| Teleost fish has important position in species evolution process, and is an important modelin biological study. Large yellow croaker (Larimichthys crocea) and zebrafish (Danio rerio) aretwo models of teleost fish. The study of large yellow croaker is becoming a hot topic of researchbecause of its important economic value. Meanwhile, zebrafish is easy to raise with a shortgeneration time, and is a widely used model organism in many biological research fields. Malespecific lethals (MSLs) genes play an important role of equalizing gender differences in someinvertebrates such as fruitfly (Drosophila melanogaster). But there are no reports on the functionof MSLs in any teleost fish. Structural and functional study of MSLs in teleost fish mightcontribute to reveal the evolution process of structure and function of MSLs.In this study, partial sequences of msl1and msl3were obtained from a normalized gonadalcDNA library of large yellow croaker established by our laboratory. A fragment of msl2wasobtained by RT-PCR while using the degenerate primers of msl2. SMART-RACE technique wasused to clone the full-length cDNA of Lc-msl1, Lc-msl2and Lc-msl3. The expression of thesegenes in different tissues, organs and different stages of large yellow croaker embryonicdevelopment were analyzed by quantitative real-time PCR (qrtPCR). The genome sequence ofLc-msl1was acquired to compare to msl1of other teleost fishes by bioinformatics analysis aswell as the synteny analysis of msl1, msl2, msl3in teleost fishes. The expression, location andfunction of Dr-msl1in different stages of zebrafish embryonic development were analyzed basedon the sequence of Dr-msl1in zebrafish from NCBI database. The results as follows:(1) The full-length cDNA of Lc-msl1is1768bp, consisting of a5’-terminal untranslatedregion (UTR) of140bp, a3’-terminal UTR (including a poly(A)+tail of19bp) of251bp andan open reading frame (ORF) of1377bp. The translated sequence has458amino acids in length,with a predicted molecular weight (MW) of52.561kDa, and a pI of7.57. The qrtPCR resultsshow that Lc-msl1was ubiquitously expressed in all detected adult tissues and different stages ofembryonic development. Lc-msl1expressed much higher than the other tissues in male brain(P<0.05). But the expression of Lc-msl1had no significant variation in stages of embryonicdevelopment. The genome sequence of Lc-msl1is2682bp in length (excluding UTR), with8exons and7introns, which is similar to the structure of msl1in other teleost fishes. The syntenyanalysis result of msl1reveals that there is a conservative syntenic group consisting of msl1(1of2), bc2, fbrs and srcap generally existing in teleost fish. (2) The full-length cDNA of Lc-msl2is2187bp, consisting of a5’-terminal UTR of498bp,a3’-terminal UTR (including a poly(A)+tail of17bp) of141bp and an ORF of1548bp. Thetranslated sequence has515amino acids in length, with a predicted molecular weight (MW) of55.772kDa, and a pI of9.11. The zinc finger domain of Lc-MSL2is more similar to Dm-MSL2compared with hMSL2. The qrtPCR results show that the expression of Lc-msl2was alsoubiquitous in all adult tissues examined and had a much higher significant level (P<0.05) in malebrain when compared to other tissues and organs, which was analogous to Lc-msl1. The level ofLc-msl2mRNA in Multicellular stage was low, then it is raised significantly (P<0.05) inBlastocyst stage, while there is no significant difference (P>0.05) after these two stages. Thesynteny analysis result of msl2reveals that there is a conservative syntenic group consisting ofENSDARG00000056262, stag1, pccb and msl2(2of2) in most Euteleostomi, which includingteleost fish and mammals.(3) The full-length cDNA of Lc-msl3is3386bp, consisting of a5’-terminal UTR of117bp,a3’-terminal UTR (including a poly(A)+tail of18bp) of1631bp and an ORF of1638bp. Thetranslated sequence has545amino acids in length, with a predicted molecular weight (MW) of61.997kDa, and a pI of9.14. The qrtPCR results show that Lc-msl3was ubiquitously expressedin all adult tissues examined either. The expression level of Lc-msl3was lower in muscle, liverand stomach than in gonad, eyes and heart, but there was no significant difference (P>0.05)between female and male. The mRNA level of Lc-msl3is raised gradually as the embryonicdevelopment reached Yolk bolt formation stage, then decreased. Expression level of Yolk boltformation stage was significant different (P<0.05) from the other stages examined. The syntenyanalysis result of msl3reveals that deletion of msl3has happened in zebrafish. There is aconservative syntenic group consisting of mid1(1of2), arhgap6(1of2), msl3and frmpd4(2of2) in most Euteleostomi excepting zebrafish.(4) The expression level of Dr-msl1in zebrafish rose gradually as the embryonicdevelopment reached75%epiboly stage then began to decrease in Bud stage, and later increasedin48hpf. Dr-msl1expressed much higher (P<0.05) in these first two stages than other stagesexamined. Whole mount in situ hybridization results of Dr-msl1were corresponding to itsqrtPCR results. The hybridization signal in the head of zebrafish is stronger compared to in otherbody regions. The mRNA levels of Dr-msl2, CDK1(cyclin dependent kinase1) gene Dr-cdc2and cyclin B gene Dr-cycB reduced significantly (P<0.05) in Multicellular stage and Spherestage, and the Dr-cdc2mRNA decreased significantly (P<0.05) in75%epiboly stage eitherfollowed by significant decline of Dr-msl1mRNA (P<0.05) in Multicellular stage after knockingdown Dr-msl1by siRNA.In conclusion, the results of structural and functional study of msl1, msl2and msl3in teleost fish demonstrated that MSLs genes might play important roles in genes evolution and mighthave significant function through the development of teleost fish especially early embryonicdevelopment. Obliviously, functions of msl1, msl2and msl3in teleost fish proposed in this studyare predicted based on sequence and expression. We will utilize protein approaches to furtherinvestigate the function of MSLs in teleost fish, especially their function in sex differentiation. |
PDF Full Text Request