实时荧光定量PCR检测凡纳滨对虾和罗氏沼虾卵黄蛋白原mRNA在卵巢和肝胰腺中的表达

卵黄磷蛋白作为卵黄蛋白的主要成分, 可为甲壳动物胚胎和早期幼体发育提供能量, 为研究其来源及合成规律, 实验应用SYBR GreenⅠ荧光定量PCR法检测了凡纳滨对虾和罗氏沼虾性腺不同发育时期卵巢和肝胰腺两种组织中卵黄蛋白原mRNA的表达水平。结果发现,凡纳滨对虾和罗氏沼虾的卵巢和肝胰腺中都有卵黄蛋白原mRNA的表达。随着卵巢的发育, 凡纳滨对虾卵巢中卵黄蛋白原mRNA的相对表达量在前5个阶段不断增加, 分别为1.1, 5.9, 10.4, 26.9, 85.0, 恢复期急剧减少, 为1.6。肝胰腺中的相对表达量也不断增加, 分别为1.3, 3.3, 7.1, 37.3, 51.6, 恢复期急剧减少, 为1.0。罗氏沼虾肝胰腺中卵黄蛋白原mRNA的相对表达量在前四个阶段不断增加, 分别为3.4, 12.6, 15.2, 38.9, 抱卵期急剧减少, 为2.9；而卵巢在整个发育过程中对卵黄蛋白原合成的贡献比较小, 分别为1.0, 1.3, 1.7, 4.8, 1.5。研究表明, 两种虾类的肝胰腺和卵巢均具合成卵黄蛋白的功能, 而且在不同的卵巢发育阶段呈现明显的规律性。

Quantitative analysis of vitellogenin mRNA expression in Litopenaeus vannamei and Macrobrachium rosenbergii

Vitellin, as the major constituent of the yolk protein is to provide nutrition to the developing of embryos and early larve in crustaceans. Its source and biosynthesis regularity have been the focus of crustacean research in past decades. The accurate site for vitellogenin synthesis was investigated in two representative shrimp species Litopenaeus vannamei and Macrobrachium rosenbergii with real-time PCR in this experi-ment.Based on histological observation, the ovarian development was divided into six stages in L. vannamei (oogonium proliferation, previtellogenic, primary vitellogenic, secondary vitellogenic, mature, and recovery stages) and five stages in M. rosenbergii (previtellogenic, primary vitellogenic, secondary vitellogenic, mature, and gravid stages). It was found that both L. vannamei and M. rosenbergii expressed Vg-mRNA in hepatopancreas and ovary. In L. vannamei, the relative quantity of Vg-mRNA expression of each developmental stage was 1.1, 5.9, 10.4, 26.9, 85.3, and 1.5 in ovary, and 1.3, 3.2, 7.1, 37.2, 51.6, and 1.0 in hepatopancreas respectively. In M. rosenbergii, the relative quantity of Vg-mRNA expression of hepatopancreas in each ovarian developmental stage was 3.4, 12.6, 15.2, 38.9, and 2.9 compared to 1.0, 1.3, 1.7, 4.8, and 1.5 in ovary, respectively. In all examined shrimps the Vg-mRNA expression decreased sharply to the minimum at the last development stage. The research confirmed that both penaeid shrimp(Litopenaeus vannamei) and caridea prawn(Macrobrachium rosenbergii) were able to synthesize the vitellin in ovary and hepatopancreas, and with the gonad development, the vitellin synthesis showed clear regularity.