Cloning And Tissue Expression Analysis Of MSTN And MyHC In Onchidiids

Onchidiidae is a kind of biology that can be distributed in seawater,fresh water and wetland.Their living area is distributed to shallow sea,intertidal zone and upper tidal zone.The living environment has typical amphibious type,its skin respiration ability,the strength of the muscle,the formation of the stratum corneum and the degree of development of the nervous system show different biocompatibility due to their different living conditions.Therefore,Onchidiidae is also a typical representative from water to land.In order to explore the molecular mechanism of muscle function in the process of evolution from water to land,we used four species of onchidiidae which are widely distributed and studied in China.Based on the database of transcriptome sequencing,the expression of muscle-related genes and their expression in different subspecies and different tissues were compared and deduced the evolution relationship between the four species.(1)In this study,we used rapid amplification cDNA ends(RACE)methods to obtain the full-length cDNA of Myostatin gene in Onchidium struma.The bioinformatic analysis and expression pattern of MSTN mRNA in different tissues detected by Real-time fluorescent quantitative PCR(qRT-PCR)were performed.The full length of MSTN cDNA sequence consists of 2,667 base pairs(bp),comprising a 374 bp 5′ untranslated region(UTR),a 1,643 bp 3′UTR,and a 1,650 bp open readingframe(ORF)which encodes 549 amino acids.The MSTN protein was predicted to compose of 8,776 atoms and its formula is C2774H4331N783O862S26,with a calculated relative molecular weight of 63.27 kD and p I of 6.02.The result of signal peptide prediction shows that the N-terminal has a signal peptide in 21 amino acids length.The common features were found in MSTN of O.struma,including a conservative hydrolytic site(RSRR)and 9 cysteine residues.The phylogeny analysis shows that the relationship between O.struma and Aplysia californica was the closest.According to RT-PCR results,MSTN gene was expressed in various tissues;highest expression in the hepatopancreas and lowest in foot(P<0.05).(2)Myosin as the basic component of muscle unit,its molecular diversity and composition directly influence the muscle growth and meat quality.It is a ubiquitous eukaryotic motor protein that interacts with actin to generate the force for cellular movements ranging from cytokinesis to muscle contraction.In this study,we used rapid amplification cDNA ends methods to obtain the full-length cDNA of myosin heavy chain gene in O.struma.The bioinformatic analysis and expression pattern of MyHC mRNA in different tissues detected by Real-time fluorescent quantitative PCR were performed.The full length of MyHC cDNA sequence consists of 7566 base pairs,comprising a 228 bp 5′ untranslated region,a 1443 bp 3′UTR,and a 5895 bp open readingframe which encodes 1964 amino acids.The MyHC protein was predicted to compose of 31713 atoms and its formula is C9765H15897N2849O3150S52,with a calculate drelative molecular weight of 225.28 kD and p I of 5.56.The result of signal peptide prediction shows that the N-terminal has a signal peptide in 29 amino acids length.The common features were found in MyHC of O.struma,including MYSc_class_Ⅱand Myosin_tail_l domain.The full length of MyHC cDNA sequence in Paraoncidium reevesii consists of 7598 base pairs,comprising a 276 bp 5′ untranslated region,a 1418 bp 3′UTR,and a 5904 bp open readingframe which encodes 1967 amino acids.The P.reevesii MyHC protein was predicted to compose of 31713 atoms and its formula is C9765H15894N2860O3156S56.The full length of Platevindex mortoni MyHC cDNA sequence consists of 7115 base pairs,comprising a 165 bp 5′ untranslated region,a 1046 bp 3′UTR,and a 5904 bp open readingframe which encodes 1967 amino acids.The P.mortoni MyHC protein was predicted to compose of 31730 atoms and its formula is C9785H15921N2865O3153S52.The full length of MyHC cDNA sequence in Peronia verruculata consists of 7115 base pairs,comprising a 192 bp 5′ untranslated region,a 1059 bp 3′UTR,and a 5904 bp open readingframe which encodes 1967 amino acids.The P.verruculata MyHC protein was predicted to compose of 31730 atoms and its formula is C9762H15895N2859O3157S57.(3)According to RT-PCR results,in the dorsal skin,the expression of O.struma was the highest,and the expression of P.reevesii was the lowest,and the difference was significant.The expression level of P.mortoni was the highest in the foot,the expression of O.struma was the lower,and the expression of P.reevesii was the lowest and the difference was significant.The overall expression level in the lung sac was very low,and the expression level of O.struma was the highest,and there was no significant difference.The expression of MSTN and MyHC gene was all detected in the eight tissues of O.struma,and it was proved that MSTN gene had a negative effect on the expression of MyHC.This study provided a novel myosin heavy chain gene sequence in Onchidiidae and the results indicate that the MyHC gene and the protein it encodes are important for the growth and development of animal,as well as its muscle characterization.