Study Of Bindin Gene Of Crassostrea Sikamea And Crassostrea Angulata

Oysters are marine bivalve molluscs widely distributed in world oceans. Bindin is a major protein packaged as insoluble granules in sperm acrosomes. It is responsible for species-specific recognition and binding of sperm to a glycoprotein receptor on the surface of eggs. Fucose binding lectin repeat (F-lectin repeat) is the main function domain of bindin including three conservative recognition motif (H37, R64, R70) and four Cysteines (C27, C36, C88, C105). We cloned a novel bindin gene from the Kumamoto oyster Crassostrea sikamea by 3'and 5'RACE. The full-length cDNA of C. sikamea bindin was 1134 bp with a 774 bp open reading frame encoding 257 amino acids. The deduced amino acid sequence contained a putative signal peptide of 24 amino acids. The length of the bindin genomic DNA we obtained was 5353 bp containing 4 exons and 3 introns. Eleven haplotypes of fucose lectin (F-lectin) repeat were detected. Haplotype diversity was 0.846. The ratios of nonsynonymous (dN) and synonymous (dS) substitutions per nucleotide site between haplotyes were from 0.158 to 1.9275. And the dN/dS values were from 0.9317 to 1.7175 between two species. No positive selection has been found. Five positive selected positions (p>95%) were identified among sixteen haplotypes from C. sikamea and C. gigas. Three sites were in the F-lectin repeat exon-1 (position 40,66 and 67; dN/dS= 7.59), and two were in the F-lectin repeat exon-2 (position 121 and 123; dN/dS= 7.41). They all located on the F-lectin binding face around the three recognition motif residues (H37, R64 and R70) in the 3D model. The full-length bindin cDNA of C. angulata was 1049 bp with a 774 bp open reading frame encoding 257 amino acids. The deduced amino acid sequence contained a putative signal peptide of 24 amino acids. The length of the bindin genomic DNA was 8508 bp containing four exons and three introns. Three haplotypes of F-lectin repeat were detected from seven sequences of F-lectin repeat of six male oysters. Intron-4 in the middle of F-lectin repeat is highly variable in both size and sequence. We classified intron-4 into three types (typeâ… , typeâ…¡, typeâ…¢) according to their size and the F-lectin repeat they were located in. We compared the number of nonsynonymous substitutions (dN) and synonymous substitutions (dS) per nucleotide site among 19 F-lectin haplotypes of the three species. No positive selection has been found between three species. Five positive selected positions (p>95%) are identified among nineteen haplotypes of three species. Three were in the F-lectin repeat exon-1 (position 40,66 and 67; dN/dS= 6.848), and two were in the F-lectin repeat exon-2 (position 123 and 125; dN/dS= 6.346). They are located on the F-lectin binding face around the three recognition motif residues. We assume that these five clustered amino acids are related with species specific recognition. Bindin and its cognate receptor must evolve together for recognition during fertilization. The rapid divergence of GRPs within species ultimately leads to incompatibility of sperm and egg between species. The gamete recognition systems could help us understand the complex speciation and adaptation.