DEAD-BOX基因家族在曼氏无针乌贼生殖细胞形成和分化中的作用

生殖细胞的发生是发育生物学研究的核心，本项目通过基因克隆技术获得曼氏无针乌贼生殖相关DEAD-box 家族基因（vasa基因，pl10基因和p68基因）全长序列，并对基因序列进行结构分析和功能预测；采用 荧光定量PCR方法检测DEAD-box 家族基因在成体组织、性腺发育周期以及胚胎和幼体发育中的时空表达，分析DEAD-box 家族基因表达的特异性，筛选生殖细胞分化特异性表达基因作为分子标记；以地高辛标记的 RNA 为探针，采用切片原位杂交和整体原位杂交方法检测基因在性腺发育周期以及胚胎和幼虫发育中的表达，获得曼氏无针乌贼生殖细胞发育表达图式，确定曼氏无针乌贼原生殖细胞起源和分化模式，初步建立曼氏无针乌贼生殖细胞起源及迁移至性腺的基本路径，建立头足类生殖细胞发生的基本模型，为进一步的生殖调控研究奠定基础。

Ontogenesis of germ cells is an important problem in developmental biology.This project would have the full length cDNA of DEAD-Box genes family by using RACE technology. The structure of sequence and function would be analyzed by bioinformatic system.Real-time quantitative PCR results would indicate the expression level of DEAD-Box gene family in different tissues,different growth phases of gonad, different embryo and larvae development.We want to find the specifictiy of DEAD-Box family expression, and choose the effective gene which would be used as molecular marker to establish the early developmental pattern of primordial germ cells(PGCs).The expression and distribution of DEAD-Box genes would detected during gonad growth, embryo development and larvae development by using in situ hybridization with DIG antisense RNA probe.We plan to gain the squid germ cells development pattern,make sure the origin and differentiation of promordial germ cells.These results will preliminary establish the basic pathway of orgin of PGCs and migration to gonad. And the next step we will used this model to regulated the reproduction.