中华鳖vasa基因克隆及在卵母细胞中的表达分析

研究利用中华鳖为研究模型进行爬行类生殖细胞发育分化成熟等生物学研究，克隆了中华鳖vasa基因的cDNA序列，全长3865 bp，包括5'端非编码区90 bp，3'端非编码区1699 bp，开放阅读框长2076 bp，共编码691个氨基酸。中华鳖Vasa氨基酸序列包含DEAD-box家族蛋白8个保守保守功能域，在N末端有4个RGG重复序列和2个GG富集区，与小鼠Vasa蛋白的同源性较高（72%）。荧光定量PCR的结果表明，中华鳖vasa mRNA主要精巢和卵巢中表达，其他体组织中均难检测到表达。卵巢冰冻切片原位杂交结果显示:中华鳖vasa mRNA在生殖细胞中特异表达；在卵子发生过程中的不同发育期卵母细胞中呈现动态的变化。即vasa mRNA在初级卵母细胞及生长期卵母细胞中表达最强，且均匀分布在细胞质中，随着卵母细胞的逐渐增大，信号逐渐减弱，直至在成熟的卵母细胞中几乎检测不到表达信号，说明vasa可能在中华鳖早期卵母细胞发育中起重要作用。同时，vasa基因可作为中华鳖生殖细胞分子标记物，根据其mRNA的表达水平来鉴别不同发育时期的卵母细胞。研究结果为进一步开展中华鳖胚胎生殖细胞发育及配子生成，特别是研究中华鳖，乃至爬行类原始生殖细胞（Primordial Germ Cells，PGCs）的起源、迁移、分化等研究奠定了基础。

CLONING AND EXPRESSION ANALYSIS OF VASA IN CHINESE SOFT-SHELL TURTLE OOCYTES

vasa encodes a DEAD-box RNA helicase and is a highly conserved germ cell marker across animal phyla. vasa is necessary for germ cell development during embryogenesis and gametogenesis. The Chinese soft-shell turtle (Pelidiscus sinensis) was used as a model to study germ cell development and differentiation in reptiles. Here a full vasa cDNA was cloned, 3865 bp in total, containing a 5′-UTR of 90 bp, a 3′-UTR of 1699 bp and an open reading frame (ORF) of 2076 bp, encoding a protein of 691 aa. The deduced amino acid sequence contains 8 conserved motifs of DEAD-box family protein, and possesses 4 RGG repeats and 2 GG repeats. The predicted protein is 72% identical in sequence to its homologs from mouse. The vasa mRNA was detected exclusively in the gonads of both sexes in soft-shell turtle. Chromogenic and fluorescent RNA in situ hybridization revealed that the vasa mRNA was restricted to germ cells: It was highly expressed from primary oocyte to early growing oocyte and uniformly distributed in oocyte cytoplasm. In oocytes at the late stage, the vasa mRNA was perinuclear distributed and decreased in oocytes with the vitellogenesis proceeding. The vasa mRNA signal was undetectable in the mature oocytes. The findings indicated that vasa gene also played an important role in oogenesis in the Chinese soft-shell turtle. Hence turtle vasa is a reliable germ cell marker to identify female germ cells at different stages during oogenesis. These results provide a theoretical basis to study the germ cell development and differentiation during embryogenesis and /or gametogenesis, especially to in-vestigate the initiation and migration of primordial germ cells (PGCs) in Chinese soft-shell turtle and reptiles.