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Gene expression profiling by cDNA-AFLP and identification of differentially expressed transcripts of bovine pituitary gland in growing bulls of dairy breeds

Chandra Shekhar Pareek
Institute of General and Molecular Biology, Faculty of Biology and Earth Science, Nicolaus Copernicus University, Gagarina 9, 87-100 ToruĊ„, Poland
Abstract :
The study utilized age-dependent breed-specific gene expression profiling by cDNA-AFLP technique
in identification of differentially expressed (DE) candidate genes for growth and development trait.
Results revealed 15579 transcript derived fragments (TDFs) expressed in bovine pituitary gland
tissue using 96 unique PCR reactions with primer combinations (PC) of TaqI-MseI for Polish Holstein
(PH) and Polish Red (PR) dairy breeds. Gene expression profiling by cDNA-AFLP identified 1451,
2877 and 4084 identically displayed (iDD), differentially displayed (DD) and single displayed (sDD)
TDFs, respectively. In all transcript profiles, frequencies of DD TDFs were higher than that of iDD
TDFs. A total of 60 DD TDFs bands were excised for PH (n=40) and PR (n=20) . Direct sequencing
results revealed identification of 24 and 8 DE-TDFs sequences in Polish HF and PR. Based on the
significance of BLAST and sequence alignment score, analysis 12 and 4 DE-TDFs sequences of PH
and PR were excluded. Overall, 12 Polish HF and 4 PR breed specific DE-TDFs sequence expressed
in bovine pituitary gland tissue were identified. The identified DE-TDFs sequences were represented
in all bovine chromosomes, except BTA3, 4, 6, 7, 8, 9, 11, 12, 17, 18, 21, 22, 23, 25 and 26. TDF
annotation results identified eight sequences that have BLAST hits to known annotated bovine genes
and eight sequences to unannotated contig regions in latest gene ensemble database
4,0. Two
breed-specific target genes
(PH) and bovine
arachidonate 5-lipoxygenase
5) isoform 1
(PR) were validated by qRT-PCR. Within breed the age-dependent qRT-PCR
analysis revealed that expression levels were differed significantly high (P<0.0001) with nine folds
higher expression in young bulls at the age of 6 month (
glycophorin C
) and 9 month (
5 isoform
). Between breed the qRT-PCR analyses revealed that the expression levels were highly significant
PR and
5 isoform 1
gene in both breeds. It was concluded that gene
expression profiling by cDNA-AFLP is a reliable technique for identification of trait-associated DE
candidate genes, and helpful in elucidating and understanding molecular basis of postnatal growth
and development of cattle.
Keywords :
os taurus / cattle / differential display / gene expression / pituitary / Polish Holsteins / Polish Red / r eal time PCR

Date Deposited : 01 Apr 2015 15:25

Last Modified : 01 Apr 2015 15:30

Official URL:

Volume 30, Number 2, - 2012

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