用福林酚法和复性电泳对红螯螯虾(Cherax quadricarinatus)雄性生殖系统的组织蛋白酶(Male Reproductive System Cathepsin,MRSC)进行了定性和定量分析,以探讨其在雄性生殖系统发育、精荚形成和精子释放过程中的作用。福林酚法测定结果显示:仔虾精巢蛋白酶比活力分别在pH6和pH10时出现2个高峰,而成虾精巢只在pH10时出现1个活力较高的峰。成虾雄性生殖系统的各部分在pH10～12的条件下有较强的蛋白酶比活力,于碱性缓冲液中浸泡有助于其后输精管内含物的溶解。复性电泳分离到4个蛋白酶条带,按分子量从大到小依次为:MRSC-A、MRSC-B、MRSC-C和MRSC-D。MRSC-A和MRSC-D广泛分布于雄性生殖系统各部分,其中MRSC-A为酸性蛋白酶,而MRSC-D是碱性蛋白酶;MRSC-C为酸性蛋白酶,只分布于中输精管;MRSC-B活性受pH值影响不大,仅存在于后输精管。在红螯螯虾雄性生殖系统的不同发育阶段,其组织蛋白酶的组成和活性不同。作为精荚的重要组成成分,组织蛋白酶与精荚排出体外后的硬化有直接关系,并促进随后精荚基质的水解以及精子的释放。
Abstract The digestive enzyme activity and mRNA level of trypsin during the embryonic development of Cherax quadricarinatus were analyzed using biochemical and Fluorogenic Quantitative PCR （FQ-PCR） methods. The results show that the activities of trypsin and chymotrypsin had two different change patterns. Trypsin specific activity increased rapidly in the early stages of development and still remained high in preparation for the hatch stage. However, chymotrypsin activity peaked in stage 4 of embryonic development and decreased significantly in the last stage. The mRNA level of trypsin was elevated in all stages and two peak values were observed in stages 2 and 5 respectively. The results indicate that trypsin is very important for the utilization of the yolk during embryonic development and for the assimilation of dietary protein for larvae. The gene of trypsin is probably regulated at transcriptional level. The mRNA levels of trypsin can reflect not only trypsin activity, but also the regulatory mechanism for expression of trypsin gene to a certain degree.
