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Published on: October 27, 2017
Molecular cloning, sequencing and tissue-level expression of complement C3 of Labeo rohita (Hamilton, 1822)
K Pushpa1, P Gireesh-Babu2, K V Rajendran1
1Aquatic Environment and Health Management Division, Central Institute of Fisheries Education, Versova, Mumbai 400061, India.
Insights
This study details the Labeo rohita complement C3 (LRC3) gene, finding conserved domains and motifs essential for complement function. LRC3 expression is highest in the liver and significantly increases in liver and kidney after Aeromonas hydrophila infection.
Area of Science:
- Immunology
- Genomics
- Aquatic Biology
Background:
- Complement component C3 is crucial for innate and adaptive immunity, mediating pathogen clearance.
- Understanding fish complement systems is vital for aquaculture health and disease resistance.
Purpose of the Study:
- To clone and characterize the complement C3 gene from Labeo rohita (rohu).
- To analyze the expression profile of rohu complement C3 (LRC3) in various tissues and under bacterial challenge.
Main Methods:
- Full-length cDNA cloning and sequencing of LRC3.
- Bioinformatic analysis of the deduced amino acid sequence for conserved domains and motifs.
- Real-time PCR to quantify LRC3 mRNA expression in different tissues and post-Aeromonas hydrophila infection.
Main Results:
- The LRC3 cDNA sequence is 5081 bp, encoding a 1645-amino acid protein with characteristic C3 domains.
- Conserved functional motifs, including the thiol-ester motif, were identified in LRC3.
- LRC3 mRNA showed highest expression in the liver, with detectable levels in all tested tissues.
- Bacterial challenge with Aeromonas hydrophila significantly upregulated LRC3 expression in liver and kidney.
Conclusions:
- The cloned LRC3 gene possesses conserved structural and functional features of complement C3.
- Rohu complement C3 plays a significant role in the immune response to Aeromonas hydrophila infection.
- Tissue-specific expression patterns highlight the liver and kidney as key sites for C3-mediated immunity in rohu.
Abstract:
Complement component C3 plays a central role in all known complement activation pathways. In the present study, we cloned, sequenced and analyzed the full-length cDNA sequence of Labeo rohita complement C3 (LRC3). The expression pattern of complement C3 mRNA in different tissues of healthy rohu and after challenge with Aeromonas hydrophila were evaluated using real-time PCR. The LRC3 cDNA sequence of rohu comprised of 5081 bp encoding a predicted protein of 1645 amino acids. The deduced amino acid sequence had the characteristic domain architecture. About eight domains specific to complement C3 are present in the sequence starting from signal peptide to netrin C345C (NTR) domain. The post-translational processing signal sequence (RKRR), the C3-convertase cleavage site sequence (LAR) and the canonical thiol-ester motif (GCGEQ) were found to be conserved in the LRC3. Real-time PCR analysis revealed the highest expression of C3 in liver and extra-hepatic expression of C3 was also observed in all the tissues studied. A. hydrophila challenge resulted in significant up-regulated expression of C3 transcripts in both liver and kidney at 6, 12, 24, 48 and 72 h post-infection.
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