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Abstract
Una biblioteca de cDNA se construy en el vector l TriplEx2 utilizando poli (A TH) de ARN a
partir de semillas inmaduras de Cicer arietinum. El gen de la lectina fue aislado a partir de semillas
de garbanzo a travs de la biblioteca de deteccin y RACE-PCR. El ADNc de longitud completa de
la lectina de la semilla Chichpea (CpGL) es 972 pb y contiene un marco de lectura abierto de 807
pb que codifica una protena de 268 amino cidos. El anlisis muestra que los genes CPSL tiene una
fuerte homologa con otros genes de lectina de leguminosas. El anlisis filogentico mostr la
existencia de dos grupos principales y claramente indic que CPSL perteneca a la familia
especfica de manosa de las lectinas. RT-PCR revel que el gen CAA expresa constitutivamente en
diversos tejidos de plantas incluyendo flores, hojas, raz y tallo. Cuando el nivel de ARNm de
garbanzos lectina se comprob en las semillas en desarrollo, fue superior en 10 semillas DAF y
disminuy durante todo el desarrollo de la semilla.
Keywords: Cicer arietinum, expression analysis, leguminosae, lectin gene, nucleotide sequencing
Introduction
Las lectinas son un grupo grande y heterogneo de
protenas que tienen la capacidad de unirse
reversiblemente a un mono-o-oligosacrido especfico
(Van Damme et al. 1998). Se distribuyen de forma ubicua
en la naturaleza y son ms abundantes en las plantas, en
las que se pueden encontrar en las semillas, hojas,
cortezas, bulbos, rizomas, races y tubos en funcin de
los tejidos de las plantas (Lis y Sharon 1986; Gupta et al.,
2004). Sin embargo, la mayora de estudios sobre lectinas
han llevado a cabo en las legumbres, en particular, las
semillas en el que comprenden hasta 15% de la protena
total. Sobre la base de las relaciones evolutivas y
estructural, siete familias de lectina se han distinguido, de
los cuales lectinas de leguminosas se caracterizan mejor
(Peumans y Van Damme 1998). Aunque las lectinas se
han estudiado ampliamente con respecto a los hidratos de
carbono especificidad de unin y utilidad potencial para
el aislamiento y caracterizacin de glicoconjugados, las
Correspondence: K. R. Koundal, NRC on Plant Biotechnology, I.A.R.I., New Delhi 110012, India. Tel: 91 11 25848783. Fax: 91 11 25843984.
E-mail: kirparam@rediffmail.com
*The nucleotide sequence reported in this paper have been submitted to the GenBank and is available under the accession no. AY221982.
ISSN 1042-5179 print/ISSN 1029-2365 online q 2007 Informa UK Ltd.
DOI: 10.1080/10425170601060608
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Figure 1. AD. A, B. Agarose gel electrophoresis and Southern hybridization of restricted chickpea genomic DNA, Lane M. Marker l/ Hind III, 1.
Chickpea genomic DNA digested with Bam HI, 2. Chickpea genomic DNA digested with Eco RI; C, D. Agarose gel electrophoresis and Southern
hybridization of restricted cDNA clones, Lane M. Marker l/ Eco RI Hind III, Lanes 1-3. Chickpea lectin clone digested with Sfi I.
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Figure 2. The cDNA sequence and deduced amino acid sequence of chickpea seed lectin (CpSL). The start codon is underlined and stop codon is
italicized and underlined.
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199
Figure 3. Alignment of deduced amino acid sequence of CpSL (chickpea seed lectin) with other legume lectins: CAA47011 from Pisum sativum;
CAC42124 from Lens culinaris; CAF18558 from Lathyrus sativus and CAD27484 from Vicia faba. Identical amino acid residues are indicated
with yellow. The amino acid residues identical among any two of the five lectins are indicated with gray. The amino acid residues different from other
lectins are indicated with black.
0
100
CAD274
100
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CAA429
100
CAA763
CAD288
100
CAF185
49
60
P050
BAA364
54
CAA369
Figure 4. Phylogenetic tree analysis of CpSL (chickpea seed lectin) with
other legume lectins: CAA47011 from Pisum sativum; CAF18558
from Lathyrus sativus; CAC42124 from Lens culinaris; CAD27484
from Vicia faba; CAA42937 from Medicago truncatula; CAA76366
from Medicago sativa; CAD28836 from Phaseolus vulgaris;
CAF18557 from Vigna unguiculata; P05046 from Glycine max;
BAA36415 from Robinia pseudoacacia and CAA36986 from
Erythrina corallodendron. Number shown in the node indicates the
branch was supported with bootstrap value out of 1000.
cDNA library
Of 5 mg of total RNA obtained from 5 g of immature
chickpea seeds, 2 mg was purified using oligo (dT)
201
ues of chickpea, Lane M. Marker l/ Eco RI Hind III, Lanes 15. Total RNA from: 1, flower; 2, leaf; 3, root; 4, stem; 5, seed; Lane 6, Negative control; B. Expression
M. Marker l/ Hind III, Lanes 1-6. Seeds from: 1, 10 DAF; 2, 15 DAF; 3, 20 DAF; 4, 25 DAF; 5, 30 DAF; 6, 35 DAF; Lane 7, Negative control.
Being of plant origin, lectin genes will have high degree of compatibility with the
transgenic host plants and hence expected to provide sustained protection against sap
sucking insects, which cause major losses to crops.
Acknowledgements
This work was carried out under the financial assistance from the National
Agricultural Technology Project (Mission Mode), ICAR, New Delhi. We thank Drs S.
Anandhan and Ramesh Bhat for their help during this work.
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