Molecular Characterization of Alternative oxidase and Trans-sialidase genes of Trypanosoma evansi of Camel
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Date
2016
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Rajasthan University of Veterinary and Animal Sciences, Bikaner - 334001
Abstract
The present study was carried out to isolate the Alternative
oxidase and Trans-sialidase genes of Trypanosoma evansi using PCR,
clone the amplicons in a suitable plasmid vector and then
characterization of above genes through sequencing. For this
investigation, morphologically suspected T. evansi infected camel was
confirmed by examination of Giemsa stained blood smear of camel
blood.
After confirming infection, DNA isolation from collected pellet of
Trypanosoma evansi was done as per the protocols given by ready to
use kit from Illustra blood genomic prep. mini kit with slight
modifications. The desired amplicons of aox and ts genes were then
amplified by PCR using gene specific primers. Amplified PCR products
were analyzed on 1.2% agarose gel stained with ethidium bromide and
identified on the basis of size of the aox and ts genes. The amplicons
of expected size were purified from the 1% low melting agarose gel
employing Illustra GFX PCR DNA and Gel Band Purification Kit. The
DNA fragment of interest was then ligated to the pGEM- T Easy vector
and ligated mixture was transformed into Escherichia coli JM109
strains. The cells containing recombinant plasmid could be identified on
the basis of blue/white colony selection on LB agar containing X-Gal,
IPTG and ampicillin. Screening of recombinants was done by
Restriction Enzyme digestion of plasmid DNAs using EcoRI and found
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that the release of DNA fragments around 990 bp for aox and 2241 bp
for ts gene. Colony PCR was done for quick screening of plasmid
inserts directly from E. coli colonies in the presence of insert specific
primers. After confirmation of clones of aox and ts genes, the plasmid
DNAs were sequenced and coding sequences of aox and ts genes
according to the results obtained were of 990 and 2241 bp
respectively. The phylogenetic and sequence analysis was done by
use of Praline, Clustal X and MEGA5 softwares. Tree topology of aox and ts gene is based on the Neighbor-Joining method and maximum
parsimony with 100% bootstrap values. Multiple sequence alignment of
obtained protein sequences of aox and ts genes was performed with
Praline sequence software. Identified aox and ts gene sequences
showed a close homology with other Trypanosoma spp. gene
sequences.
Description
Keywords
fruits, genetics, biological phenomena, heritability, developmental stages, yields, crossing over, genotypes, selection, sugar