Transcriptional profiling of liver tissues in chicken embryo at day 16 and 20 using RNA sequencing reveals differential antioxidant enzyme activity

Abstract

Considering the high proportion of polyunsaturated fatty acids, the antioxidant defense of chick embryo tissues is vital during the oxidative stress experienced at hatching. In order to better understand the mechanisms of the defense system during chicken embryo development, we detected the activity of antioxidant enzymes during the incubation of chicken embryo. Results showed that the activity of superoxide dismutase (SOD) and (GSH-PX) in livers were higher than those in hearts. Based on these results, liver tissues were used as the follow-up study materials, which were obtained from chicken embryo at day 16 and day 20. Thus, we used RNA sequencing (RNA-Seq) analysis to identify the transcriptome from 6 liver tissues. In total, we obtained 45,552,777-45,462,856 uniquely mapped reads and 18,837 mRNA transcripts, across the 6 liver samples. Among these, 1,154 differentially expressed genes (p<0.05, foldchange≥1) were identified between the high and low groups, and 1,069 GO terms were significantly enriched (p<0.05). Of these, 10 GO terms were related to active oxygen defense and antioxidant enzyme activity. GO enrichment and KEGG pathway analysis indicated that GSTA2, GSTA4, MGST1, GPX3, and HAO2 participated in glutathione metabolism, and were considered as the most promising candidate genes affecting the antioxidant enzyme activity of chicken embryo at day 16 and day 20. Using RNA-Seq and differential gene expression, our study here investigated the complexity of the liver transcriptome in chick embryos and analyzed the key genes associated with the antioxidant enzyme.

Fig 1. Effect of incubation day on SOD and GSH-PX activity in embryonic liver and heart.

Note: The SOD activity (A) and GSH-PX (B) of heart and liver tissues were determined at days 14 to 20. The value of each fraction was the mean± standard deviation (n = 3), different letters (a, b) above columns indicate significant differences (p<0.05) in liver tissues, different letters (A, B) above columns indicate significant differences (p<0.05) in heart tissues.

Fig 2. Effect of incubation day on POD activity in embryonic liver and heart.

Note: The POD activity of heart and liver tissues was determined at days 14 to 20. The value of each fraction was the mean± standard deviation (n = 3), different letters (a, b) above columns indicate significant differences (p<0.05) in liver tissues, different letters (A, B) above columns indicate significant differences (p<0.05) in heart tissues.

Fig 3. Volcano plot displaying DEGs within two different comparison groups.

Note: the y-axis shows the mean expression value of log₁₀(q-value), and the x-axis displays the log₂fold change value. The blue dots represent the transcripts that did not reach statistical significance (q > 0.05); the red (up-regulated) and green dots (down-regulated) represent those whose expression levels were significantly different (q < 0.05); the blue dots represent the transcripts did not reach statistical significance (q > 0.05).

Fig 4. Validation of the gene expression profile by real-time PCR.

Note: The x-axis represents the gene name, the y-axis represents the log₂Ratio (Day20/Day16), different color columns represent data from RT-PCR or RNA-Seq.