栉孔扇贝wnt4基因cDNA克隆及表达分析

Molecular cloning and expression analysis of wnt4 cDNA from the Zhikong scallop Chlamys farreri

由栉孔扇贝(Chlamys farreri)转录组数据库获得wnt4基因的一段表达序列标签(EST)序列,利用SMART-RACE技术克隆了wnt4基因cDNA全长序列,该序列长1 239 bp,其中开放阅读框为1 068 bp,可以编码355个氨基酸,推导的氨基酸序列含有WNT家族特有序列,且与沙蚕(Platynereis dumerilii)、海胆(Heliocidariserythrogramma)、人(Homo sapiens)等物种WNT4同源性都在60%以上。半定量RT-PCR结果显示,除肾脏外,wnt4基因在栉孔扇贝的精巢、卵巢、闭壳肌、肝胰腺、鳃、外套膜组织中均有表达,但表达量较低。定量RT-PCR结果表明:wnt4基因在成熟期的精卵巢中表达量最高,增殖期和休止期表达量次之,生长期表达量最低;整个生殖周期中精巢表达量高于卵巢。该基因在栉孔扇贝多个组织中的表达特性,暗示其参与了多样的生物学过程;在性腺中的表达特征表明其可能参与两性性腺的发育并在生殖细胞成熟过程中发挥作用。

An expressed sequence tag （EST） of wnt4 （Wingless-type MMTV integration site family, member 4） was obtained from the transcriptome database of the Zhikong scallop （Chlamys farreri） and its entire cDNA se- quence was acquired by SMART-RACE. The results indicated that the cDNA sequence of C. farreri wnt4 was 1239 bp in length and encoded 355 amino acids. The WNT4 amino acids sequences were specific to the WNT family. Homologous analysis of the amino acids showed that C. farreri WNT4 was over 60% similar to most spe- cies of WNT4 including Platynereis dumerilii, Heliocidaris erythrogramma and Homo sapiens. Semi-quantative RT-PCR results showed no expression in the kidney tissue; however, C. farreri wnt4 had large, wide but low ex- pression levels in testes, ovary, adductor muscle, hepatopancreas, gill and mantle tissues, qRT-PCR results indi- cated that wnt4 mRNA expression levels were highest in the testes and mature ovaries, with lower levels in the proliferative and resting stage and the lowest expression at the growing stage. The expression levels of C. farreri wnt4 were characteristically testis-predominant during the entire reproductive cycle. The multiple tissue expres- sion characteristics imply that WNT4 plays various biological roles in C. farreri. We can infer from the expression pattern in the gonads that WNT4 also contributes to the process of gonadal development and germ cell maturation.