Molecular Cloning And Expression Analysis Of Igf1 And Trim63a In Diploid And Tetraploid Misgurnus Anguillicaudatus

Misgurnus anguillicaudatus has tender flesh with high nutritional value,it is an important economic freshwater fish in China.With the increasing of the market demand,wild caught could not satisfied the market demand.This situation triggers the expansion of M.Anguillicaudatus breeding sector in China,how to promote the growth and development of cultured M.Anguillicaudatus has become the focus of researchers.We studied the gene expression of igf1 and trim63a at different embryonic development stages of diploid and tetraploid M.Anguillicaudatus embryos,and the expression in different tissues of normal and 17β-E₂ treated diploid and tetraploid M.Anguillicaudatus,in order to explore the regulation mechanism of igf1and trim63a in muscle metabolism and growth of diploid and tetraploid M.Anguillicaudatus,and provide theoretical basis for breeding new breeds.The findings of this study are as follows:1.The cDNA sequences of M.Anguillicaudatus igf1 were cloned,the cDNA sequences of the diploid and tetraploid M.Anguillicaudatus igf1 genes were basically identical,and the amino acid sequences were completely identical.The sequence analysis showed that the length of igf1 cDNA was 692 bp,which included 483 bp opening reading frame,and encoded 160 amino acids.The results of amino acid sequence alignment showed that the structures of igf1 was highly conservative and highly similar to Danio rerio.The phylogenetic analysis illustrated that the phylogenetic evolution of igf1 gene was consistent with the traditional evolutionary classification.The mRNA expression of igf1 in diploid and tetraploid embryos at different developmental stages;and the expression of igf1 in different tissues of adult polyploid were examined by qPCR.The expression of igf1 in different ploidy embroys was significantly different,and the expression of igf1 at fertilized egg stage in diploid embryos was significantly higher than other embryonic stages,and the expression of igf1 at hatching stage in tetraploid embryos was significantly higher than other embryonic stages.The expression level of igf1 at the 4-8 cell stage of tetraploid embryos was significantly higher than that in diploid,and the expression at muscular contraction stage was significantly lower than that in diploid.The igf1 was expressed in 7 tissues types of adult diploid and tetraploid.The expression level of igf1 was significantly higher in liver and followed by muscle.The results showed that igf1 may play an important role in the embryonic development of diploid and tetraploid M.Anguillicaudatus,but its adjustment mechanism were different.The igf1expression patterns were similar in tissues of diploid and tetraploid M.Anguillicaudatus,which indicated similar function of regulating the body growth.2.The cDNA sequences of M.Anguillicaudatus trim63a were cloned,the cDNA sequences of the diploid and tetraploid M.Anguillicaudatus trim63a genes were basically identical,and the amino acid sequences were completely identical.The sequence analysis showed that the length of trim63a cDNA was 1035 bp,and encoded344 amino acids.The results of amino acid sequence alignment showed that the structures of trim63a was highly conservative and highly similar to Danio rerio.The phylogenetic analysis illustrated that the phylogenetic evolution of trim63a gene was consistent with the traditional evolutionary classification.The mRNA expression of trim63a in diploid and tetraploid embryos at different developmental stages;and the expression of trim63a in different tissues of adult polyploid were examined by qPCR.The trim63a was expressed in all 13 developmental stages of diploid and tetraploid embryos,and the expression of trim63a at neuroembryonic stage in tetraploid embryos was significantly higher than other embryonic stages.The expression level of trim63a at the 4-8 cell stage of tetraploid embryos was significantly lower than that in diploid,and the expression at myomere formation stage was significantly higher than that in diploid.The trim63a were expressed in 7 tissues types of adult diploid and tetraploid,while the expression level of trim63a maybe involved in the regulation of muscle metabolism during the embryonic development of diploid and tetraploid M.Anguillicaudatus.The trim63a expression patterns with tissue-specificity were similar in tissues of diploid and tetraploid M.Anguillicaudatus,which indicated similar function of regulating muscle metabolism.3.The expression of igf1 and trim63a in heart,liver,muscle and blood at 6 h,12h,24 h,and 14 d after 17β-E₂ treatment（by intraperitoneal injection）in diploids and tetraploids were detected by qPCR.The results showed that the effective time of17β-E₂ was different.The expression of igf1 in muscle and blood was significantly up-regulated after injection while the expression of trim63a in liver and muscle were significantly up-regulated.The expression of igf1 and trim63a returned to normal level after two weeks.In summary,the igf1 and trim63a genes in M.anguillicaudatus are involved in the regulation of muscle protein metabolism.Muscle metabolism and body growth have a high correlation.Therefore,exploring the expression patterns of igf1 and trim63a is is helpful to grasp the muscle growth regularity of M.anguillicaudatus,and provide a theoretical basis for the cultivation of faster growing loach.