隆线溞孤雌溞和两性雌溞的蛋白质差异表达

本实验提取隆线溞孤雌溞和两性雌溞的可溶性蛋白进行双向电泳和质谱鉴定,分析隆线溞在两种生殖状态下蛋白质组的差异变化。聚丙烯酰胺凝胶SDS-PAGE结果表明:隆线溞在两种生殖状态下存在明显的蛋白质表达差异,孤雌溞的蛋白条带在分子量约50.6kD、36.2kD、32.1kD和25.7kD处表达量较两性雌溞明显;两性雌的蛋白条带在分子量约87.8kD、67.2kD、53.6kD和35.5kD处表达量较孤雌溞明显,其中35.5kD的蛋白条带为两性雌所特有。同时取两个样品的可溶性蛋白进行双向电泳,每个样品重复四次。双向电泳图谱经银染后利用软件分析可知,隆线孤雌平均可检测到约750个蛋白质点,两性雌溞平均可检测到约720个蛋白质点。同时利用软件对凝胶上的蛋白质点进行半定量分析,发现隆线溞从孤雌生殖转化为两性生殖后有18个蛋白质点呈现显著变化,其中14个点表达量明显下降,4个点表达量显著升高。实验结果具有较好的重复性。取4个表达量显著上升的蛋白质点进行质谱分析,得到两个蛋白质点(16号和17号)的测定结果。其中16号点为一类酸性脱氢酶(2I234),它在动物生长发育的各个阶段大量表达,这类蛋白质在隆线溞生殖转化过程中表达量变化尤为显著。本研究结果表明:隆线溞在孤雌生殖和两性生殖状态下存在明显的蛋白质表达差异。

Differential protein expression between parthenogenetic and sexual female of Daphnia ( Ctenodaphnia ) carinata

There are two different reproductive modes in daphnids: parthenogenetic and sexual reproduction, but the mechanism of reproductive switch has not been elucidated. In the present study, water-soluble proteins were isolated from parthenogenetic and sexual females of Daphnia(Ctenodaphnia) carinata respectively, then profiled by two-dimensional polyacrylamide gel electrophoresis and identified by mass spectrometry. Firstly, sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE) showed that each sample had a complex and characteristic protein distribution. More than 4 protein bands at approximately 50.6 kD, 36.2 kD, 32.1 kD and 25.7 kD were dominant in the parthenogenetic female sample. Four bands at approximately 87.8 kD, 67.2 kD, 53.6 kD and 35.5 kD were dominant in the sexual female sample, in which the 35.5 kD band appeared as unique to the sexual female. The results of two-dimensional polyacrylamide gel electrophoresis(2D-PAGE) including high resolution and reproducibility were obtained with four repeated assays. On average, about 750 and 720 spots were visualized respectively by sliver staining in 2D gels of parthenogenetic and sexual females. When the reproductive mode of D.carinata had switched from parthenogenetic to sexual reproduction, 18 proteins were shown to display significant and reproducible changes. Among them, 14 proteins were down-regulated while four other proteins were up-regulated. The four up-regulated proteins were further characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS), 2 proteins(No.16 and No.17) were further identified. No.16 protein was a branched acid d~ehy~dro~gen~ase(2I234), which is expressed at high levels at all stages of development, especially during the transition from parthenogenetic to sexual reproduction in D.carinata. The results showed that there were definite differences of protein expression differences during the change from parthenogenetic to sexual reproduction in D.carinata. .