青虾Ran基因的克隆、表达及其在卵巢发育中的功能

Molecular characterization and developmental expression of Ras-related nuclear protein in the oriental river prawn Macrobrachium nipponense and the effects of RNA interference on ovarian maturation

本研究应用RACE技术克隆了青虾(Macrobrachium nipponensis)Ran(Ras related nuclear protein,Ras相关核蛋白)基因全长cDNA序列,该基因cDNA全长1191 bp,包括218 bp的5'UTR,648 bp的开放阅读框(ORF),405 bp的3'UTR,编码215个氨基酸。青虾Ran基因属于P-loop-NTPase超级家族,拥有PTZ00132跨结构域,多肽分子量约为24.57 kDa,理论等电点7.13。系统进化树分析表明,在动物界进化中非常保守的青虾Ran多肽与罗氏沼虾(Macrobrachium rosenbergii)聚为一支,具有最近的亲缘关系。使用荧光定量PCR技术检测Ran基因在成体青虾不同组织和卵巢不同发育期的表达差异,结果显示,Ran基因在青虾不同组织中均有表达,其表达量在卵巢中最高,是精巢表达量的7~8倍;随着卵巢的发育,Ran基因的表达水平呈现上升趋势,在卵巢消退期又恢复到较低水平。RNA干扰后,实验组Ran基因表达量显著低于对照组(P<0.05),同时卵巢发育关键基因Vg(vitellogenin)在卵巢中的表达量也显著低于对照组(P<0.05),推测Ran基因参与雌性卵巢发育过程并对Vg基因的表达起到调控作用。

Ras-related nuclear protein （Ran） is a small GTPase with many functions, such as hydrolysis of GTP, control of cell development, replication of DNA, and RNA transcription. In this study, the cDNA-encoding Ran of the oriental river prawn （Macrobrachium nipponense） was cloned using expressed sequence tag （EST） analysis and a rapid amplification of cDNA ends （RACE） approach. The full-length cDNA of Ran was 1191 bp, comprising a 5＇ untranslated region of 218 bp, a 3＇ untranslated region of 405 bp, and an open reading frame of 648 bp. The deduced protein had 215 amino acid residues with a molecular mass of 24.6 kD, and 7.13 point of theoretical isoelectric. Ran belongs to the P-loop NTPase super family. Ran has a PTZ00132 model that crosses multiple do- mains. The members of P-loop NTPase super family have extremely conservative nucleotide sequences. Phylogenetic analyses indicate evolution of Ran proteins within the animal kingdom is very conservative, with that of M. nipponense most closely related to that of M. rosenbergii. Quantitative real-time RT-PCR analysis revealed the Ran gene was expressed in testis, ovary, brain, muscle, eyestalk, abdominal nerve, heart and gill tissues. The ovary has the highest level of expression and the eyestalk has the lowest level of expression （P〈0.05）. The Ran gene expression of ovary is seven-eight times higher than that of testis, and the expression level of Ran gene increased with the development of ovary, After ovulation in ovarian regression period, the expression level of Ran gene was at a low level. After RNA interference （RNAi）, expression of Ran gene in an experimental group of adult females was significantly lower than in the control group （P〈0.05）. After RNA interference （RNAi） in the mature female prawns, the expression of Ran gene in experimental group （injected dsRNA solution into the shrimp＇s pericardial cavity） was significantly lower than in the control group （injected equal amount of DEPC water into the pranw＇s pericardial cavity） （P〈0.05）. The expression of Ran gene changes with the development of ovary. The expression of Ran gene increased from the early stage of ovarian development to the mature stage and decreased rapidly after ovulation in the ovary. Expression of Ran in ovarian tissues in the experimental group was also significantly lower than that of control group （P〈0.05）, indicating RNA interference was effective. Expression of vitellogenin was significantly affected by RNA interference, with expression in the experimental group significantly lower than in the control group （P〈0.05）. We speculate that the Ran gene plays a regulatory role in expression of the Vg gene, and that the Ran gene is involved in female ovary development.