摘要TILLING（Targeting-induced local lesions in genomes）技术作为反向遗传学的一个重要研究工具，目前在众多的模式生物分子生物中得到广泛应用，其中CELⅠ酶是TILLING技术的核心。本实验通过硫酸铵沉淀、亲合层析、阴阳离子交换柱层析等常规的蛋白质纯化过程，从芹菜中粗纯化了具有较高活性的稳定的CELⅠ酶。并以杂合双链DNA为底物，对不同纯化程度的CELⅠ酶以及在不同温度、处理时间、有无Taq DNA多聚合酶的条件下进行了CELⅠ酶的错配切割活性分析。结果发现，所获得的CELⅠ酶得到了一定的纯化；CELⅠ酶的反应最适温度范围为40℃～50℃；长时间酶切特异性好；在含有Taq酶的酶切体系中，CELⅠ酶错配切割效果更佳，这与已报道的实验结果基本一致。与其他单位提供的CELⅠ酶活性相比，本实验分离纯化的CELⅠ酶活性值略高。通过对CELⅠ酶的提取及活性分析，为TILLING技术大规模和低成本的应用于家蚕功能基因组研究提供了保障。
Abstract：Targeting-induced local lesions in genomes (TILLING), as an important research technology in reverse genetics has been widely applied to study in molecular biology of many model organisms. CELⅠ is the key enzyme of this technology. In this study, we extracted the endonuclease CELⅠ with high activity from celery by (NH4)2SO4 deposition, affinity chromatography, anion and cation chromatography. Using heteroduplex DNAs as the substrates, we further investigated the various activities of CELⅠ under different conditions, the various combinations of different purified levels, digestion temperatures, treatment time, presence or absence of Taq DNA polymerases. The results indicated that the CELⅠin our test is much purified, the optimum temperature is between 40℃ and 50℃, well special digestion for mismatch bases in a long time, and that the activity of CELⅠ would be higher when Taq enzyme is present in the reaction system. In conclusion, our results will provide technology supports for application of TILLING in silkworm with large scale and low cost.
收稿日期: 2007-03-27
通讯作者:
夏庆友
引用本文:
林英 陈冬妹 赵萍 杨瑜 林立鹏 夏庆友. CELⅠ酶的粗纯化及活性分析[J]. , 2007, 15(6): 0-.
. Crude Purification and Activity Analysis of the CELⅠNuclease. , 2007, 15(6): 0-.
