208
Short Communication
RIBOSOMAL DNA SEQUENCE ANALYSIS OF DIFFERENT
GEOGRAPHICALLY DISTRIBUTED ALOE VERA PLANTS: COMPARISON
WITH CLONALLY REGENERATED PLANTS
A. Yagi1, Y. Sato1, Y. Miwa2, Amal Kabbash3*,
S. Moustafa3, K. Shimomura4 and A. El-Bassuony5
‫(إنداإاادناإ قي د إ‬ITS region 1)‫عند إجردء مإارنة دلإعادتإ قب نتدلإل) ا داصإ قيناداإنداإ البن دن إ قد دااإ ق نعد إ‬
‫إ ق ا د إ ة اً دنيإ نددًاإ ن د إ مرنددن إ ق يرددي لإندداإ دداإادداإ قًننددن إ‬Aloe vera‫قنددي اإ قءو ددي ااإل ددنصإقن ددن إ ق د نةإ‬
‫إ‬254‫إ إ‬252‫قياون إ ق بي لإ ماءوكًلإ ا ءإنر إ) ًاإأ هإوير إ يعن إااإ قب نتلإ قنًي اًي)ً اإ ه نإ م جإ قرنع ولإ‬
‫إإ‬254‫إ إ‬252‫إ ق يبياإعاتإ م جإ قرنع ودلإ‬A. vera‫إنإ هإناإ ن إإإإإإإ‬ITS region 1‫إ نننمإعاتإهذ إ ا بشنفإناإااناإ‬.
ITS ‫ت إ) ًاإأ إهننكإ)شننهنيإ)ننلخًنيإاننبدنيإ إ هدذ إود اإعادتإأ إهندنكإادًن لإعنقًدلإنداإت دلإتنعد لإ البن دن إنداإ ق ادناإ‬
‫إنداإعًندن إاداإ م ة لإ قبداإح داإ‬252‫إ اعإذقكإنإ إ إلحالا إ قرنع ولإلءو لإ قب نتدلإنداإ قد جإ قرنعد اإ‬.region 1
‫إتد إقديحأإأ دهإوبي رد إحدياإ‬micropropagated‫عاًهنإاداإ قن دًاإ ق دالإل قرنلداصإ اداإ قن ن)دن إ ق بكدناءلإ ق نقردلإ ق درءإ‬
‫إ ق بي قد إ ة اًدنيإ‬A.vera‫إةن نإ دن إ ا يدء فإ ق دًيإنداإ دن إ ق د نةإ‬.201-199‫إ إ‬99‫إ إ‬66‫اانا إاي تعإ قنًي اًي)ً إ‬
‫إإ ناإهذهإ ق ة للإرءىإ ) نعإطءوردلإ‬.‫إ دي صإ قخاًلإناإحنا إ قن ًاإ قرنلا‬regrenation‫هيإن لإاءحالإااإ قبا و إ‬
‫إناإ قي إ قني اإل نصإ قءو ي ااإ ي لمإر وئداإاداإأرداإ قبنءودنإندًاإ دن إ‬ITS region 1‫) نتلإت لإ قرنع لإقا اناإ‬
‫إااإ م ي عإ مدءىإااإ نحًلإ قبي عإ قارء نداإ قبا ود إ قدية ااإقن ن)دن إ ق د نةإ إ عاًدهإنإ ندنإ برد إأ إ‬A.vera‫ق نةإ‬
‫إةن دنإوكدي إ ن)ادنيإعداإجدبالندن إنداإ ق ي اداإ قرذ لًدلإ ق ًئًدلإنداإرنداإ‬ITS region 1‫إلحالا إناإت لإ قرنع لإناإااناإ‬
‫ إ‬. ‫اء حاإ)طيةإ قن ن‬
‫إ‬
A comparison of the sequences in an internally transcribed spacer (ITS) 1 region of rDNA
between clonally regenerated A.vera and the same species in Japan, USA and Egypt revealed the
presence of two types of nucleotide sequences, 252 and 254 bps. Based on the findings in the ITS
1 region, A.vera having 252 and 254 bps clearly showed a stable sequence similarity, suggesting
high conservation of the base peak sequence in the ITS 1 region. However, frequent base
substitutions in the 252 bps sample leaves that came from callus tissue and micropropagated
plants were observed around the regions of nucleotide positions 66, 99 and 199-201.The minor
deviation in clonally regenerated A.vera may be due to the stage of regeneration and cell
specification in cases of the callus tissue. In the present study, the base peak sequence of the ITS
1 region of rDNA was adopted as a molecular marker for differentiating A.vera plants from
geographically distributed and clonally regenerated A.vera plants, and it was suggested that the
base peak substitutions in the ITS 1 region may arise from the different nutritional and
environmental factors in cultivation and plant growth stages.
