栉孔扇贝sox9基因的cDNA克隆及其在不同发育时期性腺中的表达特征

Molecular cloning of sox9 cDNA and its expression characteristics in gonads at different developmental stages of Chlamys farreri

SOX9是SOX家族的SOXE亚族成员,在脊椎动物骨骼发育、胰腺发育、性别决定与分化以及肿瘤形成中发挥重要的作用。sox9在不同种类脊椎动物性腺中的表达存在差异,在无脊椎动物性腺中的表达特征尚不清楚。本研究利用RACE技术克隆了栉孔扇贝(Chlamys farreri)sox9(Cf-sox9)全长的c DNA序列,其长度为2835 bp,开放阅读框为1413 bp,编码470个氨基酸。预测的氨基酸序列具有SOX家族的HMG-box和SOXE亚族的高保守区域,但没有脊椎动物羧基端的Pro-Gln-Ser rich区域。原位杂交和免疫组织化学技术确定Cf-sox9 m RNA和Cf-SOX9蛋白均定位在栉孔扇贝精巢和卵巢的所有生殖细胞中,并且在不同发育时期性腺中呈现了类似的表达规律,即在精巢中,阳性信号在精母细胞中最强,在精子中最弱;在卵巢中,阳性信号在卵原细胞、卵母细胞以及成熟卵中呈现逐渐减弱的趋势。这一表达特征与脊椎动物性腺中多样的性别差异表达特征不同,提示sox9在贝类性腺发育和配子发生中的作用可能与大多数脊椎动物不同。

SOX9is a member of the SOXE group in the SOX family,which plays an important role in sex determination and differentiation of vertebrates.sox9is specifically expressed in the testes of mammals,birds,and reptiles,and is expressed in both the testes and ovaries in amphibians and fishes.To measure the expression of sox9in the gonads of invertebrates,we cloned a2835bp full-length cDNA of the scallop Chlamys farreri sox9(Cf-sox9)which contains a1413bp open reading frame(ORF)encoding a470-amino acid sequence with the HMG-box of the SOX family and the highly conserved region of the SOXE group,although there is no Pro-Gln-Ser-rich region at the C-terminus of vertebrate SOXE proteins.Cf-sox9DIG-labeled RNA probes and Cf-SOX9polyclonal antibodies were produced based on the Cf-sox9full-length cDNA sequence.ISH and immunohistochemistry detection showed that Cf-sox9transcripts and Cf-SOX9proteins were located in all germ cells of C.farreri testes and ovaries at different developmental stages.The expression patterns between sox9mRNA and SOX9protein were similar.In the testes,the intensity of positive signals was highest in the spermatocytes and lowest in the spermatozoa.In the ovaries,the intensity of positive signals gradually decreased across the oogonia,oocytes,and mature oocytes.The expression characteristics of Cf-sox9in C.farreri gonads were different from that most of vertebrates,which show variability in expression between sex,suggesting its function of gonad development and gametogenesis might be different from vertebrates.