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제안 방법
- Figure 4. Expression of glutamate-cysteine ligase catalytic subunit (GCLC) and glutamate-cysteine ligase modifier subunit (GCLM) genes determined by real-time quantitative polymerase chain reaction analysis (RT-qPCR) in horses before and 30 minutes after exercise. (A) Expression of the GCLC gene in muscle and blood leukocytes of Thoroughbred horses (n = 3, * p<0.
- In this study, we performed bioinformatic analyses of horse GCLC and GCLM genes, and investigated their expression in muscle and blood in response to exercise stress to gain insights into their potential application as biomarkers for ROS responses in horses.
- To investigate the evolutionary relationships of GCLC and GCLM in horses, we obtained gene sequences from eight species of mammals (human, macaque, orangutan, cow, pig, horse, rat, and mouse) from Ensembl 62 and conducted a phylogenetic analysis (Figure 2). The amino acid similarity of the two genes in the eight mammals is over 90% (Tables 2, 3).
- But there were no differences in both of GCLC gene and GCLM gene expression in the horse tissues studied in this study (Figure 3). To reveal the relationship between exercise induced stress and transcriptional activity of glutathione synthesis related genes, we prepared total RNAs from skeletal muscle and blood of Thoroughbred horses and performed RT-qPCR analysis for GCLC and GCLM genes. As result, it was found that the expression of both of GCLM and GCLC genes was up-regulated in skeletal muscle as well as blood leukocytes.
대상 데이터
- Two stallions and one mare Throughbred horses aged 5, 9, and 10, weighing from 500 to 700 kg which were maintained at Ham-an Racing Horse Resort and Training Center were used to obtain the blood and skeletal muscle samples before and after exercise. Exercise was performed by trotting at the speed of 13 km/h for 30 min and cantering through lunging and long-reining (circular bridge lunging).
데이터처리
- Both T-test and analysis of variance statistical test was conducted to determine significance levels. Data were shown by mean± standard error of mean.
이론/모형
- All measurements were performed in triplicate for all specimens, and the comparative method used was the 2-ΔΔCt method [10].
- 1), horse (F6WKI2) was obtained for GCLM. Amino acids were aligned using Multiple Sequence Comparison by Log-Expectation (MUSCLE) (http://www.ebi.ac.uk/Tools/msa/muscle/). Phylogenetic analysis was performed using Neighbor-Joining method [11] with pairwise deletion, 1,000 bootstrap replication, and Kimura 2 as described previously [12].
- Phylogenetic analysis was performed using Neighbor-Joining method [11] with pairwise deletion, 1,000 bootstrap replication, and Kimura 2 as described previously [12].
- Quantitative analysis was performed using the 2-ΔΔCt method.
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