单环刺螠Vasa基因的早期发育表达图式

Developmental expression pattern of Urechis unicinctus Vasa gene

Vasa基因编码DEAD-box家族成员中一种ATP依赖的RNA解旋酶,是决定生殖系发育的重要调控因子之一。采用同源克隆策略及SMART-RACE技术,克隆了海洋经济螠虫动物单环刺螠Vasa基因的全长cDNA,该序列长4 080 bp,开放阅读框2 322 bp,编码733个氨基酸,具有DEAD-box蛋白家族共有的全部9个保守域。整体原位杂交和半定量RT-PCR结果显示,Vasa基因在未受精卵、受精卵、2～8细胞的早期胚胎中均有明显的表达,显示其母源性提供的特点;从囊胚开始,表达量明显降低,在原肠胚表达信号主要集中在内中胚层细胞中;至担轮幼虫,表达信号进一步集中在消化道处;当发育至体节幼虫时,阳性细胞分布于消化道和体节的隔膜处,进入蠕虫状幼虫,信号仅在头部的腹刚毛附近以及后肠周围的细胞中表达。实验结果为探知刺螠动物生殖系的起源和分化以及低等型生殖腺的发生提供重要的数据。

The Vasa gene encoding an ATP-dependent RNA helicase protein member of the DEAD-box family,is one of the important regulatory factors which determines the germline cells development.In the present study,homolog cloning strategy and SMART RACE technique were used to isolate and clone the Vasa gene of U.unicinctus,a sole commercial species of Xenopenusta.The full length of the Vasa cDNA sequence is 4 080 bp,comprising an open reading frame（ORF）of 2 322 bp encoding a polypeptide of 773 amino acids,sharing nine conserved motifs of all DEAD-box family proteins.Phylogenetic analysis showed the deduced amino acids sequence contributes to the VASA-relate proteins.The temporal expression analysis of Vasa mRNA using RT-PCR and in situ hybridization techniques indicated Vasa mRNA was present in unfertilized eggs,fertilized eggs and 2-8 cell embryos,suggesting a maternally-provided characteristic.The expression level decreased obviously at blastula stage,and kept the low level from blastulas to worm-like larvae and juveniles.In situ hybridizations howed that Uu-Vasa mRNA is distributed equally in the cytoplasm of early embryos from unfertilized egg,fertilized egg to blastula.At gastrula stage,Uu-Vasa mRNA concentrated on endoderm and mesoderm of gastrula.In rtochophore,the Vasa positive signals are located in cells of alimentary canal.During the somite larvae,the hybridization signals appear in the cells of body segment membrane and alimentary canal.When developing to worm-like larvae,Un-Vasa is detected in abdominal bristle of the head region and posterior region of intestine.The location of positive signals in the posterior region of intestine is coincident with that of gonad in the future,and it was deduced that U.unicinctus gonad maybe initializes to form in worm-like larva.Our results provide primary data to generate new insight into the origin and differentiation of germ cells as well as the gonad genesis and differentiation in U.unicinctus.