拟赤梢鱼卵巢早期发育与sox3基因cDNA克隆及表达分析

Early Ovary Development,sox3 Gene cDNA Cloning and Expression Analysis of Pseudaspius leptocephalus

为初步阐明拟赤梢鱼(Pseudaspius leptocephalus)卵巢发育特征及sox3(sry related high mobility group box 3)基因在其卵巢发育过程中的作用,本研究通过组织切片观察了拟赤梢鱼卵巢早期发育过程,利用RACE技术克隆了该鱼sox3基因c DNA全长序列,并进行生物信息学分析;利用实时荧光定量PCR技术分析了sox3基因在拟赤梢鱼不同组织及性腺不同发育时期的表达模式。结果显示,拟赤梢鱼卵巢在孵化后45 d分化形成,160 d时由Ⅰ期进入Ⅱ期,孵化后360 d,卵巢仍处于Ⅱ期。拟赤梢鱼sox3基因cDNA全长为1800 bp(GenBank登录号:MT952206),编码299个氨基酸,存在保守的HMG(histidine,methionine,glycine-rich)结构域。氨基酸序列比对和系统进化树分析结果显示,拟赤梢鱼SOX3蛋白与斑马鱼(Danio rerio)和翘嘴鲌(Culter alburnus)亲缘关系最近。此外,sox3基因在拟赤梢鱼卵巢中表达量最高,其次是脑和眼睛,在精巢和其他组织中微量表达;在性腺分化过程中,sox3基因在卵巢中表达量显著高于未分化性腺和精巢,在卵巢发育阶段表达量不断升高,而在精巢中持续低水平表达。综上,本研究推测sox3基因主要参与拟赤梢鱼卵巢的分化和发育。

Pseudaspius leptocephalus is the famous indigenous fish in Northeast China region,with very important economic and research value.In recent years,environmental pollution and overfishing has caused it to become increasingly scarce.We performed P.leptocephalus conservation research,including clarifying the characteristics of ovarian development and the role of the sox3 gene(SRY-related high-mobility-group-box 3)in the ovarian development of P.leptocephalus.The histological characteristics of P.leptocephalus ovarian development were investigated using the paraffin section technique.The full length of the cDNA of the sox3 gene was cloned and analyzed using Rapid Amplification of cDNA End(RACE)technology.The expression pattern of the sox3 gene in different tissues and at different gonad development stages were analyzed using real-time PCR.The results revealed the ovarian cavity was clearly visible in the elliptical gonads,indicating ovary formation occurs around 45 d.At 160 d,the nucleoli of the oocytes in the ovaries were distributed along the nucleus and the testis was preparing to form seminiferous lobules and began to enter early stage Ⅱ development.At 260 d of ovarian development,the yolk nucleus appeared in the cytoplasm of some ovary oocytes,and the ovaries entered the middle stage Ⅱ phase.Also at 260 d,the number of secondary spermatocytes in the testis increased,and remained at stage Ⅱ until 360 d(when the study ceased).The differentiation of the testis began later than that of the ovaries.This result is consistent with the gonadal development in most fish.The results indicate the cDNA of the sox3 gene in P.leptocephalus is 1800 bp(GenBank:MT952206),and encodes 299 amino acids.Among them,the protein contains an HMG conserved domain.The alignment of the amino acid sequence and the phylogenetic tree analysis indicates the SOX3 sequence in P.leptocephalus contains highly conserved regions.These regions are highly consistent with the SOX3 sequences in Danio rerio and Carassius auratus.Furthermore,the RT-PCR demonstrated that sox3 was highly expressed in the ovary,followed by the brain and eyes.Between the sexes,the expression level of the sox3 gene only varied significantly in the gonads,with the sox3 gene expression in the ovary being significantly higher than that in the testis.In the early stage of gonadal differentiation,the expression of sox3 in the ovaries is obviously higher than that in undifferentiated gonads.The lowest expression levels were detected in the testis.In the developmental stage after gonadal differentiation,sox3 was continuously expressed throughout ovarian development,while the expression in the testis remained at a low level.Therefore,the sox3 gene might play an important role in ovary differentiation and development when compared to the testis,but its specific function and mechanism require further research.