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Physicochemical, enzymatic and molecular characterisation of the storage protein of aerial tuber, Dioscorea bulbifera Linn

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Background: The storage protein of the aerial tuber of Dioscorea bulbifera was purified and its physicochemical, enzymatic and molecular properties determined with a view to comparing its functionality and genetic relatedness with other storage proteins. Results: The purified protein had molecular weight of 21 kDa. The protein showed carbonic anhydrase, trypsin inhibitory, dehydroascorbate reductase and monodehydroascorbate reductase activities. Amplifications with polymerase chain reactions resulted in the detection of two genes encoding the storage protein. The deduced amino acid sequence of the shorter and larger genes had homologies with the storage proteins of members of the Dioscorea family. Conclusion: The study concluded that the storage protein of the aerial tuber of D. bulbifera had similar properties with those of other Dioscorea species and may be suitable for development as functional food.
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R E S E A R C H Open Access
Physicochemical, enzymatic and molecular
characterisation of the storage protein of
aerial tuber, Dioscorea bulbifera Linn.
Olukemi Adetutu Osukoya
1
and Adenike Kuku
2*
Abstract
Background: The storage protein of the aerial tuber of Dioscorea bulbifera was purified and its physicochemical,
enzymatic and molecular properties determined with a view to comparing its functionality and genetic relatedness
with other storage proteins.
Results: The purified protein had molecular weight of 21 kDa. The protein showed carbonic anhydrase, trypsin
inhibitory, dehydroascorbate reductase and monodehydroascorbate reductase activities. Amplifications with
polymerase chain reactions resulted in the detection of two genes encoding the storage protein. The deduced
amino acid sequence of the shorter and larger genes had homologies with the storage proteins of members of the
Dioscorea family.
Conclusion: The study concluded that the storage protein of the aerial tuber of D. bulbifera had similar properties
with those of other Dioscorea species and may be suitable for development as functional food.
Keywords: Aerial potato, Aerial yam, Bulb, Dioscorin, Storage protein, Protein purification
Introduction
Yam, a dioecious plant belonging to the Dioscorea genus,
is an important staple crop in many areas of the tropics
and sub-tropics [1]. There are 8 genera and 880 species of
yam plant. They produce edible tubers, bulbils, corms or
rhizomes [2]thatarebasicallymadeupofcarbohydrates
and are important sources of proteins and micronutrients
[3]. Yam tubers are widely utilized as food due to their
compositions [4]. Yam tubers also contain functional
components such as mucin, dioscin, diosgenin, allantoin,
choline and polyphenol oxidases [5] and, in addition, min-
erals and vitamins such as calcium, zinc, phosphorus, cop-
per, iron, sodium, potassium, β-carotene, thiamine,
riboflavin and niacin [6]. About 80% of the proteins in
yam are storage proteins [7], which are usually affected by
factors such as cultural practices, climate, soil fertility, ma-
turity at harvest and length of storage time [8].
Plants accumulate storage substances such as starch,
lipids and proteins in certain phases of development.
The major role of storage proteins is to act as stores of
nitrogen, sulphur and carbon, which are accumulated in
both vegetative and reproductive tissues. Thus, they
serve as a reservoir for later stages of plant development
[9,10]. Storage proteins provide nutrients to support the
growth of new plants as seedlings (from seeds) or shoots
(from tubers). They are localized in specific organs, cell
types and subcellular compartments in discrete deposits
(protein bodies) where they facilitate high-level accumu-
lation without any adverse effects on other cellular func-
tions. They also allow plants to survive periods of
adverse conditions between growing season [11]. Plant
storage proteins are grouped into two classes: seed stor-
age proteins that accumulate to high levels in seeds dur-
ing the late stages of seed development, and vegetative
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* Correspondence: akuku@oauife.edu.ng;adenikekuku@yahoo.com
2
Department of Biochemistry and Molecular Biology, Obafemi Awolowo
University, Ile-Ife, Nigeria
Full list of author information is available at the end of the article
Journal o
f
Genetic Engineerin
g
and Biotechnology
Osukoya and Kuku Journal of Genetic Engineering and Biotechnology
(2020) 18:29
https://doi.org/10.1186/s43141-020-00040-y
storage proteins, which accumulate in vegetative tissues
such as leaves, stems and tubers [12].
Dioscorins, yam storage proteins, isolated from differ-
ent Dioscorea species have been shown to have various
biological activities, which include enzymatic (α-carbonic
anhydrase, trypsin inhibitory), antioxidant, antihyperten-
sive and immunomodulatory activities. They are thus
worth developing as healthy or functional foods. Dios-
corea bulbifera belongs to the family Dioscoreaceae
assigned to the order Dioscorales. It is commonly known
as air potato, potato yam, air yam, or bulbil-bearing yam.
It is native to Africa and Asia but widely grown and con-
sumed in the tropics [13], the Caribbean Islands, South
East Asia, South Pacific and West Indies. The unculti-
vated form is bitter, not edible and may be poisonous.
Air potato plants produce aerial tubersthat are at-
tached closely to the axil. These aerial tubers (bulbils)
are usually round or spherical with mostly smooth sur-
faces. The aerial tuber (from where the name air potato
is derived) serves as the main storage organ of D. bulbi-
fera [13,14]. The aerial tubers of D. bulbifera are com-
monly consumed especially in South Eastern Nigeria and
serve as a good source of calories and minerals [15]. The
plant has many health benefits and is used in folk medi-
cine as analgesic, aphrodisiac, diuretic and rejuvenative
tonic. It is also used as a folk remedy to treat conjunctiv-
itis, diarrhea and dysentery [16,17]. Despite all its medi-
cinal and agricultural uses, D. bulbifera is widely
characterized as an organism that outcompetes and
smothers native vegetation and is usually considered as
weed by farmers. It is thus paramount to investigate the
properties of some of the bioactive molecules such as
the major storage protein. The study aimed at isolating
and purifying the storage protein from the aerial tubers
of Dioscorea bulbifera, determining some of its physico-
chemical properties, identifying and sequencing the gene
encoding the storage protein and establishing the phylo-
genetic relatedness with the storage proteins from other
Dioscorea species.
Methods
Materials
Dioscorea bulbifera aerial tubers were obtained from a
farmland in Amichi, Nnewi South Local Government
of Anambra State, Nigeria. The plant was identified
in the IFE Herbarium of the Department of Botany,
Obafemi Awolowo University, Ile-Ife, Nigeria, where
the specimen copy was deposited and voucher num-
ber IFE-14754 was given.
All chemicals and reagents used were purchased from
either Sigma Chemical Co. (St. Louis, MO, USA), Phar-
macia Chemicals (Uppsala, Sweden) or Bio-Rad Lab
(Hercules, CA, USA).
Preparation of crude extracts
The crude extract of the aerial tubers of Dioscorea bulbi-
fera was prepared at different pH and varying
temperature, in order to ascertain conditions at which
most of the proteins in the aerial tuber are solubilized.
