用改进的TRAP法测定树木端粒酶活性

Detection of Tree Telomerase Activity by a Modified TRAP Assay Method

TRAP法是一种常用的测定端粒酶活性的方法,但并不太适合测定木本植物的端粒酶活性.因此以油松针叶为实验材料,对TRAP法进行改进.参照前人的研究通过对比实验证明,在端粒酶提取过程中将PEG8000加入上清液,可获得高效的端粒酶.在此基础上,对PCR先导引选择TS、模板浓度选择100 ng,最终获得油松针叶高质量的端粒酶PCR产物.应用SYBR Green I替代银染液对电泳凝胶进行染色,获得条带清晰、重复性好的电泳结果.通过对油松针叶、银杏叶片、胡杨愈伤组织、沙冬青悬浮细胞等4种木本植物实验材料进行测定,证明该改进的技术体系可能是适合于木本植物端粒酶活性测定的稳定的、灵敏度高的可行方法.

Telomeric repeat amplication protocol （TRAP） is a common method for detecting telomerase activity. However, it is not completely suitable for the detection of telomerase activity in tree plants whose development differs in fundamental ways from that of herbaceous plants. So, we modified the TRAP assay to make it stable and highly sensitive for woody plants. According to the previous research, PEG 8000 was added to the telomerase extracts at a final concentration of 10%. Comparative telomerase assay results indicated that PEG 8000 might reduce the concentration of inhibitors to Taq polymerase or telomerase that were commonly found in crude extracts. TS was choosed as the primer and 100 ng of protein sample as the template concentration for PCR. 14 μL aliquot of each PCR product was applied to a 12% non-denaturing polyacrylamide gel and electrophoresed. The gels were stained using the SYBR Green I and photographed under ultraviolet light using an electronic gel documentation system. The results indicated that the modified TRAP assay was fast, safe and highly sensitive for detection of telomerase activity in Pinus tabulaeformis, Ginkgo biloba, Populus euphratica and Ammopiptanthus mongolicus. Fig 5, Tab 1, Ref 17