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Cloning and expression of lectin gene from cowpea and colocasia

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Title Cloning and expression of lectin gene from cowpea and colocasia
 
Creator Spurthi N.Nayak
 
Contributor Spurthi N.Nayak
 
Subject Plant Bio-Technology
 
Description Lectins are carbohydrate binding proteins of non-immune origin that
agglutinate cells, which are known to have insecticidal properties. Lectins, being of
plant origin have an added advantage over other exotic prokaryotic genes as there is
no problems related to codon bias, RNA processing and protein stability. In the
present study, an attempt was made to clone lectin genes from cowpea (Vigna
unguiculata) and colocasia (Colocasia esculentum). A 1kb DNA fragment was
amplified using heterologous primers designed from the lectin sequences of Arachis
hypogaea in cowpea and a 800bp amplicon resulted from gene specific primers in
colocasia. The amplicons were cloned to pTZ57R/T and transformed into E.coli
DH5a.
Sequence data showed recombinant clones pSKR0601 and pSKR1904 to
contain 994bp and 795bp respectively. The pSKR0601 nucleotide sequence has 97
per cent homology with A.hypogaea and the translation product has 97 per cent
homology with mannose/glucose binding lectin precursor, showing the novelty of the
gene. Colocasia lectin sequence has 95 per cent homology with the published
colocasia lecin sequence. The amino acid sequence has 99 per cent homology with
globulin G1 precursor of C. esculentum. The conserved domain search indicated
single Lectin legB domain in cowpea lectin and two B_lectin domains (bulb mannose
specific) in C. esculentum.
The confirmed clones were further cloned into prokaryotic expression vector
(pET28a (+)) and plant transformation vector (pHS100). SDS-PAGE analysis of
pSKR1803 and pSKR2105 showed the expression of 29kDa and 26kDa proteins
respectively. The recombinant clones pSKR2804 and pSKR2606 were mobilized into
Agrobacterium tumefaciens LBA4404 by tri-parental mating and Agrobacterium
(pSKR2804) mediated transformation of tobacco was carried out.
 
Date 2016-10-17T13:32:03Z
2016-10-17T13:32:03Z
2005
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/80702
 
Format application/pdf
 
Publisher UAS, Dharwad