Dioscorea bulbifera aerial tubers were peeled, sliced
and homogenized with 4 volumes (w/v) of buffers at dif-
ferent pH: 0.5 M citrate/phosphate buffer (pH 46), 0.5
M Tris-HCl buffer (pH 7 and pH 8.3) and glycine-NaOH
buffer (pH 9 and 10). The mixtures were stirred for 4 h
and centrifuged at 13,500 rpm for 30 min at 4 °C. The
supernatants collected were stored as crude extracts.
Also, approximately 100 g portions of yam slices were
boiled in 1 L of water at 25, 30, 40, 50, 60, 70, 80, 90 and
100 °C for 10 min. The treated aerial tubers were
drained, cooled, weighed and homogenized with 50 mM
Tris-HCl (pH 8.3) at 1:4 (w/v). The mixture was stirred
for 4 h and centrifuged at 13,500 rpm for 30 min at 4 °C.
The supernatants were collected as crude extracts.
Protein content of extracts was determined by Lowry
method using 1 mg/mL bovine serum albumin (BSA) as
standard.
Purification of protein
The crude extract obtained at pH 8.3 and 25 °C (which
had the highest protein concentration) was used for fur-
ther studies. Purification of the storage protein of Dios-
corea bulbifera aerial tuber was carried out following the
method of Hou et al. [18] with a little modification.
The crude extract of the aerial tuber of Dioscorea bul-
bifera was subjected to 70% ammonium sulphate pre-
cipitation, stirred and kept overnight at 4 °C. The
mixture was centrifuged at 13,500 rpm for 30 min and
the precipitate recovered. The precipitate was dissolved
in 10 volumes of 50 mM Tris-HCl buffer, pH 8.3 and di-
alyzed exhaustively against distilled water.
Ion-exchange chromatography on DEAE Sephadex A-25
The dialyzed protein solution (7.5 mg/mL; 2.5 mL) was
loaded on DEAE Sephadex (A-25) ion exchange column
(1.5 × 20 cm) previously equilibrated with 50 mM Tris-
HCl buffer, pH 8.3. Unadsorbed proteins were eluted
with 50 mM Tris-HCl buffer, pH 8.3, and adsorbed pro-
teins were eluted stepwise with 150 mM NaCl in 50 mM
Tris-HCl buffer, pH 8.3, at a flow rate of 15 mL/h. Frac-
tions of 5 mL each were collected, and elution was moni-
tored at 280 nm. The adsorbed protein fractions, which
correspond to the major storage protein of the aerial
tuber of D. bulbifera, were pooled and concentrated.
Gel filtration on Sephadex G-75
Adsorbed protein sample (1.5 mg/mL; 5 mL) obtained
from ion-exchange chromatography was further purified
by gel filtration on Sephadex G-75 column (1.5 × 40 cm)
Osukoya and Kuku Journal of Genetic Engineering and Biotechnology (2020) 18:29 Page 2 of 14
previously equilibrated with 50 mM Tris-HCl buffer, pH
8.3. The column was eluted with 100 mM Tris-HCl buf-
fer (pH 7.9) containing 100 mM NaCl at a flow rate of
27 mL/h. Fractions of 3.6 mL each were collected. The
purified protein was collected, concentrated and stored
at 20 °C for further use. Protein concentration was de-
termined after each purification step.
Non-SDS polyacrylamide gel electrophoresis
The protein samples were subjected to polyacrylamide
gel electrophoresis in the absence of sodium dodecyl
sulphate (SDS) according to the modified method of
Shiu et al. [19] to monitor the purity of the protein ob-
tained after each purification step. Electrophoresis was
performed on a 10% discontinuous gel system under
non-denaturing conditions and stained with Coomassie
Brilliant Blue.
Determination of molecular weight
The native molecular weight of the protein was deter-
mined by gel filtration on a Bio gel P-200 column (1.5 ×
63 cm) using the following protein markers: lysozyme (Mr
14,000), α-chymotrypsinogen A (Mr 25,000), egg ovalbu-
min (Mr 45,000) and bovine serum albumin (Mr 66,000).
Each protein (5mL) was applied on the column and run
separately using 10 mM phosphate buffer pH 7.0 as eluant
at a flow rate of 10 mL/h. Fractions of 5 mL were col-
lected, and the elution was monitored at 280 nm. The void
volume (V
o
) of the column was determined using Blue
dextran (elution monitored at 620 nm).
The purified storage protein was subjected to SDS-
polyacrylamide gel electrophoresis for subunit molecular
weight determination following the modified method of
Shiu et al. [19] using the following protein markers: ov-
albumin (Mr 45,000), carbonic anhydrase (Mr 29,000),
trypsinogen (Mr 24,000), trypsin inhibitor (Mr 20,000)
and α-lactalbumin (Mr 14,200).
Detection of protein-bound carbohydrate
The presence of covalently-bound carbohydrate in the
storage protein was investigated by staining the gels with
periodic acid-Schiffs reagent (PAS) after electrophoresis,
as described in the Pharmacia Manual of Laboratory
Techniques, revised edition. The protein sample was
subjected to electrophoresis under non-denaturing con-
ditions using phosphate-buffered system. After electro-
phoresis, the gel was fixed in 7.5% acetic acid at room
temperature for 1 h. The fixed gel was transferred into a
beaker containing 0.2% aqueous periodic acid and kept
at 4 °C for 45 min. Afterwards, the gel was removed and
transferred into a beaker containing Schiffs reagent,
kept at 4 °C for 45 min. The gel was destained in 10%
acetic acid. Glycoprotein band (if present) will stain
purplish red.
Amino acid composition of the protein
The storage protein was subjected to amino acid content
analysis using methods described by Ekeanyanwu [20].
The sample was hydrolysed, evaporated in a rotary evap-
orator and loaded into the Technicon Sequential Multi-
Sample Amino Acid Analyzer (TSM).
Enzymatic activities of storage protein of Dioscorea
bulbifera
Determination of carbonic anhydrase activity
Carbonic anhydrase activity of the protein was measured
by hydrolysis of 4-nitrophenyl acetate resulting in an in-
crease of absorbance at 348 nm [21]. The activity of the
tuber storage protein was compared with that of car-
bonic anhydrase from bovine erythrocytes. The reaction
mixture contained 0.3 mL of freshly prepared 3 mM 4-
nitrophenyl acetate in aqueous 3% acetone and 0.7 mL
of 15 mM Tris sulphate buffer, pH 7.6. Exactly 10 μL
purified protein solution (1 mg/mL) was added, and the
catalyzed reaction was monitored by measuring the in-
crease in absorbance at 348 nm for 5 min.
Determination of dehydroascorbate reductase activity
Dehydroascorbate (DHA) reductase activity of the pro-
tein was carried out according to the method of Hou
et al. [18]. In this reaction, 10 mg of DHA was dissolved
in 5 mL of 100 mM phosphate buffer of different pH
values (pH 6.0, 6.5 and 7.0). The reaction was carried
out at 30 °C; 100 μL purified protein solution (1 mg/mL)
was added to 0.9 mL DHA solution with or without 4
mM glutathione. Increase in absorbance at 265 nm was
recorded for 5 min. Non-enzymatic reduction of DHA in
phosphate buffer was measured in a separate cuvette.