Key words: Aloe-vera, clonally regenerated A.vera, ITS 1 region, micropropagated.
1
Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama
University, Gakuen-cho, Fukuyama, Hiroshima 729-0929, Japan.
2
Faculty of Life Science and Biotechnology, Department of
Marine Biotechnology, Fukuyama University, Gakuen-cho,
Fukuyama, Hiroshima 729-0929, Japan. 3 Faculty of Pharmacy,
Tanta University, Tanta 8130, Egypt. 4 Faculty of Life Sciences,
Toyo University, 1-1-1,Izumino, Itakura, Gunma, 374-0193,
Japan 5 Graduate School of Science, Hiroshima University,1-3-1
Kagamiyama, Higashi-Hiroshima 739-8526, Japan.
*
To whom correspondence should be addressed.
Email: amkabbash_99@yahoo.com
Saudi Pharmaceutical Journal, Vol. 14, Nos. 3-4, July-October 2006
Introduction
Within the Asphodelaceae, the subfamily
Alooideae includes seven genera (Aloe, etc.). The
Alooideae is recognized as a taxonomically difficult
subfamily, because the succulent leaf morphology
significantlydepends on environmental conditions (1).
The initial taxonomic and biosynthetic investigations
were demonstrated in an Aloe section, Pachydendron,
on the basis of phytochemical studies of the leaf-
RIBOSOMAL DNA SEQUENCE ANALYSIS
exudatecompounds (2). However, recent experiments
disclosed some discrepancies between the chemical
composition ofexudates and morphological characters
of Aloe series, Asperifoliae (3). Variations in the
quality of A.vera were reported, especially with
regard to its gel. Owing to frequent adulteration of
carbohydrate fraction, one of the most important
ingredients, there was a great need to authenticate A.
vera as a medicinal plant. Apart from technical
inconsistencies, it appears that the range of carbohydrate types in the gel may relate to plants of
different geographical origin or possible varieties
(4).‫إ‬Recently, random amplified polymorphic DNA
method was used to differentiate between A.species
and this method provided an unequivocal detection
of A.species (5). The ITS 1 region is widely applied
in authentication and phylogenetic analysis on
Dendrobium species (6) and is comparable in Aloe
species. In the present study, the ITS 1 region of
A.vera was adopted as a molecular marker for accurate identification of Aloe plants, because the ITS 1
region appears to evolve under weak selection
constraints, and blocks of sequence homology occur
between related species on angiosperm phylogeny (7).
Materials and Methods
Voucher specimens of A.vera and the callusderived and micropropagated leaves (8) were
collected from the medicinal garden at Fukuyama
Univer- sity (the herbarium number of A.vera: 54-3),
the Plantations in Lyford Hilltop and Harlingen,
Texas, USA and the botanical garden, Cairo, Egypt.
Total DNA was isolated using a modified Apt
method from each sample leaf (0.7 g) (9).
Polymerase chain reaction(PCR):
The PCR reaction volumes were 50 µl and
contained 5 µl of 10x Ex Taq buffer [20 mM
(NH4)2SO4, 75 mM Tris-HCl, pH 8.8, 0.01% Tween
20, 2 mM MgCl2, 50% glycerol], 0.2 µl of Ex Taq
polymerase (1U), 0.2 mM dNTPs mixture, 0.5 µl of
100 pmol forward and reverse primers (synthesized
by Takara Biomedicals Co.) Japan and approximately
70-80 ng of genomic DNA. The primers used for
amplification of the ITS 1 region were aloe 18S20F
(5`-AAGTCGTAACAAGGTTTCCG-3`) and aloe
5.8S19R (5`-T- CTTCATCGACGCGAGAGC-3`)
primers, which were designed based on sequences
Saudi Pharmaceutical Journal, Vol. 14, Nos. 3-4, July-October 2006
209
alignment of 18S rDNA and 5.8S rDNA in several
Aloe species. Thirty cycles of PCR amplification ( 95


C, 30 sec; 50 
C, 2 min) were
performed on GeneAmp 9600 (Applied Biosystem).
