大鼠正加速度高耐力相关基因的分离

The Separation of Genes from Rats Related to High Tolerance to Positive Acceleration

为从基因水平上揭示正加速度 (+Gz)高耐力产生机理及寻找 +Gz高耐力相关功能性蛋白 ,利用抑制消减杂交技术分离 +Gz高耐力相关基因 .雄性SD大鼠在离心机上处理后 ,选取耐受终点在高、低两个极端的动物 ,立即取全脑 ,分离mRNA .以高耐力者为Tester ,低耐力者为Driver,利用抑制消减杂交技术进行 +Gz耐力处于高、低两个极端动物脑组织间基因表达差异显示 ,获得 +Gz高耐力大鼠脑组织相关cDNA .以高、低耐力大鼠脑组织mRNA来源的cDNA为探针 ,对获得的cDNA克隆进行斑点杂交 .分别以杂交筛选出的阳性克隆为探针 ,对高、低耐力大鼠脑组织总RNA进行Northern杂交分析 .两次杂交结果均选择高耐力组杂交信号是低耐力组 3倍以上的cDNA克隆 .经过斑点杂交筛选 ,从大鼠脑组织中获得了 6 7个在 +Gz高耐力大鼠脑组织中上调表达的cDNA克隆 .Northern杂交分析发现 ,钙离子 钙调蛋白依赖性蛋白激酶Ⅱβ亚基 (Camk2b)和一未知基因在 +Gz高耐力大鼠脑组织中的表达量增加 .结果提示 ,+Gz耐力处于高、低两个极端的大鼠脑组织基因表达有明显差异 ,这些差异表达的基因很可能与 +Gz高耐力的产生有关 ,且钙离子 钙调蛋白依赖性蛋白激酶Ⅱβ亚基和一未知基因是初步获得的与

In order to discover the molecular mechanism by which high tolerance to positive acceleration (+Gz ) may produce and search for the functional protein related to high tolerance to +Gz, the genes related to high tolerance to +Gz were separated using suppression subtractive hybridization(SSH). Brains from rats with high and low tolerance to +Gz were isolated immediately and poly A + RNA of those were extracted after male SD rats were treated on animal centrifuge. With poly A + RNA of brains from rats with high tolerance to +Gz serving as tester and that from rats with low tolerance to +Gz serving as driver, the cDNA clones related to high tolerance to +Gz were obtained using SSH to display differentially expressed genes between brains from rats with high and low +Gz tolerance. cDNA probes reverse transformed from poly A+ RNA of brain samples from rats With high and low tolerance to +Gz were hybridized with the replicate cDNA dot blots. With positive clones, Northern blot analysis was made of total RNA isolated from brain samples with rats of high and low tolerance to +Gz. The clone whose signal intensity was three times higher in brains from rats with high tolerance +Gz than that in brains from rats with low tolerance +Gz was selected after the two hybridizations. Sixty seven up regulated cDNA clones in brains from rats with high tolerance to +Gz were obtained and the expressions of Camk2b (Ca 2+ /cadmodulin dependent protein kinase Ⅱ,βsubunit)and an unknown gene were increased in brains from rats with high tolerance to +Gz. The expressions of genes were significantly different between brains from rats with high and low +Gz tolerance and the differentially expressed genes were probably related to the high tolerance to +Gz. It was preliminarily found that Camk2b and an unknown gene were genes specifically related to high tolerance to +Gz.