Determination of monodehydroascorbate reductase activity
Monodehydroascorbate (MDA) reductase activity of the
protein was assayed according to the method described
by Hou et al. [18] by monitoring the decrease in absorb-
ance at 340 nm due to NADH oxidation. MDA free radi-
cals were generated by ascorbate oxidase in the assay
system. The reaction mixture contained 50 mM phos-
phate buffer (pH 6.0, 6.5 and 7.0); 0.33 mM NADH; 3
mM ascorbate, ascorbate oxidase (0.9 U); and 200 μL
purified protein solution (200 μg protein) in a final vol-
ume of 1 mL. Distilled water was used to replace protein
solution in blank solutions. One unit of MDA reductase
is defined as the amount of protein required to oxidize
1μmol of NADH per min.
Determination of trypsin inhibitory activity
Trypsin inhibitory activity of the protein was determined
according to the method of Xue et al. [22] by monitoring
the inhibition of trypsin-catalyzed hydrolysis of N-ben-
zoyl-L-arginine-4-nitroanilide (substrate) in 0.1 M Tris-
Osukoya and Kuku Journal of Genetic Engineering and Biotechnology (2020) 18:29 Page 3 of 14
HCl buffer (pH 8.2). Different concentrations of the pro-
tein were pre-incubated with 20 μM trypsin at room
temperature for 15 min. The substrate (100 μg/mL) was
added to give a final volume of 1 mL for an additional
20 min. The absorbance at 405 nm was measured. The
inhibitory activity is calculated as the percentage de-
crease in substrate hydrolysis rate, which is directly pro-
portional to increase in absorbance at 405 nm. The
result was expressed as micrograms of trypsin inhibited.
Molecular characterization of the storage protein of
Dioscorea bulbifera
Genomic DNA extraction
The aerial tuber was peeled, cut into bits and ground
into fine powder with a mortar and pestle under liquid
nitrogen. Genomic DNA was extracted using QIAGEN
DNeasy Plant Mini Kit. DNA concentrations were deter-
mined with a Nanodrop spectrophotometer (Beckman
Coulter) and adjusted to 25 ng/μL for PCR amplification.
Primer design for polymerase chain reaction
Sequences of some Dioscorin genes from various Dioscorea
sp. were obtained from NCBI nucleotide database (http://
www.ncbi.nlm.nih.gov/nuccore). These sequences were
inserted into the input window of the web-based polyacryl-
amide chain reaction (PCR) primer designing program,
Primer3 (https://primer3plus.com/cgi-bin/dev/primer3plus.
cgi). The primer minimum and maximum sizes were set to
100 and 900 nucleotides, respectively. The DNA was
subjected to PCR amplifications using the designed
Dioscorin-specific primers (5-CTCCTCTCCTCCCT
CCTCTT-3(forward primer) and 5-GGGGGT
ACAATGGAGAAGT G-3(reverse primer)). The
amplification was conducted in a final reaction vol-
ume of 25 μLcontaining5μLofDNAsample,2.0μL
MgCl
2
,0.2μLTaq polymerase, 2.5 μL 10 X reaction buf-
fer, 1 μLdNTPs,1μL each of forward and reverse primers,
2.0 μL Tween 20 and sterile deionized water in a 96-well
microtiter plate and carried out in a GeneAmp PCR Sys-
tem 9700 (Applied Biosystems). The PCR cycles were
made up of initial denaturation of DNA template at 94 °C
for 3 min, followed by 36 cycles of denaturation at 94 °C
for 1 min, annealing at 60 °C for 1 min and extension at
72 °C for 2 min. The final extension step was at 72 °C for
7min.
Electrophoresis of PCR products
The PCR products obtained were detected by agarose
gel electrophoresis. An aliquot (3 μL) of 5 × loading dye
(0.25% bromophenol blue, 0.25% xylene cyanol FF and
13% Ficoll in water) was added to the 10 μL of the PCR
product, and 6 μL of the mixture was loaded onto a 1.5%
agarose gel pre-stained with ethidium bromide. TBE
(0.5X) was used as running buffer, and DNA ladder
(markers) was loaded for fragment sizing. Electrophor-
esis was conducted at 1500 V for 3 h, and the gels were
viewed under ultraviolet rays.
Gel extraction, DNA sequencing
The resulting DNA fragments generated from amplifica-
tions were purified by excising bands from the agarose gel
after electrophoresis. The DNA was recovered using QIA-
quick gel extraction kit (Qiagen). The nucleotide sequences
of the purified dioscorin genes were obtained with a genetic
analyser. The sequencing amplifications were performed in
a20-μL reaction mixture consisting of 400 ng of DNA to
be sequenced, 10 pmole of dioscorin-specific primers 5-
CTCCTCTCCTCCCTCCTCTT-3(forward primer) and
10 pmole of 5- GGGGGTACAATGGAGAAGTG-3(re-
verse primer) and 4 μL of Reaction Dye Terminator Premix
(Qiagen) with standard sequencing conditions. Amplifica-
tion was performed in a thermowell microtitre plate (Costa
Corporation) using Perkin Elmer programmable Thermal
Controller model 9600. The cycling program was 36 cycles
of 94 °C for 1 min for denaturation, 60 °C for 1 min for an-
nealing ofprimersand7Cfor2min forextension.Amp-
lification products were stored at 4 °C before use. One
microlitre of 125 mM EDTA, 1 μL 3 M sodium acetate (pH
4.8), 25 μL 100% ethanol (20 °C) and 50 μL70%ethanol
(20 °C) were added to the amplification products, mixed
and centrifuged for 10 min at 10,000 rpm at 4 °C. DNA pel-
let was dried at room temperature and re-suspended in
5μL sterile deionized distilled water. One microlitre of the
re-suspended DNA was added to 9 μL Hi formamide,
mixed and denatured for 3 min at 94 °C. It was placed in-
side ABI PRISM 3130 X1 genetic analyser, which carried
out the automated sequencing analysis using a standard se-
quencing module with Performance Optimized Polymer
and 50-cm array.
Sequence analysis
The nucleotide sequences of the purified dioscorin genes
were subsequently translated to protein sequence using
bioinformatic resource tool from CLC Genomics Work-
bench software (CLC Bio Denmark). The nucleotide and
translated protein sequences were further subjected to
computer-based homology search with NCBI BLAST pro-
gram. Phylogenetic analysis was carried out to compare
the relationship of the major storage protein of D. bulbi-
fera with the storage proteins of other Dioscorea spp.
Results
Crude extracts
The protein concentration of crude extracts of the aerial
tuber of Dioscorea bulbifera extracted at different
temperature and varying pH is as shown in Fig. 1. The
result showed that maximum protein concentration was
obtained at pH 8.3 and at room temperature, 25 °C.
