不同发育阶段草鱼肾脏蛋白质组差异的初步分析

PRELIMINARY PROTEOMIC DIFFERENCE ANALYSIS ON THE KIDNEY OF GRASS CARP,CTENOPHARYNGODON IDELLUS,IN VARIOUS DEVELOPMENTAL STAGES

为了研究草鱼在不同发育阶段抗病能力差异的发育遗传学原因,以1龄草鱼和3龄草鱼的免疫器官肾脏为材料研究其在蛋白质组水平的差异。提取1龄草鱼和3龄草鱼肾脏的全蛋白,用二维聚丙烯酰胺凝胶电泳进行蛋白质的分离,染色后,扫描成像,经PDQUST软件分析,分别检测到大约900个蛋白质点。这些蛋白质点主要分布在pH4.5—7之间,其分子量大部分在66.2kDa以下。在3龄草鱼中检测到了3个在1龄草鱼中没有的差异蛋白质点,和20个上调、下调的差异点。经MALDI-TOF质谱分析和数据库搜索,证明在3龄草鱼肾脏中上调的差异点中有4个是与免疫或肾脏发育相关的蛋白质。这些初步的研究结果提示草鱼在不同发育阶段抗病能力的差异可能具有其发育遗传学基础。

It is well known that the infectious diseases caused by virus and bacterium frequently occur in one year and two years old grass carp but rarely in the three years old grass carp. This phenomenon suggests that the grass carp can establish the ability to resist the virus and bacterium by itself. However, some genes that are interrelated to the immune system would not be expressed and the grass carp would not complete the immune system until develop for more than two years. To determine the developmental genetic cause of different disease-resistant ability in grass carp of various developmental stages, we investigated the proteomic difference between kidneys of one year and two years old grass carp with methods of proteomics. The kidney of fish contains pronephros and mesonephros. The main function of the pronephros is immunity, and the mesonephros can produce blood cells including the leukocytes and erythrocytes that are also associated with immunity. Therefore, the whole kidney rather than head kidney was utilized for proteomic experiment to reflect the developmental genetic backgrounds. After extraction, the total kidney proteins both one year old and three years old grass carp were separated by two-dimensional polyacrylamide gel electrophoresis. The silver stained gel images were analyzed with PDQUST software and about 900 protein spots were determined. Most protein spots were distributed in the gel area of pH4.5-7 and Mr〈66.2kDa. 23 differential protein spots were detected. Among them, 3 protein spots were observed only in three years old grass carp, and other 20 spots were up-or down-regulated protein. Four differential proteins were identified by matrix assisted laser desorption/ionisation-time of flight-mass spectrometry and database analysis, These proteins are Novel Protein Similar to Novel Immune-Type Receptors 2, Recombination Activating Gene 1, MHC Class Ⅱ Beta Chain and Wnt4. The preceding three proteins are directly associated with the immunity and the forth protein is associated with the kidney development. Since these identified proteins are not the antibody proteins that are induced by antigens but the proteins that have something to do with the development of the immune system or kidney, they might play a role in perfection of the disease-resistant system of the three years old grass carp. These results suggested that the different disease-resistant ability in various developmental stages of grass carp might have its developmental genetic cause.