The resulting PCR products were analyzed by
electrophoresis on a 2% agarose gel. The main
products were subcloned into the pGEM-T vector
(Promega). The recombinant plasmid DNA isolated
using the automatic DNA extraction machine, PI-50
(Kurabo Co. Ltd,), was linearlized with restriction
enzyme ScaI.
DNA sequencing and sequence analysis
The sequencing was performed by the dideoxy
chain termination method (10) using fluorescent Big
Dye Terminator cycle sequencing FS Ready Reaction
kit in a reaction mixture containing the purified
linearized plasmid DNA (50 ng) and analysis of the
reaction products on an ABIPRISMTM 310 Genetic
analyzer
(Applied
Biosystems). The sequencing
primer, pGEM-F from plasmid pGEM-T vector was
used:
5`-CGACGTCGCATGCTCC- GGC-3`. The
sequence was analyzed
using
the Genetyx-Mac
software package (Version 10: software Development
Co. Ltd.). Determination of the bp sequence was
carried out on at least more than five independent
clones for each same sample. Gene bank accession
numbers of bps 254 and 252 in sample numbers 1-6
are AB090282-AB090293.
Results
A comparison of the sequence in the ITS 1
region between clonally regenerated A.vera and the
same species in Japan, USA and Egypt revealed the
presence of two types of nucleotide sequences, 254
and 252 bps (Figs. 1 and 2). This suggests that 254
and 252 bps were highly conserved with a stable
sequence similarity in the ITS 1 region. While the
other A.vera tested including the clonally regenerated
plant leaves from callus tissue and micropropagated
plants from shoot tips of A. vera showed both 254
and 252 bps in the ITS 1 region, this difference may
arise from the parent chromosomes. Frequent base
substitutions in 252 bps of the micropropagated and
callus-derived plants were observed around the
region of nucleotide positions 66, 99, 184 and 199201, 240 (Figure 3).
210
KABBASH ET AL
Fig.1. Organization of the ITS region in Aloe vera. Arrows
indicate orientation and approximate position of primers
used in this study.
ated with plant tissue cultures (11) and micropropagation (12). The minor deviation in clonally
regenerated A.vera in Figure 3 may be due to the
stage of regeneration and cell specification in the
callus tissue cases. DNA content of micropropagated
A.vera was compared to that of normally cultivated
plants by cytological analysis and the decrease in
DNA content was observed in micropropagated
A.vera (13). The results corresponded to our
previous findings showing the decrease in glycoprotein, verectin, (8) and barbaloin contents (unpublished
data) in clonally regenerated A.vera. This suggests
that clonally induced mutations are associated with
phenotypic variation in A.vera.
In conclusion, we report the possible use of ITS 1
region of rDNA as a molecular marker for differentiating
A.vera from geographically different and
clonally regenerated A.vera and the base peak
sequences in ITS 1 region of rDNA were highly
conserved in geographically different A.vera.
Acknowledgments
We are grateful to Professors M.Fukunaga and
A. Nakata for advice and to Aloecorp (Broomfield,
CO USA) for providing A.vera plants.
Fig.2. Alignment of ITS 1 254 base peak sequences
Sample 1:Herbal garden in Fukuyama University;
2:Micropropagated plant; 3:Plantation in Harlingen,
Tx,USA; 4:Plantation in Lyford, Tx,USA; 5:Botanical
garden in Cairo, Egypt; 6:Callus-derived plant
Discussion
Plants clonally regenerated from callus cultures
and micropropagation possess a vast array of genetic
changes. DNA methylation polymorphisms are AssociSaudi Pharmaceutical Journal, Vol. 14, Nos. 3-4, July-October 2006
Fig.3. Alignment of ITS 1 252 base peak sequences
Sample codes are same as those in Fig.2.
RIBOSOMAL DNA SEQUENCE ANALYSIS
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