Osukoya and Kuku Journal of Genetic Engineering and Biotechnology (2020) 18:29 Page 4 of 14
Purification of storage protein
The crude extract of the aerial tuber of Dioscorea bulbifera
(379.60 mg) when subjected to 70% ammonium sulphate
precipitation gave 130.80 mg protein which corresponds to
34.46% yield. The elution profile of the ion-exchange chro-
matography on DEAE-Sepahdex A-25 of the dialyzed pro-
tein sample is as presented in Fig. 2a. Two protein peaks
were obtained from the DEAE-Sephadex A-25 column, one
unadsorbed peak and the adsorbed protein peak which was
eluted with 150 mM NaCl. The adsorbed peak which was
pooled and further purified by gel filtration on Sephadex
G-75 column is as shown in Fig. 2b. At the end of purifica-
tion, the amount of protein recovered was 13.20 mg, corre-
sponding to 3.48% of the starting material.
Molecular weight of D. bulbifera storage protein
The molecular weight of the native storage protein of the
aerial tuber of D. bulbifera as determined by gel filtration
on Bio-gel P-100 was 22,000 Da. The subunit molecular
weight, which was determined by SDS-PAGE under de-
naturing conditions, was estimated to be 21,095 Da (Fig. 3).
Detection of protein-bound carbohydrate
The storage protein of the aerial tuber did not stain
purplish-red with Schiffs reagent suggesting that it has
no covalently linked carbohydrate molecule and thus is
not a glycoprotein.
Amino acid composition
The amino acid composition of the storage protein of
the aerial tuber of D. bulbifera is presented in Table 1.
The amino acid composition is characterized by an
abundance of neutral and charged polar amino acids, es-
pecially tyrosine, arginine, glutamate and cysteine, which
constituted about 58% of the total concentration amino
acids of the protein (g/100 g protein). Among the non-
polar amino acids, proline and phenylalanine were
present in relatively high concentration. Of the sulphur-
containing amino acids, concentration of cysteine was
higher when compared with methionine. Tryptophan,
which was probably destroyed during acid hydrolysis of
the protein, was not detected.
Fig. 1 Protein concentration of the crude extracts of Dioscorea bulbifera aat different temperature and bat different pH
Osukoya and Kuku Journal of Genetic Engineering and Biotechnology (2020) 18:29 Page 5 of 14
Carbonic anhydrase activity
The protein had low carbonic anhydrase activity (0.202
units/mg) as compared with standard carbonic anhy-
drase from bovine erythrocytes.
Dehydroascorbate reductase activity
The storage protein of the aerial tuber of D. bulbifera
exhibited dehydroascorbate reductase activity. The pro-
tein was able to regenerate ascorbate from dehydroas-
corbate in the presence and absence of glutathione as
shown in Fig. 4a, b. In the presence of glutathione, the
specific activities of dehydroascorbate reductase for the
protein were 4.14 and 6.01 μmol ascorbic acid produced/
min/mg protein at pH 6.5 and pH 7.0, respectively. In
the absence of glutathione, the specific activities were
2.07 and 2.76 μmol ascorbic acid produced/min/mg pro-
tein at pH 6.5 and pH 7.0, respectively. No activity was
observed at pH 6.0.
Monodehydroascorbate reductase activity
The storage protein from the aerial tuber of D. bulbifera
showed monodehydroascorbate reductase activity. The
protein reduced monodehydroascorbate to ascorbate
coupled with NADH oxidation. At pH 6.0, the activity
was 0.0017 units/mg which implies that the amount of
protein required to oxidize 1 μmol of NADH per min at
pH 6.0 was 0.0017 units/mg. At pH 6.5 and pH 7.0, the
activity was 0.00038 units/mg and 0.00051 units/mg re-
spectively. Monodehydroascorbate reductase activity was
higher at pH 6.0 than at other pH as shown in Fig. 4c.
Trypsin inhibitory activity
Different amounts of the protein were used to determine
trypsin inhibitory activity, and the activity was expressed
as micrograms of trypsin inhibited as shown in Fig. 5.A
positive correlation (r
2
= 0.9752) was found between
trypsin inhibitory activity and amounts of storage pro-
tein from the aerial tuber of D. bulbifera. The storage
protein of the aerial tuber of D. bulbifera exhibited low
trypsin inhibitory activity with an average of 0.94 μg
trypsin inhibited per 100 μg of the protein.
Presence of dioscorin genes
Screening of the primer sets designed for the study with
the genomic DNA samples revealed some of the specific
primers were able to detect the dioscorin gene in the
Fig. 2 Column chromatography profile of Dioscorea bulbifera. aIon-exchange chromatography of dialyzed 70% ammonium sulphate precipitate
of the extract of the aerial tuber of Dioscorea bulbifera on DEAE Sephadex A-25 column (elution buffer: 0.05 M Tris-HCl buffer pH 8.3. Adsorbed
protein was eluted with 0.05 M Tris-HCl buffer pH 8.3, containing 150 mM NaCl). Column size (1.5 × 20) cm; flow rate 15 ml/h; fraction size 5 ml. b
Gel filtration of adsorbed peak from ion-exchange on Sephadex G-75 column (elution buffer: 100 mM Tris-HCl pH 7.9 with 100 mM NaCl). Column
size (1.5 × 40) cm; flow rate 27 ml/h; fraction size 3.6 ml
Osukoya and Kuku Journal of Genetic Engineering and Biotechnology (2020) 18:29 Page 6 of 14
genomic DNA sample of Dioscorea bulbifera. Target se-
quences are readily obtained by polymerase chain reac-
tion if the flanking sequences of the target sequences are
known. The presence of dioscorin gene was thus estab-
lished in the genomic DNA extracted from the aerial
tuber of Dioscorea bulbifera. Sequence analysis of dios-
corin gene DNA marker produced two DNA fragments
and nucleotide sequence sizes which were DBSPOOA1-
556 bp and DBSPOOA2-913 bp, respectively (Fig. 6a).
Homologous similarities of the genes and sequence
alignments
BLAST homology search using nucleotide sequence of
DBSPOOA1-556 gave significant alignments of 100%
nucleotide identity with Dioscorin B from Dioscorea
alata (dioB-1), 96% nucleotide identity with Dioscorea
oppositifolia microsatellite Dios23 sequence and 88% nu-
cleotide identity with Arabidopsis thaliana chromosome
3. DBSPOOA2-913 gave significant alignments of 91%
nucleotide alignment with D. alata voucher GZY109
ribulose-1,5-bisphosphate carboxylase large subunit
(rbcL) gene and 81% nucleotide identity with D. japonica
voucher Hsu 231 ribulose-1,5-bisphosphate carboxylase
large subunit (rbcL) gene (Table 2).
