油松端粒酶活性的测定方法

端粒酶具有修复延伸端粒的功能,对于维持真核生物染色体的完整性至关重要,因此对其活性的测定有着重要意义。通过大量的摸索实验,将常用于动物细胞端粒酶活性检测的端粒末端重复序列扩增法（TRAP法）运用到对植物材料的检测中来,对北方常见木本植物油松进行了端粒酶活性测定。基于TRAP法的原理,对体外延伸、引物选择、退火温度、电泳检测等实验条件进行优化。结果表明,油松端粒酶活性测定的最佳体外延伸条件为30℃延伸1 h,最适前导链引物和反向引物分别为TS21和ACX,PCR退火温度为61.8℃,电泳检测使用Gene Finder核酸染料染色效果更好,建立了一种有效测定木本植物油松松针叶端粒酶活性的测定方法。

Optimization Telomerase Activity Assay for Pinus tabulaeformis Carr.

Telomerase is essential to maintain the integrity of eukaryotic chromosomes because of its function for restoring and extending telomere length. Though there are well established methods for measuring telomerase activity in animal cells, telomerase activity assay for plant cells is far away from effective, method to determine the telomerase activity is of great significance. In this study, we adopted and optimized TRAP method to determine the telomerase activity of the P. tabulaeformis. The optimization was made to the steps of the extension condition, the primer, the annealing temperature and the electrophoresis detection. The results showed that the best extension condition was one hour for 30℃, the optimum guide primer was the TS21 and the reverse primer was the ACX, the annealing temperature was 61.8℃, and the Gene Finder nucleic acid dye was selected to stain the polyacrylamide gel, respectively. Based on our data, optimized TRAP assay could effectively detect telomerase activity of the P. tabulaeformis, made it a valuable tool in testing telomerase activity in plant, an effective method for measuring the telomerase activity in P. tabulaeformis was established.