Abstract:

Bone morphogenetic protein 6 gene(bmp6) plays an important role in regulating the formation and development of intermuscular bones in fish.In order to explore the sequence characteristics and expression characteristics of bmp6 gene in Carassius auratus var.Qihe,the coding sequence(CDS) of bmp6 gene was cloned and analyzed by bioinformatics.Through Quantitative Real-time PCR (qRT-PCR) detection,the expression differences of bmp6 gene in different tissues and developmental stages were identified.The results showed that the bmp6 gene of C.auratus var.Qihe was composed of two homologous genes,each with three alleles,namely bmp6a-1,bmp6a-2,bmp6a-3 and bmp6b-1,bmp6b-2,bmp6b-3,with full-length CDS of 1 245,1 257 bp,encoding 414 and 418 amino acids,respectively.Both bmp6a and bmp6b were unstable hydrophobic secretory proteins containing a signal peptide sequence and lacking a transmembrane structure.bmp6a was mainly distributed in mitochondria,nucleus and cytoplasm,while bmp6b was mainly distributed in nucleus and mitochondria.The secondary structure of bmp6a and bmp6b was mainly random coil,consistent with the results of tertiary structure prediction,and both contained one TGF-β-rel family conserved domain.The amino acid sequence homology analysis showed that the amino acid sequence of bmp6a had higher similarity with bmp6 of Megalobrama amblycephala,but lower similarity with mammals.The amino acid sequence of bmp6b had higher similarity with that of C.auratus and C.gibelio.Phylogenetic tree analysis showed that the bmp6a of C.auratus var.Qihe and the bmp6a of Cyprinus carpio were clustered in the same clade,while the bmp6b of C.auratus var.Qihe and the bmp6b of C.auratus and C.gibelio were clustered in the same clade.The results of qRT-PCR showed that bmp6a and bmp6b genes were expressed in brain,muscle,gill,spleen,intestine,ovary,liver and kidney tissues of C.auratus var.Qihe,but the expression levels were higher in liver,gill and kidney tissues.The expression patterns of bmp6a and bmp6b were similar in different developmental stages of juvenile C.auratus var.Qihe.The expression levels of bmp6a and bmp6b were the highest before the ossification of intermuscular spines,and then decreased with the development of intermuscular spines.To sum up,the CDS of bmp6 gene of C.auratus var.Qihe was obtained by cloning,and bioinformatics analysis and qRT-PCR detection were carried out,providing a reference for further creating a new strain of C.auratus var.Qihe without intermuscular bones.

Key words: Carassius auratus var.Qihe, Bone morphogenetic protein 6(bmp6), Gene cloning, Sequence analysis, Gene expression