BLAST homology search using translated amino acid
sequences from the nucleotide sequences of
DBSPOOA1-556 and DBSPOOA2-913 also produced se-
quence homology with known protein sequences from
Dioscorea spp. DBSPOOA1-556 conceptual amino acid
sequence has 75% amino acid sequence identity with
Dioscorin B from D. alata, 73% identity with the pre-
dicted S-type anion channel SLAH1-like from Solanum
tuberosum (potato), 71% identity with the hypothetical
protein OsJ_01095 of Oryza sativa Japonica (rice) group,
68% identity with S-type anion channel SLAH1 of A.
thaliana and 60% amino acid sequence identity with the
storage protein of Dioscorea cayenensis. The minimum
molecular weight calculated from translated sequence of
DBSPOOA1-556 using Protparam online server (https://
web.expasy.org/protparam/) is 20,456.38 Da, which is
similar to what was obtained for the subunit molecular
weight of the storage protein by SDS-PAGE (21,095 Da).
DBSPOOA2-913 conceptual amino acid sequence also
showed homology with other known proteins, such as
ribulose-1,5-bisphoshate carboxylase/oxygenase large
subunit of D. bulbifera, putative carbonic anhydrase of
Neosartorya fischeri and putative Dioscorin from O.
sativa Japonica group (Table 3).
Relationship among the storage protein gene from the
aerial tuber of D. bulbifera (DBSPOOA1-556 and
DBSPOOA2-913) obtained in this study and the storage
protein genes from other Dioscorea spp. was revealed by
CLCBio homology nucleotide sequence alignment un-
weighted pair-group method arithmetic (UPGMA)
Fig. 3 Electrogram of SDS-polyacrylamide gel electrophoresis of the
storage protein of the aerial tuber of D. bulbifera. PAGE was carried
out in the presence of SDS using the discontinuous Tris-glycine
buffer system. The separating gel was 10%, and stacking gel was
3.5% acrylamide. Other conditions were as described in text. Lane A:
standard proteins; B: gel filtration pooled fractions; C: ion exchange
pooled fractions; D: ammonium sulphate precipitate; E: crude extract
Table 1 Amino acid composition of the storage protein of
Dioscorea bulbifera
Amino acid Concentration (g/100 g protein)
Lysine 3.50
Histidine 4.43
Arginine 11.87
Aspartic acid 4.41
Threonine 3.97
Serine 4.10
Glutamic acid 9.92
Proline 16.20
Glycine 4.39
Alanine 5.79
Cysteine 9.66
Valine 4.25
Methionine 3.75
Isoleucine 4.56
Leucine 4.09
Tyrosine 23.11
Phenylalanine 12.29
Tryptophan ND
Osukoya and Kuku Journal of Genetic Engineering and Biotechnology (2020) 18:29 Page 7 of 14
analysis. The analysis revealed that DBSPOOA1-556 and
DBSPOOA2-913 are distinctly different. However,
DBSPOOA1-556 formed a cluster with other Dioscorin
genes from different Dioscorea spp. from Asian coun-
tries, but DBSPOOA2-913 was distinctly different from
all known Dioscorin genes as shown in Fig. 6b.
Discussion
The crude extracts of fresh aerial tuber of D. bulbifera
prepared at 25 °C and at pH 8.3 showed optimum pro-
tein concentration, which is similar to the results ob-
tained from previous reports [23,24] on the effect of
heating temperature and pH on the major storage pro-
tein of various yam species. There was reduction in
protein concentration of Dioscorea alata L. var. pur-
purea at increasing temperature, and at temperature
above 90 °C, there was complete denaturation of the pro-
tein. Protein concentrations of D. alata L. var. Tainung
No. 2 and D. japonica Thunb. Var. pseudojaponica
showed similar trend with D. alata L. var. purpurea with
increasing heating temperatures except that the storage
proteins were not extractable at temperatures above
80 °C. Protein concentrations of the yam storage pro-
teins were not changed after heating at temperatures be-
tween 30 and 40 °C [23]. Protein concentration of D.
bulbifera storage protein was highest at pH 8.3 which
compares reasonably with what was obtained for the
major storage proteins of other yam tubers [18,2426].
Fig. 4 Dehydroascorbate reductase activity of storage protein of the aerial tuber of D. bulbifera at pHs 6.5 and 7 with (a) or without (b)4mM
glutathione and (c) monodehydroascorbate reductase activity of the storage protein of the aerial tuber of D. bulbifera at pHs 6, 6.5 and 7
Osukoya and Kuku Journal of Genetic Engineering and Biotechnology (2020) 18:29 Page 8 of 14
At acidic medium, low protein concentrations were
observed for D. alata L. var. purpurea,D. alata L.
var. Tainung No. 2 and D. japonica Thunb. Var.
pseudojaponica [24].
The major storage protein of the aerial tuber of D. bul-
bifera obtained was about 87% of the total protein of the
aerial tuber which is similar to the percentage concen-
tration of the major storage proteins from other yam tu-
bers [27]. DB2, the major storage protein of Dioscorea
batatas, accounted for 50% of the total protein of the
tuber [21]. The methods of purification of the storage
proteins follow similar trends of ammonium sulphate
precipitation followed by ion-exchange chromatography
and hydrophobic or gel filtration chromatography, or a
combination of any two of these steps [21,23]. However,
some researchers purified the major storage proteins
from the yam tubers using a one-step purification proto-
col either by ion exchange (most especially on DE-52
column) or gel filtration on Sephadex G-75 [28,29].
The native molecular weight of D. bulbifera major
storage protein was estimated to be 22,000 Da while the
subunit molecular weight was 21,000 Da, suggestive of a
monomeric structure for the protein. This result is in
contrast with those obtained for other underground yam
tuber storage proteins. Dioscorins purified from other
yam tubers showed a number of isoforms of about 31,
000 and 32,000 Da [11,30]. The storage protein isolated
from the tuber mucilage of D. batatas had molecular
weight above 250,000 Da while that from D. cayenensis
was 31,000 Da [29]. The dioscorins isolated from D.
batatas showed two bands (28,000 and 82,000 Da) on
non-reducing SDS-PAGE and only one band (32,000
Da) under reducing condition [25]. On the other hand,
D. alata was reported to have four subunits with mo-
lecular weight of 32,000 Da [31], while the storage pro-
tein of Dioscorea opposita was a monomeric protein
with molecular weight of 32,000 Da [32]. Wang et al.
[33] also purified a 32,000-Da storage protein from D.
purpurea. Different yam cultivars have therefore been
reported to behave differently in protein composition
and structure [34].
D. bulbifera storage protein is not glycosylated as
shown by periodic acid Schiffs reagent (PAS) staining
technique. Storage proteins from D. batatas and Dios-
corea rotundata were also reported not to be glycosyl-
ated with PAS staining method [21]. On the contrary,
the yam storage proteins from D. batatas,D. alata cv.
Tainong No. 1 [27] and D. japonica [22] were reported
to be glycosylated using conA-peroxidase staining
method. The protein from D. opposita was also shown
to be glycosylated with PAS staining [32].
