斑鱾dmrt1的克隆及其表达分析

Cloning and expression of dmrt1 gene in Girella punctata

克隆了斑鱾(Girella punctata)dmrt1(doublesex and mab-3 related transcription factor 1)基因,用DNAMAN 9软件比较了Dmrt1蛋白的氨基酸同源性,用MEGAX软件构建了Dmrt1系统进化树,并应用实时荧光定量PCR方法检测dmrt1 mRNA在斑鱾雌雄性腺和不同胚胎发育阶段的表达。结果显示,斑鱾dmrt1的开放阅读框(ORF)全长894 bp,编码297个氨基酸,氨基酸序列含有典型的DM结构域。斑鱾Dmrt1氨基酸同源性比对分析发现,它与黄金鲈(Perca flavescens)的同源性最高,为85.71%;与人类(Homo sapiens)同源性最低,为32.89%。斑鱾Dmrt1与其他鲈形目鱼类在系统进化树上聚为一支,与传统分类地位一致。斑鱾dmrt1在精巢中有较高表达(P<0.05),在卵巢中微弱表达。此外在胚胎发育过程中,dmrt1的表达水平呈先升后降的趋势,在桑葚期表达水平最高(P<0.05)。综上所述,dmrt1可能参与了斑鱾性腺分化和早期胚胎发育。

Doublesex and mab-3 related transcription factors(dmrt)is a family of genes,which are highly homologous with Drosophila melanogaster doublesex(dsx)and Caenorhabditis elegans male abnormal-3(mab-3).Dmrt proteins have a highly conserved doublesex and male aberrant-3 relative domain(DM domain),which can combine with the specific DNA sequences and determine the regulation of the expression of downstream genes.The doublesex and mab-3 related transcription factor 1(dmrt1)is one of dmrt family and has the classical DM domain.This gene is widely present in mammals,birds,reptiles and fish,and determines gender differentiation.In this study,3 male and 3 female Girella punctata were killed humanely using tricainemethanesulfonate(MS-222,Sigma,St.Louis,MO,USA)for samples of the gonad tissues;in addition,embryos at different developmental stages were observed,and collected for samples.All experimental procedures were carried out by the Guidelines for the Care and Use of Laboratory Animals in China,and the protocol was approved by the Experimental Animal Ethics Committee of Guangdong Ocean University,China.Embryo samples and part of fresh gonad tissues were frozen immediately in liquid nitrogen to extract total RNA,and the rest of the ganod tissues were fixed in Bouin’s solution for subsequent dehydration with alcohol,transparent with xylene,embedded in paraffin,sectioned,and stained with hematoxylin-eosin(HE)for histological observation.Total RNA was extracted using Trizol reagent kit(Invitrogen,Carlsbad,CA,USA),and synthesized the fist-strand cDNA following the PrimeScript^(TM) RT reagent kit with gDNA eraser manufacturer’s protocol(TaKaRa,RR047A,Japan).The relative expression of dmrt1 gene in different tissues was calculated usingβ-actin as an internal reference.The specific primers of dmrt1 gene were designed by Primer Premier 5.0.The dmrt1 gene of G.punctata was cloned and bioinformatics were analyzed in its base sequence.The full-length open reading frame(ORF)of dmrt1 gene of G.punctata was 894 base pairs,encoding 297 amino acids.It encoded Dmrt1 protein with a total number of 4414 atoms,structural formula of C_(1374)H_(2154)N_(406)O_(457)S_(23),a theoretical molecular weight of 32.41 ku,a theoretical isoelectric point(pI)of 7.01,an instability coefficient of 64.19,greater than the threshold of 40,and an unstable property,and a lipid solubility index of 53.60,the grand average of hydropathicity(GRAVY)was-0.638,which was a hydrophilic protein,located in the nucleus,without a signal peptide and transmembrane region,and contained many functional sites.Sequence analysis revealed that Dmrt1 protein contained a typical DM domain,and more than half of the amino acids in the second structure were involved in the formation of random curly structures.Comparison of Dmrt1 identity and the construction of phylogenetic tree was performed using DNAMAN 9 and MEGAX software,respectively.Multiple amino acid sequence alignment showed that Dmrt1 protein of G.punctata had the highest identity with Perca flavescens(85.71%),and the lowest with Homo sapiens(32.89%).However,the DM domain of Dmrt1 protein had the highest identity with P.flavescens and other Perciforme fishes(96.30%),and higher with Homo sapiens(87.10%),the high identity of DM domain indicated its evolutionary conservation.Phylogenetic analysis revealed that the Dmrt1 protein of G.punctata was the closest to P.flavescens and other Perciforme fishes,consistent with the traditional taxonomic status.Histological observations of both male and female gonads found that the spermathecae were at stage Ⅲ and the ovaries were at stage Ⅱ.The spermatogonial cells were nearly round or oval,and had a large round nucleus in the center,which was dyed dark blue by hematoxylin,and the cytoplasm was dyed light red by eosin;the oocytes were irregular in shape,sub-circular,polygonal or pear-shaped,with a deviated nucleus that accounted for a larger proportion of the oocytes.Observation of embryo development showed that the zygote was terminal yolk.The diameter of the zygote was(973.92±21.46)μm(n=36)and had one oil sphere,whose diameter was(221.55±11.68)μm.Embryo development stage were mainly as follows:countable cell stages,multi-cellular stage,morula stage,blastula stage,gastrula stage,embryo body stage,muscle burl stage,optic capsule stage,pre-hatching stage,newly-hatched larvae.The whole embryonic development process lasted for 24 h and 36 min.The real-time quantitative PCR(RT-qPCR)was performed to explore the relative expression of dmrt1 mRNA in gonads and embryos at different stages.Results showed that dmrt1 gene was weakly expressed at stageⅡof the ovary,and was highly expressed at stage Ⅲ of the testis(P<0.05).Throughout the G.punctata embryo development period,dmrt1 gene expression rose first and then fell.The dmrt1 gene expression level was the highest at the morula stage(P<0.05),decreased at the blastula stage,and showed almost no expression at the initial incubation stage.In brief,dmrt1 gene might be involved in gonadal differentiation and early embryonic development in G.punctata.The present study analyzed the expression pattern of dmrt1 gene in female and male gonads and at different embryonic development stages,provided some basic information for studying the regulation of embryo development and sex differentiation of G.punctata,and laid a certain foundation for breeding of G.punctata in the future.