MOLECULAR CHARACTERIZATION OF COMPLEMENT COMPONENT C9 OF ROHU Labeo rohita

Complement component 9 (C9) is a major molecule of the complement system which plays a significant role in the formation of the membrane attack complex (MAC) on the targeted cell surface. The current study is dealt with molecular characterization of C9 gene from rohu, Labeo rohita, an important cultured carp species of India. A partial mRNA sequence of 2073 bp encoding an open reading frame (ORF) of 1998 bp was amplified by polymerase chain reaction (PCR) that encodes a polypeptide of 666 amino acids. The polypeptide contains a signal peptide of 19 amino acids and a mature peptide of 647 amino acids. SMART domain architecture analysis revealed two TSP1 domains, a LDLa, a MACPF domain, and an EGF-like domain. Multiple sequence alignment revealed a primitive C9 sequence of rohu which showed maximum similarity with common carp, grass carp and zebrafish. Evolutionary analysis resulted in clustering of rohu C9 sequence with common carp, gold fish, bighead carp, grass carp and zebrafish. C9 was highly expressed in liver tissue and constitutively expressed in wide array of tissues viz., spleen, anterior and posterior kidneys, intestine, stomach, heart, gills, brain, skin and muscle except in eye in healthy rohu juveniles. C9 was expressed during early developmental days of rohu as well. Milt showed significantly high level of expression of C9. Upon Aeromonas hydrophila challenge, a slight up-regulation of the gene was noticed at 6 h and 72 h in liver and spleen tissues without significant change in its expression in gills. Whereas a significant increase in transcript level was noticed in liver (3-fold), spleen (30-fold)and gills (30,000-fold) after 24 h, 15 d and 6 h of poly I:C  induction, respectively. Similarly, during Argulus siamensis infection expression of C9 transcripts was increased in liver tissue from 12 h to 15 d (3-fold to 70-fold), spleen (16-fold) at 72 h and anterior kidney (4-fold)at 12 h post-infection whereas significant down-regulated was observed in skin tissue. Constitutive expression of C9 in different organs of rohu during ontogeny and in response to bacterial, viral and parasitic exposures along with high degree of sequence homology with other fish species proved it as an important primitive immune molecule of the complement system lytic pathway.