Amino acid composition analysis of D. bulbifera stor-
age protein revealed that it is characterized by high con-
tent of tyrosine, proline, phenylalanine, cysteine,
glutamic acid and arginine. The high content of cysteine
residues showed some similarity with the dioscorins
from D. batatas and D. japonica [22,25,34] with high
half-cystine content. In contrast, dioscorins from four
cultivars of D. alata (Tainung No. 1, Tainung No. 2,
Dasan and Chanhon) had only trace amounts of cysteine
[35]. Cysteine, a sulphur-containing amino acid, even
though non-essential is required in the diet to meet the
bodys requirement. Sulphur is an important element ne-
cessary for normal growth and metabolism. Cysteine has
been implicated in anti-ageing, promoting healthy hair
and skin and also boosts the immune system. Cysteine
Fig. 5 Trypsin inhibitory activity of the major storage protein of the aerial tuber of D. bulbifera
Osukoya and Kuku Journal of Genetic Engineering and Biotechnology (2020) 18:29 Page 9 of 14
residues are also very important especially in crosslink-
ing proteins, increasing the rigidity of proteins and also
conferring proteolytic resistance. The storage protein of
D. bulbifera contains high amounts of essential amino
acids, approximately 40.9% of the total concentration of
amino acids (g/100 g protein), especially phenylalanine
and arginine.
The 22 kDa storage protein of the aerial tuber of D.
bulbifera exhibited carbonic anhydrase activity, albeit
low. Carbonic anhydrases are zinc metalloenzymes that
catalyze the simple interconversion of CO
2
and HCO
3
.
They are pH regulatory and metabolic enzymes found in
almost all organisms. In higher plants, carbonic anhy-
drases play a vital role in CO
2
fixation during photosyn-
thesis [36]. In mammals, they are involved in respiration
[37]. Carbonic anhydrases are found in many tissue
where they participate in many biological processes such
as acid-base regulation, respiration, carbon dioxide and
ion transport, bone resorption, ureagenesis, gluconeo-
genesis, lipogenesis and electrolyte secretion. Thus, they
are important therapeutic targets for treatments of de-
rangements such as edema, glaucoma, obesity, cancer
and epilepsy [38]. Six genetically distinct carbonic anhy-
drases gene families have been identified (α-, β-, γ-δ-, z-
and η-carbonic anhydrases) [39,40]. Hou et al. [25]
showed that the major storage protein of D. batatas had
carbonic anhydrase activity, which could not be detected
in another study by Gaidamashvili et al. [21]. The dis-
crepancies in the two reports, albeit, in the same yam
species could not be explained. Also, carbonic anhydrase
activity was detected in the major yam storage proteins
from different species of Dioscorea,D. alata (var. Tai-
nong 1, var. Tainong 2, var. Zhongguochang) and D.
pseudojaponica var. Keelung [27]. Xue et al. [41] also
Fig. 6 aPCR amplification of D. bulbifera Dioscorin genes (DBSPOOA) with Dioscorin specific primer set (5-CTCCTCTCCTCCCTCCTCTT-3and 5-
GGGGGTACAATGGAGAAGTG-3). PCR products were resolved on agarose gels and stained with ethidium bromide. The last lane shows DNA
ladder containing DNA fragments of defined length for sizing the bands in the experimental PCRs. The bands outlined in green and red
represent DBSPOOA1 and DBSPOOA2, respectively. bPhylogenetic relationship between DBSPOOA genes and other Dioscorin genes
Osukoya and Kuku Journal of Genetic Engineering and Biotechnology (2020) 18:29 Page 10 of 14
revealed that yam storage proteins, dioscorins, catalyse
reactions assumed by carbonic anhydrases.
Ascorbic acid (vitamin C) is a plant secondary metab-
olite involved in a number of physiological processes.
The main role of ascorbic acid is to neutralize free radi-
cals and prevent against oxidative damage [42]. It also
functions as a cell signalling modulator in cell division,
growth regulation and senescence in plants [43,44]. Be-
cause of the deleterious effects of reactive oxygen species
(mostly as a result of salt imbalance), plants usually have
well-developed enzymatic and non-enzymatic antioxi-
dant defense system [45]. In plants, enzymes involved in
the ascorbate-glutathione pathway (ascorbic acid-specific
peroxidase, monodehydroascorbate reductase, dehy-
droascorbate reductase and glutathione reductase) assist
in peroxides (formed as by-products of normal metabol-
ism or as a result of environmental stresses) detoxifica-
tion [46]. In its role as an antioxidant, ascorbic acid is
univalently oxidized to monodehydroascorbate, an en-
dogenous index of oxidative stress, which in turn rapidly
dissociates to form ascorbic acid and dehydroascorbate
in a reaction catalysed by mondehydroascorbate reduc-
tase [46]. Thus, monodehydroascorbate reductase and
dehydroascorbate reductase are important in the regulat-
ing ascorbic acid level and its redox state during oxida-
tive stress [47]. The major storage protein of the aerial
tuber of D. bulbifera was shown to have both dehydroas-
corbate reductase and monodehydroascorbate reductase
activities. The dehydroascorbate reductase activity was
higher in the presence of gluthathione. Dehydroascor-
bate reductase activity was also detected without glutha-
tione but was lower when compared to the activity in
the presence of gluthathione. The activity was also found
to be pH dependent. At pH 6.0, there was no activity de-
tected without gluthatione. This is similar to the report
of the yam storage proteins, dioscorins, of D. batatas
tuber which displayed both dehydroascorbate reductase
and monodehydroascorbate reductase activities with and
without gluthathione [18]. These activities might repre-
sent an important defense in the cytoplasm of yam cells
in response to environmental oxidative stress [18].
Most storage proteins have been reported to play pro-
tective roles against environmental stresses, such as act-
ing as protease inhibitors [48]. Protease inhibitors in
plants are usually termed as anti-nutritional compounds
because of their ability to inhibit digestive enzymes.
