Molecular Cloning And Expression Analysis Of Smydl And Myostatin Genes From Mandarin Fish (Siniperca Chuatsi)

As a methyltransferase, Smydl is essential for myofibril maturation and muscle contraction, which plays a catalytic role in the methylation modification of histone. Smydl is mainly catalyzed lysine methylation of histone H3, and most of acting sites on K4, K9, K27, K36and K79(H3K4, the4th lysine of histone H3), but the histone H4is K20. Methylation modification of the histone played an important role in the regulate activity of the gene for it’s not only link with the forming of biological chromatin, the inactivation of X chromosome and the activation or inactivation of transcription, but also promoted growth and differentiation of the muscle. As far as we know, the Smydl family had been found has the same isomer such as Smydl a and Smydlb in fishes. Myostatin, is originally known as growth differentiation factor-8(GDF-8), belonging to the transforming growth factor-β (TGF-(3) superfamily, has been established as the most potent negative regulator of skeletal muscle growth and development in several vertebrates. In current study, we first cloned the Smydl a&b and Myostatin genes of mandarin fish, then analysis the characteristic of there genes and the tissue and developmental stage specific expression of there genes, in the end, we explored the effect of nutrient restriction and re-feeding(nutrition stress). Results are as follows:1. The full length cDNA of Smydl a and Smydlb in mandarin fish were cloned by common PCR and RACE method, and their sequences were deposited in the GenBank with accession numbers of KF744041and KF744042. The size of the full-length Smyd1a and Smyd1b cDNA was1862bp and1802bp, respectively. At the same time, the Smydla and Smydlb both composed with a5’non-coding region, an open reading frame (ORF) and a3’non-coding region. Smydla included an open reading frame (ORF) of1440bp encoding479amino acids (aa),5’non-coding region of149bp and3’non-coding region of273bp whereas the Smydlb was1431bp(476AA),207bp and164bp, respectively. The characteristic of the Amino acid sequences were contained domains of SET and MYND.2. With common PCR, RACE method and Tail-PCR technology, we obtained the ORF and3’non-coding region sequences and the promoter of the Myostatin gene. The accession number of Myostatin gene was JF896453. The Myostatin cDNA with3347bp contained an open reading frame(1131bp) encoding376amino acids (aa), a promoter region (863bp) and3’non-coding region (1353bp). The character of Myostatin promoter contains several regulatory elementals, such as three E-boxes, two CAAT-boxes, and one I-box. The ORF consists of a signal peptide of22amino acids, a TGF-beta functional peptide area, a RARR (264-267aa) motif which is the site of the protease hydrolysis and consistent with RXXR, and a conservative cysteine knot of9cysteines.3. The expression of Smydla, Smydlb and Myostatin were reveal the three genes were mainly expressed in the muscle tissues which is significantly higher than in other tissues (p<0.05).The expression level of Smydla gene appeared to be up-regulated with the developmental stages, and reached the maximal value at150days (P<0.05). However, the expression of the Smydlb doscoverd no obvious change at the entire stage of development (P>0.05), but increased at days50of post-embryonic development (P<0.05). The Myostatin expression was molto basso and no significant differences in the early stage of post-embryonic development, only significantly increased at30days (P<0.05), but follow back to the early levels later and lastly arrival to the maximal level until150days (P<0.05).4. When the mandarin fish were fast for7days and re-fed to apparent satiety for14days. The expression of Smydla and Smydlb both significantly raised after the meal of1day, and Smydla keep high expression in the skeletal muscle at all stages in the following, whereas the expression of Smydlb was down-regulated significantly after3to5days of meal (P<0.05), until14days later it just returned to the initial values. Expression level of Myostatin was down-regulated within5days of re-feeding, which appeared significantly up-regulated at7days and keep to end.Otherwise, the juvenile mandarin fish were fasted for1week and fed a single meal to Satiation, the expression level of Smydla was significantly up-regulated observed at6h postprandially (P<0.05), soon returning to initial values, but Smydlb have no significant change after feeding (P>0.05). Expression of Myostatin was significantly reduced in response to feeding(P<0.05) with significant down-regulation observed at6h,12h and24h after the single meal, and exhibited significantly up-regulated in48h postprandially, when it returning to initial values after96h.