MOLECULAR STUDIES ON HEAT SHOCK PROTEIN 70 GENE IN DOMESTIC FOWL AND DUCK
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Date
2011
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR
Abstract
A study has been carried out to analyse the promoter and 3’UTR of
hsp70gene in different breeds of chicken (Naked neck (NaNa), Kadaknath (KAD),
White Leghorn (WL), Rhode Island Red (RIR) and White Plymouth Rock
(WPR)) and duck (Kuttanadu (KUT) and White Pekin (WP)). Plasma
corticosterone level was also estimated in two different (summer and rainy)
seasons.
Promoter and 3’ UTR of Hsp70 gene were amplified by PCR and the
products were sequenced. Sequence analysis of the promoter region showed 100%
homology between three breeds of chicken, WL, RIR and KAD and the duck
breeds. These breeds exhibited only 99.7% identity with WPR and NaNa
Sequence alignment (with Gene bank accession no. J02579) revealed a
point mutation in the CAAT box, where an ‘A’ is deleted at position -312 in all
the breeds of chicken and duck except NaNa. Another ‘A’ deletion could also be
detected at position -329 from a heat shock element in WPR. In all the breeds
under study, TATA box (TATAA) was found at position -134. Three inverted
CAAT boxes, ‘ATTG’ and two variants of GC boxes, one having GGCG motif (6
numbers) and another having GGGCGG motif (2 numbers) were identified in
both chicken and duck sequences.
Two variants of consensus heat shock elements (HSE), NGAAN (single
unit) and ‘NGAAGAAN’ (double unit) were detected in the promoter region of all
breeds of chicken and duck under study except WPR, in which a point mutation
(‘A’ deletion) was noted in one of the double units.
Restriction analysis showed that only Bgl І has a site in the amplified
region of the promoter of both the species. Due to the presences of point
mutations, three alleles for the promoter region of the gene could be detected.
Sequence analysis of 3’ UTR showed varying levels of sequence
polymorphism between chicken breeds, where only 80 to 95.8 % identity could be
observed. Between duck breeds, KUT and WP, 97.7 % identity was observed.
Analysis between chicken and duck revealed 79 to 99.5% identity where 97 to
99.5% identity was observed between duck breeds and WPR.
Two consensus, CAAC sequence and a variant of poly adenylation signal
sequence could be identified in 3’ UTR. A variant of poly adenylation signal
sequence, TATAAA could be identified in all breeds except RIR, in which two
point mutations (transversion) were observed (TAAAAA) when compared to the
consensus sequence (AATAAA). A second poly adenylation signal sequence,
which was again a variant (AATAAT) was detected only in NaNa. The number
and position of CAAC motifs varied (2 to 4 numbers) between species and breeds
under study. In both the species, rather than a consensus sequence, a variant of K
box (GGTGAT) and Brd box (TGCTTA) could be identified. Several AT (AU in
mRNA) rich regions could be identified in the 3’ UTR of both chicken ad duck
breeds. However, an additional ATATA motif is also detected in RIR and NaNa.
Restriction enzyme analysis of 3’ UTR revealed that NaNa, RIR and WL
have no cutting site for any of the common enzymes while a single cutting site for
Bgl ІІ was observed in KAD, showing two alleles in chicken breeds. Duck
breeds also showed two alleles where the enzyme, Bam HI has a restriction site at
different positions.
Plasma corticosterone level in chicken and duck showed considerable
variation in different breeds of chicken both in summer and rainy seasons. Among
the chicken breeds WPR showed the lowest level of corticosterone in summer
season, which did not differ significantly from that of rainy season. Comparison
between seasons showed a highly significant (P<0.01) increase in summer in all
breeds except WPR. Duck breeds also showed a similar trend. Significant
(P<0.01) increase in the plasma corticosterone level was noticed in summer.
However, between breeds, no significant variation was observed in each season.
Hormone responsive elements (HRE) for corticosterone could not be
detected in the promoter region of any of the breeds under study. Any correlation
between the sequences and the plasma corticosterone level could not be detected
in both chicken and duck breeds under study. Although, two point mutations were
detected in the promoter region of WPR, it could not be correlated with the
plasma corticosterone level.
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