Met基因在黏虫生殖中的功能分析

保幼激素(juvenile hormone, JH)是由咽侧体分泌的倍半萜类化合物,可以调控昆虫的很多生理过程,如发育、变态、生殖等。作为bHLH-PAS(helix-loop-helix-Per-ARNT-Sim)转录因子家族成员之一的methoprene-tolerant (Met)是JH的受体,在JH的信号传导过程中有非常重要的作用。本研究通过实时荧光定量PCR结合RNAi技术测定了沉默Met基因后黏虫Mythimna separata的Vg基因表达、卵巢发育和生殖行为,旨在探究Met基因在黏虫生殖过程中的功能。结果表明当Met基因沉默后,与卵巢发育密切相关的卵黄原蛋白(vitellogenin, Vg)基因表达量下调了50%,从而显著抑制了卵巢发育,并导致产卵显著延迟、产卵历期显著缩短,产卵量显著下降。结果表明Met基因是黏虫生殖发育过程中的关键受体基因,它通过调节后续Vg的表达和沉积,来达到控制卵巢发育,从而调控生殖作用。     

Molecular Characterization of the Von Willebrand Factor Type D Domain of Vitellogenin from Takifugu flavidus

The von Willebrand factor type D (VWD) domain in vitellogenin has recently been found to bind tetrodotoxin. The way in which this protein domain associates with tetrodotoxin and participates in transporting tetrodotoxin in vivo remains unclear. A cDNA fragment of the vitellogenin gene containing the VWD domain from pufferfish (Takifugu flavidus) (TfVWD) was cloned. Using in silico structural and docking analyses of the predicted protein, we determined that key amino acids (namely, Val¹¹⁵, ASP¹¹⁶, Val¹¹⁷, and Lys¹²²) in TfVWD mediate its binding to tetrodotoxin, which was supported by in vitro surface plasmon resonance analysis. Moreover, incubating recombinant rTfVWD together with tetrodotoxin attenuated its toxicity in vivo, further supporting protein–toxin binding and indicating associated toxicity-neutralizing effects. Finally, the expression profiling of TfVWD across different tissues and developmental stages indicated that its distribution patterns mirrored those of tetrodotoxin, suggesting that TfVWD may be involved in tetrodotoxin transport in pufferfish. For the first time, this study reveals the amino acids that mediate the binding of TfVWD to tetrodotoxin and provides a basis for further exploration of the molecular mechanisms underlying the enrichment and transfer of tetrodotoxin in pufferfish.     

中华鲟血清卵黄蛋白原水平的初步观察

通过碱不稳定性蛋白结合磷法测定不同年龄中华鲟(Acipenser sinensis)血清中磷含量,以磷的含量变化反映卵黄蛋白原(VTG)变化情况。实验鱼为人工养殖0.5、1、2、3、4、5、6、8、9、10龄中华鲟和野生成熟中华鲟,共81尾,其中8尾野生雌鱼在人工繁殖前后各取样2~4次,共测97个血液样本。结果显示:低龄中华鲟VTG含量较低,最低值在5龄,含磷量为(4.6±1.6)mg/L,5龄后增加,成熟后达到最大,含磷量为(52.0±4.5)mg/L。在性腺不同发育时期,雌性个体在Ⅱ~Ⅴ期VTG的含量增加,Ⅱ~Ⅲ期增加速度较快,随后变缓,Ⅴ期达到最高水平,Ⅵ期降低;而雄性个体在Ⅱ~Ⅳ期VTG的增加量很少,Ⅳ期达到最高水平,含磷量为(28.7±12.2)mg/L。繁殖雌鱼产前VTG含量最高,含磷量为(62.5±8.7)mg/L,产后低于产前。此外,发现产后鲟鱼卵巢液中含量低于其血清中含量,但高于其他年龄组血清中的水平。本实验表明淡水养殖的中华鲟有可能达到性成熟。     

己烯雌酚和辛基酚对真鲷幼鱼的雌激素效应研究

采用静态暴露方式研究己烯雌酚(DES)和辛基酚(OP)对真鲷幼鱼的雌激素效应.当真鲷幼鱼分别暴露于一定浓度的DES和OP,42 d后,真鲷幼鱼的肥满度均极显著下降;血浆中卵黄蛋白原被诱导产生,肝胰脏指数和血浆蛋白总量极显著升高.试验结果表明,真鲷幼鱼的肥满度、肝胰脏指数、血浆蛋白总量等可作为评估DES和OP等环境雌激素毒性效应的生物指标;真鲷幼鱼血浆中的卵黄蛋白原可作为生物标志物,用于监测海洋水体DES和OP等环境雌激素的污染.     