However, their presence in plants is often as a result of
an evolutionary adaptation which allows plants to sur-
vive under natural conditions [49]. In plants, protease in-
hibitors may be important in regulating and controlling
endogenous proteinases, serving as storage proteins, and
acting as protective agents against insect and microbial
proteases. Protease inhibitors have also been classified
under potential cancer-protective micro-components, by
controlling misfunctioning of certain proteases in cancer
progression [49]. The N-terminal amino acid sequences
Table 2 Dioscorin gene DNA marker (DBSPOOA1-556) nucleotide sequence and deduced amino acid sequence alignments and
homologous details
DBSPOOA1 gene
Nucleotide sequence (556 bp) CCTCTCTCTCACTCAACTTTTGCCGCATCCCCCCACCACTCCCCTCCCAGCACTCCCTCTCCTCTCCTTGCCCCCC
TTCTACCCTCCTGCCCCCCCGTTCCAAAACTTCTTCTATTCCCCATCTGTTTTTACCAAAGATGATGATCCTTAACT
TCTTCTTCCTCCACTTCTCCATTGTACCCCCTTAATTTTACGGCCAGTGGTTCACCAAAGGAAGAAAATTCTTATT
GGTCGCCGCAAATCCGACGAGCTTGTTGAGCGTCATAGCCAATCTTGCCGGAGCAAGAGCTGCG
GCAAGAATGGGATGGAAAGAGAGTGCGGTTTGCATGTTTTCACTAGCCATGACTCATTACCTCGTGTTATTCGTA
ACCTTGTATCAGCGTCTACAAGGCAGCAACAGCCTTCCGGCGATGCTCCGTCCGGCTTTCTTCCTCTTCTTCGCCG
CACCGAGCATGGCCAGTTTCACCTGGGTGTCAATTTCCGGCGAATTCGACATCTCATGCAAAATGCTTTTCTTCCTT
TCTCTTTTCCTCTTCACTTCTCCATTGTACCCCCT
Translated amino acids sequence
(184 amino acids)
SLSLNFCRIPPPLPSQHSLSSPCPPSTLLPPRSKTSSIPHLFLPKMMILNFFFLHFSIVPPFYGQWFTKGRKFLLVAANPT
SLLSVIANLAGARAAARMGWKESAVCMFSLAMTHYLVLFVTLYQRLQGSNSLPAMLRPAFFLFFAAPSMASFTWVS
ISGEFDISCKMLFFLSLFLFTSPLYP
Nucleotide sequence alignments 1. Dioscorea alata Dioscorin B (dioB-1) mRNA, complete cds; 100% identity; Accession (AF243526.1)
2. Dioscorea oppositifolia microsatellite Dios23 sequence; 96% identity; Accession (JQ955632.1)
3. Dioscorea cirrhosa isolate PS5034MT03 ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) gene,
partial cds; chloroplast; 95% identity; Accession (HQ637837.1)
4. Dioscorea alata voucher GZY110 ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL)
gene, partial cds; chloroplast; 95% identity; Accession (JX139768.1)
5. Ipomoea batatas voucher GZY116 ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit
(rbcL) gene, partial cds; chloroplast; 95% identity; Accession (JX139773.1)
6. Arabidopsis thaliana chromosome 3, complete sequence; 88% identity; Accession (CP002686.1)
Translated amino acids sequence
alignments
1. Dioscorin B [Dioscorea alata]; 75% identity; Accession (AAF44711.1)
2. PREDICTED: S-type anion channel SLAH1-like [Solanum tuberosum]; 73 % identity; Accession
(XP_006342228.1)
3. Hypothetical protein OsJ_01095 [Oryza sativa Japonica Group]; 71% identity; Accession (EAZ11241.1)
4. S-type anion channel SLAH1 [Arabidopsis thaliana]; 68% identity; Accession (NP_176418.2)
5. Storage protein [Dioscorea cayenensis]; 60% identity; Accession (CAA53781.1)
Osukoya and Kuku Journal of Genetic Engineering and Biotechnology (2020) 18:29 Page 11 of 14
of storage proteins purified from yam bean (Pachyrhizus
erosus), YGB1 and YGB2, showed high homology to
cysteine protease, but both of them exhibited low prote-
ase activities using azocasein as substrates [50]. The
storage protein from the aerial tuber of D. bulbifera had
low trypsin inhibitory activity as compared with those
from sweet potato roots [48]. However, just like the re-
sult obtained from this study, dioscorin from D. batatas
showed only a weak trypsin inhibitory activity, with
1.9 μg of trypsin inhibited per 100 μg of the protein [25].
Large amounts of these storage proteins could provide a
significant protective role in the aerial tuber even with
this low trypsin inhibitory activity.
Two dioscorin gene DNA markers were amplified
from the total genomic DNA of the aerial tuber of D.
bulbifera, DBSPOOA1-556 and DBSPOOA2-913 using
primers designed with NCBI primer BLAST tool and
Primer 3. In a study done by Barman et al. [51], dios-
corin gene was also amplified from RNA extracted from
different species and cultivars of Dioscorea using forward
and reverse primers designed with NCBI primer BLAST
tool and Primer 3 software. The deduced amino acid se-
quences from D. bulbifera genes gave sequences of 184
and 304 amino acid residues respectively. The deduced
amino acid sequences from DBSP00A1-556 was highly
homologous to the amino acid sequences from the stor-
age proteins of D. alata (dio-B) (75% identity) and D.
cayenensis (60% identity). The deduced amino acid se-
quences for the two genes did not show any similarities
with tuber storage protein genes of patatin and spora-
min. Proteins of the same family normally perform the
same biochemical function and may be related phylo-
genetically [52]. Amino acid sequence analyses have
been found necessary for unambiguous evidence of
structural relationship among proteins. Proteins with
structural similarities tend to have evolutionary and
functional similarities. In addition to having sequence
similarities with the storage proteins from D. cayenensis
and D. alata (dio-B), the translated amino acid sequence
from DBSPOOA1-556 had homologous sequences with
the predicted S-type anion channel SLAH1-like from So-
lanum tuberosum, and S-type anion channel SLAH1
from Arabidopsis thaliana. The predicted S-type anion
channel from Solanum tuberosum is a hypothetical pro-
tein OsJ_01095 from Oryza sativa Japonica group and
the S-type anion channel SLAH1 (SLAC1-homolog pro-
tein 1) from Arabidopsis thaliana is a slow and weak
voltage-dependent S-type anion efflux channel which is
involved in the maintenance of anion homeostatis. Also,
the translated amino acid sequence from DBSPOOA2-
924 was highly homologous with a putative dioscorin
from Oryza sativa Japonica group and putative carbonic
anhydrase from Neosartorya fischeri NRRL 181. Of the
two genes of the storage protein from the aerial tuber of
Table 3 Dioscorin gene DNA marker (DBSPOOA2-913) nucleotide sequence and deduced amino acid sequence alignments and
homologous details
DBSPOOA2 gene
Nucleotide sequence (913 bp) ACCCTCATGGGTGTCGGTGAGGAGAAGGTGACCCGGCAGCGGAGTTTGTTCGCAGGAGATAGAGGAGTCCAGGGAGA
CCTACGGTGGGCTGTTTATGAATGTCTACGTGGGGGACTTGATTTTACCAAAGATGATGACGCTGATGAAGGGGGGGAA
AAGCACGGGAGGGTGAGTCATGGCTGGTGAAAACTTGCTAACCGCACTCTCTTTCCATCCCATTCTGGCCGCAGCT
CTTGCTCCGGCAAGATTGGCTATGACGCTCAACAAGCTCGGCGGATTTGCGGCGACCAATAAGAATTTTCTTCCTTTG
GGGAACCACTGGTCGTAGATTTTTACGTCGAGGACAATTACCGGGAGCGAAAAGAAGAGACACACAAGAATGAAC
GGAGGAGATTGAGGATCAAGAAAAGGAGTTGATTGGAGAAGGAGCAAGAAAGAATTCCAAGGTGCGAATAGATAGT
TCATGCCGATATAGTCAGACAACTCGGCGCGAACATGGTGGAACTGGCGGAGGCACCGGAGGAGGAAGAGGAGGG
AGGAGAGGAGAACTATTTCTCTGCTTTACCTCCAATTATCATCTAATGTGATTCCCTCCTTCCATAAAATCCATCTTATCT
TTATTAAAATGGGTGCTCTTCTACCTGCTGCTAATATTCCATAAACCACTTGATAACCTGAATTTATTTATCTTTATCCTA
