlastP results showed the presence of EFh (EF-hand) domains as specific hits. This EFh, calcium binding motif is a diverse superfamily of calcium sensors and calcium signal modulators. Ca2+ binding induces a conformational change in the EF-hand motif, leading to the activation or inactivation of target proteins.[141516]

Results suggest that the secondary structures of PvCaM protein are mainly alpha helices (60%) [Figure 2]. The predicted 3D structure of the PvCaM protein is based on the best template, 1RFJ. This template is of potato (Solanum tuberosum) CaM protein which shows the highest (97%) similarity (figure not shown) with PvCaM protein when compared with other templates available in a database of protein.[17] The reported, potato CaM structure was determined using X-ray diffraction data in the resolution range 8.0–2.0 A.[18] Therefore, we strongly believe that the 3D structure predicted for PvCaM protein in this study should be closer to its real structure. However, we suggest the further wet lab experimental work to validate the predicted structure.

Calmodulin is an important protein; because it decodes Ca2+-dependent and-independent signals and could be helpful in in-depth understanding of the biological molecular mechanisms and signals.[19202122]

However, very little is known about PvCaM. Therefore, further research is required to understand more about PvCaM protein.

CONCLUSION

This brief-study has annotated the basic features of PvCaM gene cDNA and deduced protein. Comparative molecular modelling suggests that the deduced PvCaM protein is analogous to Potato CaM protein. However, in order to have a comprehensive understanding of PvCaM protein further studies are required to validate the predicted 3D structure, and to understand its expression and regulation in beans.