Carvalho Klébea, Ribeiro Lupis, Moraes Jorge, Roberto da Silva José, Costa Evenilton P, Souza-Menezes Jackson, Logullo Carlos, Nunes da Fonseca Rodrigo, Campos Eldo
Laboratory of Integrated Biochemistry-Hatisaburo Masuda, Universidade Federal do Rio de Janeiro, Núcleo em Ecologia e Desenvolvimento Sócio Ambiental de Macaé, Avenida São José do Barreto, 764, São José do Barreto, Macaé, RJ CEP 27965-045, Brazil.
Nacional Institute of Science and Technology-Molecular Entomology, Rio de Janeiro, RJ CEP 21941-590, Brazil.
Int J Mol Sci. 2015 Mar 24;16(4):6631-44. doi: 10.3390/ijms16046631.
Polyphosphates have been found in all cell types examined to date and play diverse roles depending on the cell type. In eukaryotic organisms, polyphosphates have been mainly investigated in mammalian cells with few studies on insects. Some studies have demonstrated that a pyrophosphatase regulates polyphosphate metabolism, and most of them were performed on trypanosomatids. Here, we investigated the effects of sPPase gene knocked down in oogenesis and polyphosphate metabolism in the red flour beetle (Tribolium castaneum). A single sPPase gene was identified in insect genome and is maternally provided at the mRNA level and not restricted to any embryonic or extraembryonic region during embryogenesis. After injection of Tc-sPPase dsRNA, female survival was reduced to 15% of the control (dsNeo RNA), and egg laying was completely impaired. The morphological analysis by nuclear DAPI staining of the ovarioles in Tc-sPPase dsRNA-injected females showed that the ovariole number is diminished, degenerated oocytes can be observed, and germarium is reduced. The polyphosphate level was increased in cytoplasmic and nuclear fractions in Tc-sPPase RNAi; Concomitantly, the exopolyphosphatase activity decreased in both fractions. Altogether, these data suggest a role for sPPase in the regulation on polyphosphate metabolism in insects and provide evidence that Tc-sPPase is essential to oogenesis.
迄今为止,在所有已检测的细胞类型中均发现了多聚磷酸盐,并且根据细胞类型的不同发挥着多种作用。在真核生物中,多聚磷酸盐主要在哺乳动物细胞中进行研究,对昆虫的研究较少。一些研究表明,一种焦磷酸酶调节多聚磷酸盐代谢,其中大多数研究是在锥虫中进行的。在这里,我们研究了红粉甲虫(赤拟谷盗)中sPPase基因敲低对卵子发生和多聚磷酸盐代谢的影响。在昆虫基因组中鉴定出一个单一的sPPase基因,在胚胎发育过程中,该基因在mRNA水平上由母体提供,且不限于任何胚胎或胚外区域。注射Tc-sPPase双链RNA后,雌性存活率降至对照(dsNeo RNA)的15%,产卵完全受损。对注射Tc-sPPase双链RNA的雌性卵巢小管进行核DAPI染色的形态学分析表明,卵巢小管数量减少,可观察到退化的卵母细胞,生殖腺减少。在Tc-sPPase RNA干扰中,细胞质和细胞核部分的多聚磷酸盐水平升高;与此同时,两个部分的胞外多聚磷酸酶活性均降低。总之,这些数据表明sPPase在昆虫多聚磷酸盐代谢调节中起作用,并提供了证据证明Tc-sPPase对卵子发生至关重要。