The objective of this study was to determine the molecular characteristics of the horse vascular endothelial growth factor alpha gene ($VEGF{\alpha}$) by constructing a phylogenetic tree, and to investigate gene expression profiles in tissues and blood leukocytes after exercise for develo...
The objective of this study was to determine the molecular characteristics of the horse vascular endothelial growth factor alpha gene ($VEGF{\alpha}$) by constructing a phylogenetic tree, and to investigate gene expression profiles in tissues and blood leukocytes after exercise for development of suitable biomarkers. Using published amino acid sequences of other vertebrate species (human, chimpanzee, mouse, rat, cow, pig, chicken and dog), we constructed a phylogenetic tree which showed that equine $VEGF{\alpha}$ belonged to the same clade of the pig $VEGF{\alpha}$. Analysis for synonymous (Ks) and non-synonymous substitution ratios (Ka) revealed that the horse $VEGF{\alpha}$ underwent positive selection. RNA was extracted from blood samples before and after exercise and different tissue samples of three horses. Expression analyses using reverse transcription-polymerase chain reaction (RT-PCR) and quantitative-polymerase chain reaction (qPCR) showed ubiquitous expression of $VEGF{\alpha}$ mRNA in skeletal muscle, kidney, thyroid, lung, appendix, colon, spinal cord, and heart tissues. Analysis of differential expression of $VEGF{\alpha}$ gene in blood leukocytes after exercise indicated a unimodal pattern. These results will be useful in developing biomarkers that can predict the recovery capacity of racing horses.
The objective of this study was to determine the molecular characteristics of the horse vascular endothelial growth factor alpha gene ($VEGF{\alpha}$) by constructing a phylogenetic tree, and to investigate gene expression profiles in tissues and blood leukocytes after exercise for development of suitable biomarkers. Using published amino acid sequences of other vertebrate species (human, chimpanzee, mouse, rat, cow, pig, chicken and dog), we constructed a phylogenetic tree which showed that equine $VEGF{\alpha}$ belonged to the same clade of the pig $VEGF{\alpha}$. Analysis for synonymous (Ks) and non-synonymous substitution ratios (Ka) revealed that the horse $VEGF{\alpha}$ underwent positive selection. RNA was extracted from blood samples before and after exercise and different tissue samples of three horses. Expression analyses using reverse transcription-polymerase chain reaction (RT-PCR) and quantitative-polymerase chain reaction (qPCR) showed ubiquitous expression of $VEGF{\alpha}$ mRNA in skeletal muscle, kidney, thyroid, lung, appendix, colon, spinal cord, and heart tissues. Analysis of differential expression of $VEGF{\alpha}$ gene in blood leukocytes after exercise indicated a unimodal pattern. These results will be useful in developing biomarkers that can predict the recovery capacity of racing horses.
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제안 방법
Therefore, in this study, we investigated the molecular structure of horse VEGFα gene, constructed a phylogenetic tree, and examined the expression profiles in various horse tissue samples.
We also showed that synonymous substitutions per site (Ks) and non-synonymous substitutions per site (Ka) in VEGFα genes for individual species. In addition, pairwise distance was calculated by analyzing the nucleotide and amino acid similarity.
As not much is known about the horse VEGFα gene, we retrieved horse VEGFα sequences from the Ensembl database and analyzed its molecular structure.
To investigate the relationship between VEGFα gene expression level and time length after exercise, we performed qPCR analysis using blood leukocyte samples obtained before and after exercise (30 min, 60 min, 90 min, 120 min) from three horses (Figure 4A and B).
대상 데이터
Blood samples were obtained from three male Thoroughbred horses (2 through 4 year-old; two horses: 2 year-old, one horse: 4 year-old) maintained at Ham-An racing Horse Resort and Training Center. Horses were placed on a treadmill and the treadmill exercises were performed at the speed of 10 to 15 km/h.
데이터처리
, 2001). Statistical significance was calculated by t-test. A p values of less than 0.
이론/모형
The retrieved sequences were aligned in BioEdit with the CLUSTERW option. Phylogenetic analysis was conducted using MEGA version 5.0 (Arizona State University, AZ, USA) (Tamura et al., 2011) The Neighbor Joining method (Saitou et al., 1987) was used with the following options: pairwise-deletion, 1,000 bootstrap replications and Kimura 2-parameter. Pairwise deletion was chosen to retain all sites initially, then excluding them as necessary in the pairwise distance estimation.
Pairwise deletion was chosen to retain all sites initially, then excluding them as necessary in the pairwise distance estimation. Substitutions of nucleotides were obtained by using the Kimura 2-parameter model. We also showed that synonymous substitutions per site (Ks) and non-synonymous substitutions per site (Ka) in VEGFα genes for individual species.
All samples were measured in triplicate to ensure reproducibility, and Ct value was calculated using the 2−ΔΔCt method (Livak et al., 2001).
성능/효과
In terms of expression, horse VEGFα was found to be ubiquitously expressed but regulated by exercise in blood leukocytes. Especially, expression kinetics obtained in this study showed that the transcript level gradual increased up to 60 min (p[0.05), and then remained at a slightly higher level than at time 0, which is not statistically significant (p]0.05). It is not certain how VEGFα expression is upregulated and reached the highest level after 60 min of exercise.
후속연구
Further study is warranted to explore the relationship between expression levels of VEGFα in muscle and blood with physiological changes.
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