【摘要】 以中华绒螯蟹(Eriocheir sinensis)肌肉、鳃和精巢为对照,从线粒体标志酶SDH活性、线粒体特异性荧光探针(Rh123)以及线粒体16S rDNA PCR扩增3个途径,探讨了该蟹精子线粒体的存在状况。研究发现,肌肉和鳃SDH酶活性较高,精巢SDH酶活性显著低于肌肉和鳃,而精子SDH酶活性则无法检测到。使用Rh123检测发现,精巢中有较强的荧光,而精子中则无法观察到。通过GenBank中的中华绒螯蟹线粒体16S rDNA序列设计引物,利用PCR扩增方法,以beta-actin做内参,检测了中华绒螯蟹肌肉、鳃、精巢和精子中线粒体16S rDNA扩增情况,发现各组织或细胞beta-actin扩增片段大小、条带宽度和亮度基本一致,表明各模板DNA量基本一致;而在同一DNA浓度下,16S rDNA的扩增片段长度虽一致,但其产物量存在明显差异,精子16S rDNA产物量显著低于其他3种组织,其条带宽度和亮度很弱;16S rD-NA扩增产物经测序分析及比对证明与已有序列完全吻合。上述结果表明,中华绒螯蟹精子中存在线粒体,但其线粒体的数量极显著低于精巢和其他组织,这也是利用灵敏度较低的SDH酶活性和Rh123探针以及常规的组织学和超微结构观察法难以检测到其精子线粒体的原因。[中国水产科学,2007,14(1):139-143]更多还原

【Abstract】 Three methods,which were SDH activity,specific fluorescent probe(Rh123) and mitochondrial 16S rDNA gene amplification,were used to detect sperm mitochondria of Eriocheir sinensis,and three tissues—muscle,branchia and testis,were investigated as control.SDH activity in testis was only 28.06 U/mg prot,which was significantly lower than that in muscle(114.55 U/mg prot) and in branchia(93.72 U/mg prot),while it was too low to be detected in sperm.Strong fluorescence was detected in testis by Rh123 but nothing was observed in sperm.Primers used in PCR were designed based on the sequence of mitochondrial 16S rDNA(AJ250642 and AF312718) and beta-actin(AY910691)of Eriocheir sinensis which had been deposited in GenBank.PCR analysis using total DNA from muscle,branchia,testis and sperm indicated that 16S rDNA exhibited different expression in the four samples.Beta-actin gene was detected plentiful in all the four tissues/cells and exhibited the same expression pattern.16S rDNA gene amplification was detected at high levels in muscle and branchia,appreciable low in testis and very low in sperm.The PCR products of 16S rDNA gene were sequenced and aligned with the sequence of 16S rDNA from GenBank(AJ250642 and AF312718),and the alignment result showed there was no difference between them.All the results suggest that there are mitochondria in Eriocheir sinensis sperm but much fewer than those in testis and other tissues,and this may be the reason why SDH activity and Rh123 as well as common histology and ultrastructure research can not detect the existence of mitochondria in Eriocheir sinensis sperm.[Journal of Fishery Sciences of China,2007,14(1):139-143]更多还原