斑马鱼Zar1和Zar1-like基因的克隆及其表达分析

MOLECULAR CLONING AND EXPRESSION PATTERN OF ZAR1 AND ZAR1L IN THE ZEBRAFISH

合子阻滞基因1(Zygote arrest 1,Zar1)及Zar1-like(Zar1L)作为母源基因,在小鼠卵子-合子转变过程中发挥着极其重要的作用。研究以斑马鱼为对象,采用cDNA末端快速克隆(RACE)技术获得了Zar1L cDNA全长,并采用RT-PCR技术检测Zar1和Zar1L在斑马鱼不同组织器官、卵母细胞和胚胎发育时期的表达情况。Zar1L cDNA的全长为1146 bp,编码311个氨基酸残基,5′非编码区20 bp,3′非编码区190 bp。多重序列比对结果显示,C-末端的序列非常保守,相似性高达74%,具有非典型的PHD基序(Plant homeo domain),并具有两个C4类型的锌指结构。在基因结构上,Zar1和Zar1L均由四个外显子构成,两个基因的每一个外显子在大小上也比较相近。分子进化表明斑马鱼Zar1和Zar1L属于两个不同的基因,可能是同一来源的祖先基因重复和进化的结果。RT-PCR结果表明斑马鱼Zar1和Zar1L的表达模式相似,都是卵巢特异表达基因,在卵母细胞及早期胚胎中可检测到大量转录本的存在,囊胚、原肠胚期后下降,但在整个胚胎发育期都可检测到转录本。斑马鱼Zar1和Zar1L mRNA的表达水平的一致性,似乎暗示着两个基因的功能基本一致。表达模式分析表明Zar1和Zar1L可能与斑马鱼卵子-合子转变有关,也可能与其他的胚胎发育过程有关。

Maternal mRNAs,which are expressed in oocytes,play an important role in the success of early embryonic development.Zygote arrest 1（Zar1） and Zar1-like（Zar1L） are the oocyte-specific maternal genes that function at the oocyte-to-embryo transition in mouse.Zar1 and Zar1L are conserved in evolution,but the expression patterns of Zar1 and Zar1L are diverse in animal kingdom.In this experiment,the full length of Zar1L cDNA was cloned from the ze-brafish ovary by RACE.The expression patterns of Zar1 and Zar1L in different tissues,developing oocytes and em-bryogenesis were checked by RT-PCR.The full-length cDNA of Zar1L was 1146 bp,which comprised a 20 bp 5′ un-translated region（UTR）,a 190 bp 3′-UTR;a 936 bp open reading frame（ORF） encoding a 311 amino acids peptide.Structurally,the Zar1L protein contained an atypical PHD motif（plant homeo domain） and two C4 type zinc finger in the C-terminal,and showed 74% homology to known counterparts from mammals and birds.In gene structure,Zar1 and Zar1L had four exons,each of the two genes exons were relatively similar in size.It showed that zebrafish Zar1 and Zar1L gene belonged to two different genes,and it might be the ancestors of the same source gene duplication and evolution.Zar1 and Zar1L were expressed at a similar pattern,and both of them were ovary specific.The expressions of Zar1 and Zar1L did not change during oogenesis.Both Zar1 and Zar1L mRNA were maternal deposited,and the ex-pression levels of both genes were high in the early embryos till gastrulation.A significant decrease of Zar1L occurred at gastrula,while Zar1 decreased apparently at neurula.However,both Zar1 and Zar1L were expressed in the whole process of embryonic development till hatching.The consistency expression level of Zebrafish Zar1 and Zar1L implied that the function of the two genes were basically the same.The results suggested that Zar1 and Zar1L were maternal factors for mid-blastula transition,and Zar1 and Zar1L might have other important roles during embryonic development.