论文部分内容阅读
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。
In this study, the RACE technique was used to clone the full-length cDNA of Ras related nuclear protein (Ran related nuclear protein) gene. The full length cDNA of this gene is 1191 bp in length, including 218 bp 5’UTR and 648 bp Open reading frame (ORF), 405 bp 3’UTR, encodes 215 amino acids. The Pseudostellaria reticulata Ran gene belongs to the P-loop-NTPase superfamily and possesses the PTZ00132 trans-domain. The polypeptide has a molecular weight of about 24.57 kDa and a theoretical isoelectric point of 7.13. Phylogenetic tree analysis showed that the Ran polygamy Ran polypeptide, which is highly conserved in the evolution of the animal kingdom, is clustered with Macrobrachium rosenbergii and has the closest genetic relationship. Quantitative real-time PCR was used to detect the differential expression of Ran gene in different tissues and stages of adult oviparous shrimp. The results showed that Ran gene was expressed in different tissues of freshwater shrimp and its expression was the highest in ovary, 7 to 8 times higher than that of normal ovaries. With the development of ovary, the expression level of Ran gene showed an upward trend and returned to a lower level during ovarian regression. After RNA interference, the expression of Ran gene in experimental group was significantly lower than that in control group (P <0.05), and the expression of vitellogenin in ovary was also significantly lower than that in control group (P <0.05) The gene is involved in the development of female ovary and regulates the expression of Vg gene.