CTGTACAAGGCATCTTAGAAAAAGCGTTGTTTCCTTCTTTCTACCACATCCAACTTGGATTGTTATTCCCTTCCTTTGCA
AAATTTATATAAGATTTTTTTTTCCTCTCGAATAACCCTGTGACCCCTTTAGCTGGGTGAACTTTTCACCATTCGGACAT
GCTCTCCTGTACCATATTTTTCTTTGTGCCTGCTTCCTATTTACCCCTCCCACAA
Translated amino acid sequence
(304 amino acids)
TLMGVGEEKVTRQRSLFAGDRGVQGDLRWAVYECLRGGLDFTKDDDADEGGEKHGRVSHGWKLANRTLFPSHSGRSS
CSGKIGYDAQQARRICGDQEFSSFGEPLVVDFYVEDNYRERKEETHKNERRRLRIKKRSLEKEQERIPRCEIVHADIVRQL
GANMVELAEAPEEEEEGGEENYFSALPPIIICDSLLPNPSYLYNGCSSTCCYSINHLITIYLSLSYCTRHLRKSVVSFFLPHPT
WIVIPFLCKIYIRFFFPLEPCDPFSWVNFSPFGHALLYHIFLCACFLFTPPT
Nucleotide sequence alignments 1. Dioscorea japonica voucher Hsu 231 ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit
(rbcL) gene, partial cds; chloroplast; 89% identity; Accession (JQ733767.1)
2. Dioscorea alata voucher GZY109 ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL)
gene, partial cds; chloroplast; 91% identity; Accession (JX139767.1)
3. Dioscorea nitens voucher YSL 2628 ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit
(rbcL) gene, partial cds; chloroplast; 89% identity; Accession (JQ733810.1)
Translated amino acid sequences
alignments
1. Putative Dioscorin [Oryza sativa Japonica Group]; 41% identity; Accession (BAC99799.1)
2. Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit [Dioscorea esculenta]; 94% identity;
Accession (AFC89185.1)
3. Carbonic anhydrase, putative [Neosartorya fischeri NRRL 181]; 45% identity; Accession (XP_001267068.1)
4. Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit [Trebouxia simplex]; 94% identity;
Accession (AIJ50559.1)
Osukoya and Kuku Journal of Genetic Engineering and Biotechnology (2020) 18:29 Page 12 of 14
D. bulbifera, DBSPOOA1-556 formed a cluster with
other dioscorin genes from different Dioscorea spp. from
Asian countries, but DBSPOOA2-913 was distinctly dif-
ferent from all known dioscorin genes. This could be be-
cause it is an aerial tuber and not an underground tuber.
It could also be because of the different geographical lo-
cations in which the yam species are cultivated. Several
factors such as root-crop species, local climate and
fertilization pattern have been reported to directly influ-
ence the composition of root crops [35]. The yam spe-
cies used in this study was cultivated in Africa in
contrast to various reported studies of other yam spe-
cies, D. alata,D. cayenensis, and D. japonica which are
cultivated in Asian countries.
Conclusion
In conclusion, a storage protein was isolated from the
aerial tuber of Dioscorea bulbifera for the first time. The
storage protein has similar functional properties and
structural homology with the storage proteins of other
Dioscorea species. The storage protein is heat stable and
exhibited carbonic anhydrase, dehydroascorbate reduc-
tase and trypsin inhibitory activities. It is also a good
source of essential amino acids; thus, the protein may be
suitable for development as functional food.
Authorscontributions
AK designed, developed and conceptualised the study. OAA carried out all
experiments, analysis and interpretation of data in this study as her PhD
research project under the supervision of AK and wrote the first draft of the
manuscript. Both authors read and approved the final manuscript.
Funding
This research did not receive any specific grant from funding agencies in
the public, commercial, or not-for-profit sectors.
Availability of data and materials
The datasets used and/or analysed during the current study are available
from the corresponding author on reasonable request.
Ethics approval and consent to participate
Not applicable
Consent for publication
Not applicable
Competing interests
The authors declare that they have no competing interest.
Author details
1
Department of Chemical Sciences, Afe Babalola University, Ado Ekiti, Nigeria.
2
Department of Biochemistry and Molecular Biology, Obafemi Awolowo
University, Ile-Ife, Nigeria.
Received: 13 November 2019 Accepted: 5 June 2020
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... In the presence of alcoholic extracts of Dioscorea bulbifera, stimulation of osteogenesis activity of osteoblastic cells has been seen in vivo condition on rat skeletons. [59] The extract of bulb is used in different indigenous system of medicines to cure cardiac and neurological disorders, [60] throat infection, [61] antiinflammatory actions, [62] antihyperglycemic activity [63] and also used as antileishmanial, [64] diuretic, [65] analgesic, [66] antihelminthic, [67] Antitumor, [68] and Antihypertensive [69] agent. ...
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Dioscorin was purified by DE-52 ion exchange chromatography from two yam species, Dioscorea alata L. cv. Tainong 1 (TN1) and Dioscorea batatas Decne (imported from Japan, JP). By different in vitro antioxidant tests, including DPPH radical and hydroxyl radical scavenging activity assay, a reducing power test, an anti-lipid peroxidation test, DNA damage protection, and inhibition of dihydrorhodamine 123 oxidation by peroxynitrite, it was shown that dioscorins from the two species exhibited different scavenging activities against DPPH and hydroxyl radicals, even after heating 100°C for 5 min. Dioscorins from TN1 were hydrolyzed by pepsin for different durations and the peptic hydrolysates exhibited DPPH radical scavenging activities. Peptic hydrolysates separated by Sephadex G-50 (F) gel filtration were tested for anti-DPPH radical activity. Results showed that fractions of smaller molecular weight still have antioxidant activities.
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A novel protein, designated as DOI, isolated from the Chinese yam (Dioscorea opposita Thunb.) could be the first protein drug for the treatment of menopausal syndrome and an alternative to hormone replacement therapy (HRT), which is known to have undesirable side effects. DOI is an acid- and thermo-stable protein with a distinctive N-terminal sequence Gly-Ile-Gly-Lys-Ile-Thr-Thr-Tyr-Trp-Gly-Gln-Tyr-Ser-Asp-Glu-Pro-Ser-Leu-Thr-Glu. DOI was found to stimulate estradiol biosynthesis in rat ovarian granulosa cells; induce estradiol and progesterone secretion in 16- to 18-month-old female Sprague Dawley rats by upregulating expressions of follicle-stimulating hormone receptor and ovarian aromatase; counteract the progression of osteoporosis and augment bone mineral density; and improve cognitive functioning by upregulating protein expressions of brain-derived neurotrophic factor and TrkB receptors in the prefrontal cortex. Furthermore, DOI did not stimulate the proliferation of breast cancer and ovarian cancer cells, which suggest it could be a more efficacious and safer alternative to HRT.