中华卵索线虫vasa基因的克隆及其表达模式分析

Molecular cloning and expression analysis of vasagene inOvomermis sinensis

为明确vasa基因在中华卵索线虫Ovomermis sinensis生殖系发育以及原始生殖细胞增殖过程中的作用,采用同源克隆与cDNA末端快速扩增技术(RACE),从中华卵索线虫中克隆得到vasa基因全长cDNA序列(3053bp),编码813个氨基酸,具有DEAD-box家族蛋白全部9个保守基序。经同源比对与系统进化树分析,确定其属于DEAD-box家族的VASA亚家族成员。利用反转录PCR(reverse transcription PCR,RT-PCR)以及实时荧光定量PCR(real time PCR)分析,vasamRNA在中华卵索线虫性腺中专一性表达,其它部位如头、尾、体壁均未检测到vasa表达信号,且在生殖腺发育初期表达量最高。此外,vasa基因转录本在中华卵索线虫胚胎发育期卵中表达量较高,证实其为母源性基因,调控中华卵索线虫胚胎发育的过程。

In order to understand the function of vasa gene in the development of germ line and the reproduction of primordial germ cells in Ovomermis sinensis, the full-length cDNA sequence of Os-vasa was cloned from the mermithid by rapid amplification of cDNA end （RACE） methods. The full length of Osvasa cDNA is 3 053 bp, encoding 813 amino acids. The putative amino acid sequence possesses all nine conserved motifs of the DEAD-box family. High identity of VASA in amino acid sequence （more than 58% ） was observed between O. sinensis and the other organisms through blast analysis, indicating that the cloned cDNA belonged to the subfamily of VASA. Expression of Os-vasa was detected by reverse transcription PCR （RT-PCR） and real time PCR. The results showed that the Os-vasa gene specifically expressed in the gonads of both male and female, and there was no expression in other body parts, such as head, tail and body wall. The expression of Os-vasa in the early development stage of gonads was much higher than those in other development stages. Moreover, the transcription of vasa mRNA was relatively high in embryo stage, suggesting that vasa gene might be a maternal factor for the embryogenesis and plays an important role in the early embryo development.