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Results and Discussion

Nucleic acid analysis

Using the software BIOEDIT, predicted gene sequences of the rhizome of Ginger and Bamboo were subjected to nucleotide analysis (Figure 1a and 1b). According to this, the lengths of the isolated gene were 2211 bp and 339 bp respectively. The number of amino acids coded by the genes was 736 and 112 respectively. The nucleotide number and molecular percentage of the gene sequences are shown in Table 1.

Nucleotide composition of rhizome coding gene in bamboo (Figure 2a)

DNA molecule: Gene 1

Length = 2211 base pairs

Molecular Weight = 668964.00 Daltons, single stranded

Molecular Weight = 1342173.00 Daltons, double stranded

G+C content = 44.87%

A+T content = 55.13%

Nucleotide composition of rhizome coding gene in ginger (Figure 2b)

DNA molecule: Gene 2

Length = 339 base pairs

Molecular Weight = 102980.00 Daltons, single stranded

Molecular Weight = 206140.00 Daltons, double stranded

G+C content = 50.44%

A+T content = 49.56%

Protein analysis-primary structure

The physical parameters of the identified protein sequences were subjected to PROTPARAM analysis at ExPASy server. Based on the parameters predicted, the identified protein sequences were stable and the GRAVY score indicated that the proteins were hydrophilic in nature. This result indicated that the protein has a good interaction with water in the soil which is essential for the enlargement of rhizome and nitrogen translocation [14]. Accumulation of nitrogen in the nodules and rhizome region of plants is mediated by movement of water using the principle of hydraulic lift, which means in the absence of water, the movement of nitrogen is unidirectional and only in the presence of sufficient water the nitrogen gets accumulated throughout the rhizome.

Amino acid analysis

Amino acids were analyzed by using BIOEDIT software, and the composition of the identified protein sequences was depicted in Table 2a and 2b. The amino acid composition of the protein sequences is indicated diagrammatically in Figure 3a and 3b.

Amino acid composition analysis of Bamboo rhizome protein showed 49.4% of hydrophobic (Gly, 5.84%, Ala, 9.24%; Val, 7.20%; Leu 10.05%; Ile, 4.62%; Pro, 4.62%; Phe, 2.72%; Trp, 0.95%; Met, 2.85%), amino acid residues. Based on the secondary structure predictions, 36.58% of α- helices, 0.00% 3₁₀ helix, 18.75% B-turn, and 16.51% contributed the overall helical conformation of Bamboo rhizome coding gene.

Amino acid composition analysis of Ginger rhizome protein showed 49.4% of hydrophobic (Gly, 8.21%, Ala, 8.00%; Val, 6.95%; Leu 6.74%; Ile 4.84%; Pro, 4.00%; Phe, 2.53%; Trp, 2.11%; Met, 1.05%) amino acid residues. Based on the secondary structure predictions, 6.25% of α- helices, 0.00% 3₁₀ helix, 18.75% β-turn contributed the overall helical conformation of ginger rhizome coding gene.

From the amino acid composition, it was clear that the identified proteins are capable of forming α helices interconnected by loops. So, the protein can be classified under the helical protein category.

Prosite analysis

Prosite analysis of test protein sequence of rhizome coding gene in bamboo and ginger revealed the presence of two domains:

(a) Six TPR domains which is Tetratricopeptide Repeat (TPRs) (Figure 4a), a degenerate 34-amino acid repeated motif that is widespread among all organisms. In the cell, TPR containing proteins are localized in a variety of subcellular compartment, including the nucleus, the cytoplasm and mitochondria. Processes involving TPR proteins include cell-cycle control, transcription repression, stress response, protein kinase inhibition, mitochondrial and peroxisomal protein transport and neurogenesis. TPR repeats mediate proteinprotein interactions and the assembly of multiprotein complexes. The smallest functional unit that is widely used appears to be three tandem-TPR motifs

(b) Bulb Lectin super-family domain (Figure 4b) which occurs commonly in Amaryllidaceae, Orchidaceae and Aliaceae. The domain contained a ~115-residue-long domain whose overall three dimensional fold is very similar to that of

Dictyostelium discoideum comitin, an actin binding protein,

Curculigo latifolia curculin, a sweet tasting and tastemodifying protein.

Although this domain is a mannose-binding lectin in the bulb super-family, curculin is considered as a non-functional mannosebinding protein devoid of mannose-binding activity.

Secondary structure prediction

For the secondary structure analysis of bamboo rhizome, SOPMA tool was used. The results revealed that the identified protein was predominantly α-helical protein, which mainly consisted of α -helices (41.17%) and random coils (29.62%), extended stand (20.65%) (Figure 5a).

Similarly, SOPMA analysis of ginger rhizome gene revealed that was predominantly α-helical protein, which mainly consisted of α -helices (26.74%) and random coils (39.62%), extended stand (22.53%) (Figure 5b). The secondary structure prediction is in line with the amino acid composition analyzed.

Three dimensional structure of rhizome coding genes

The quality of the model generated was checked by ProSA, PROCHECK and Verify-3D and Ramachandran plot was constructed (Figure 6a and 6b). The R-plot indicated that 75% of the input residues were present in the most favourable region, 17.8% in additional allowed region and 6.7% in generously allowed region. Only one residue (0.5%) was present in disallowed region indicating the overall stereo chemical stability of the predicted structure. PROCHECK, Verify-3D and ERRAT results also confirmed the stability and reliability of the predicted 3D structure (Figure 7a and 7b). From this result, it is clear that the protein is highly stable with stabilized three dimensional structure which is highly hydrophilic in nature encompassing helical domain, which is the major domain which contributes to the function of the protein.