3种组织保存方法在进行流式细胞倍性分析和DNA含量测定中影响比较研究

Comparisons of three methods of tissue preservation in the flow cytometry on ploidy analysis and DNA content determination

流式细胞术能快速检测生物细胞的倍性和DNA含量,而水生生物多倍体的发掘与利用在多倍体育种和丰富物种多样性中具有重要意义。该研究利用濒危冷水鱼类花羔红点鲑鳍条组织,分别采用低温冻存、70%乙醇固定和活体采样3种方法对收集的组织进行短期保存并进行流式细胞检测,结果显示,花羔红点鲑为四倍体鱼类,3种保存方法测得的DNA含量大小为活体组织<乙醇固定<低温冻存,且DNA含量分别为3.67 pg/N、3.47 pg/N和6.0 pg/N。在组织保存效果中,70%乙醇固定的效果仅次于活体采样,低温冻存方法流式检测结果出现较多杂峰,表明这种保存方法不能保证细胞的完整性,在制备细胞悬液时破碎的细胞较多,并形成黏连体,检测效果不佳。所有被检测个体中,有一条花羔红点鲑为多倍体。

Flow cytometry can rapidly detect ploidy and DNA content of cells,and the discovery and utilization of aquatic organisms polyploidy are important in polyploid breeding and enriching species diversity. The study use the fin tissue of Salvelinus malma, an endangered cold - water fish, using three methods that cryopreservation, fixed with 70% ethanol and living sampling forshort - term preservation of the collected tissues, and then examined by flow cytometry. The results showed that the Salvelinusmalma are tetraploid fishes, The content of DNA measured by three kinds of preservation methods was as follows: living tissue < ethanol < Cryopreservation, and the content of DNA respectively was 3. 67pg/ N, 3. 47pg/ N and 6pg/ N. In comparing the effect of preserving tissue, the effect of 70% ethanol fixation was be next only to that of living samples,the results of Cryopreservation flow cytometry showed that there were many stray peaks, and show that this method cannot guarantee the preservation of the integrity of the cell, in the preparation of cell suspension, more broken cells, and formation of sticky conjoined, poor detection results. There is one individual is polyploidy in all of the samply.