实时定量RT-PCR方法评价壬基酚的雌激素效应

壬基酚是1种环境雌激素物质,以血清卵黄蛋白原作为生物标记物检测壬基酚的最低雌激素效应浓度高于10μg/L.利用亚成体青鱼进行不同浓度的(1、10、50、100μg/L)壬基酚暴露实验,运用实时定量RT-PCR方法,从分子水平上,对青鱼VTG-Ⅰ、VTG-Ⅱ、CHG-H和CHG-L的基因表达进行了研究,并对低浓度壬基酚的环境雌激素效应进行了分析.结果表明:1μg/L浓度的壬基酚暴露组青鱼肝脏的VTG-Ⅰ、VTG-Ⅱ、CHG-H、CHG-L基因表达均被显著诱导,说明实时定量RT-PCR能够检测1μg/L壬基酚     

Vitellogenin in tilapia (Oreochromis mossambicus): Induction of two forms by estradiol,quantification in plasma and characterization in oocyte extract

Two forms of vitellogenin were isolated by DEAE agarose ion-exchange chromatography from plasma of the tilapia, Oreochromis mossambicus. The monomers have apparent molecular masses of 200 and 130 kDa, as indicated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and a total amount of phosphorus of 1.7 and 0.1%, respectively. Antibodies specific to the two forms, designated tVTG-200 and tVTG-130, were generated in rabbits and used to develop enzyme-linked immunosorbent assays (ELISAs) and in Western blot analyses of plasma and oocyte extract. SDS-PAGE of the oocyte extract showed a major protein band at 106.6, minor bands at 26.6, 24.2, and 23.7 kDa, and very faint bands at 83.4 and 17.5 kDa. Western blots of the oocyte extract revealed that the antiserum to tVTG-200 recognized strongly the protein bands at 24.2 and 23.7 kDa, and less strongly the bands at 25.1 and 22.6 kDa, whereas the antiserum to tVTG-130 recognized mainly the protein band at 106.6 kDa. The presence of both VTGs in untreated male tilapia was detected with the ELISAs using relatively high plasma volumes. Their presence in males was confirmed by VTG-like immunoreactive materials eluting from the ion-exchange column at the same positions as tVTG-200 and tVTG-130. The concentrations of the VTGs in males were several orders of magnitude lower than in vitellogenic females. Treatment of male tilapia with estradiol-17β (E₂) induced both VTGs within 24h. After 7 days, tVTG-130 reached a maximum concentration in plasma, whereas tVTG-200 continued to increase. Our findings demonstrate that the two vitellogenins are biochemically distinct, possibly differentially regulated, and made by both sexes.     

Development of an enzyme-linked immunosorbent assay (ELISA) for vitellogenin measurement in greenback flounder Rhombosolea tapirina

Vitellogenin (Vtg) was purified from the plasma of 17-estradiol (E₂)-injected male greenback flounder,Rhombosolea tapirina. The molecular weight of the native Vtg was estimated by gel filtration as 540 kD. SDS-PAGE and Western blotting analyses indicated that this protein consisted of three bands with molecular weights of 155, 104, 79 kD, respectively. A polyclonal antibody against the highest molecular weight band of putative Vtg was generated in sheep and an indirect antibody-capture competitive enzyme-linked immunosorbent assay (ELISA) was developed. The assay was validatedfor plasma Vtg measurement in greenback flounder. Serial dilutions of plasma from vitellogenic females parallelled the standard Vtg curve, whereas no cross-reaction was observed with the plasma of males in the ELISA. The Vtg ELISA was used to assess the induction of Vtg by E₂ in vivo in males. The induction of Vtg in greenback flounder showed a time- and dose-dependent response as in other species. In E₂-treated fish, detectable levels of Vtg were first found at 48 h, and reached a peak at 96 h post-injection. Plasma levels of Vtg increased as the E2 dose increased with a threshold of 0.1 mg kg^–1.     

Haemolymph metabolic variables in relation to eyestalk ablation and gonad development of Pacific white shrimp Litopenaeus vannamei Boone

The present study analyses some biochemical variables in haemolymph proposed as predictive indicators of the maturation capability following eyestalk ablation. Haemolymph of captive females was obtained before and 8 days after eyestalk ablation, and levels of haemocyanin, total proteins, glucose, lactate, cholesterol, triacylglycerides and vitellogenin were determined. Biochemical variables were also analysed in the hepatopancreas at the end of the experiment. Females were grouped as immature (previtellogenic stage) and mature (vitellogenic and cortical stages) based on histological analysis done 8 days after eyestalk ablation. To analyse haemolymph variables before eyestalk ablation in relation to maturation capability, immature females were classified as those with a low maturation capability and mature females as those with a high maturation capability. Females of high maturation capability had significantly higher vitellogenin levels before eyestalk ablation than females of low maturation capability. No significant differences were found for the other biochemical variables. Vitellogenin was also higher in mature than in immature females at the end of the experiment. These results indicate that vitellogenin levels in haemolymph could be used as possible predictive criteria of maturation capability, possibly because they reflect the degree of ovarian development at the time of eyestalk ablation.     

17β—雌二醇对雄性金鱼卵黄原蛋白的诱导作用

采用腹腔注射17β-雌二醇的方法诱导雄性金鱼卵黄原蛋白产生,注射浓度为0.05mg·g-1BW,诱导2周后取尾静脉血,离心分离血浆,进行血浆常规聚丙烯酰胺凝胶电泳,通过对卵黄原蛋白特性基团磷、脂和糖蛋白的染色,确定了卵黄原蛋白在电泳图谱上的位置,开发了一种简便、高效的定性卵黄原蛋白的聚丙烯酰胺凝胶电泳法。电泳结果表明,在0.05mg·g-1BW的注射浓度下,2周后17β-雌二醇诱导了雄性金鱼卵黄原蛋白产生,并通过ELISA检测卵黄原蛋白的平均含量为690.2ng·mL-1,与对照组雄性金鱼平均含量为10.7ng·mL-1的差异极显著(P<0.01),比雌性对照组检出量285.5ng·mL-1高1倍多;17β-雌二醇诱导组雄鱼血浆钙离子和血总蛋白含量明显增加。