CEL Ⅰ酶的粗纯化及活性分析.pdfVIP

农业生物技术学报 Journal ofAgricultural Biotechnology 2007，15 (6)： 1006-101 I · 研究论文· CEL I酶的粗纯化及活性分析水 林 英，陈冬妹，赵 萍，杨 瑜，林立鹏，夏庆友料 (西南大学生物技术学院，农业部蚕桑学重点开放实验室，重庆400716) 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，其中CEL(celery)I 酶是TILLING技术的核心内切酶。实验通过硫酸铵沉淀、亲和层析、阴阳离子交换柱层析等常规的蛋白质纯化过程，从芹菜 (Apium graveolens)eP粗纯化了CEL I酶。以杂合双链DNA为底物，检测了CEL I酶在不同纯化程度、不同温度、不同处理时 间以及有无TaqDNA聚合酶条件下的错配切割活性。结果表明，所获得的CEL I酶得到了粗纯化；CEL I酶的最适活性温度 范围为45~50℃；处理时间长达l50 min时，CEL I酶仍能特异识别并切割错配碱基；在含有TaqDNA聚合酶的酶切体系 中，CEL I酶错配切割效果更佳。与其它单位提供的CEL I酶活性相比，实验粗分离纯化的CEL I酶的酶切条带更亮，说明 其活性更好。通过对CEL I酶的提取及活性分析，为TILLING技术大规模和低成本地应用于家蚕功能基因组研究提供了保 障。 关键词：CEL I酶；粗纯化；活性分析；targeting induced local lesions in genomes(TILLING) 中图分类号：S188 文献标识码：A 文章编号：1006．1304(2007)06．1006—06 Crude Purification and Activity Analysis of the CEL I Enzyme LIN Ying，CHEN Dong—mei，ZHAO Ping，YANG Yu，LIN Li—peng，XL～Qing-you rKeySericulturalLaboratory,MinistryofAgriculture，College ofBiotechnology,Southwest University,Chongqing400716,China) Ab由_=t Targeting induced local lesions in genomes(TILLING)．as an important research technology in reverse genetics has been widely applied in many model organisms．CEL(celery 1 I enzyme from celery(apium graveolens)is a key enzyme in TILLING technology．The endonuclease CEL 1 with high activity from celery Was crudely purified by (NH4)2SO4 deposition，affinity chromatography，anion and cation chromatography．Using heteroduplex DNAs as the substrates，the various activities of CEL 1 were further investigated under the various combinations of different purified levels，digestion temperatures，treatment time and presence or absence of Taq DNA polymerase．The results indicated that the CEL 1 was crudely purified．the optimum temperatures were between 40 to 50℃，special digestion