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浙江大学学报（农业与生命科学版）
Journal of Zhejiang University (Agriculture and Life Sciences)  2011, Vol. 37 Issue (6): 591-591    DOI: 10.3785/j.issn.1008-9209.2011.06.001
Biological sciences & biotechnology     
Effects of some factors on extraction of celery CELI nuclease
CUI Hai‐rui１ , LI Chun‐nan１ , SUN Jie１ , CUI Bi‐jun２
1 . Institute o f Nuclear‐A gricultural Sciences /Key L aboratory o f Nuclear‐A gricultural Sciences , Ministry of Agriculture ,Zhe jiang University ,Hangzhou 310029 , China ; 2 . College of Life Sciences , South China Agricultural University ,Guangzhou 510642 , China
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Abstract  In order to extract celery CEL I nuclease effectively and use it to develop the detection method for point mutations , the effects of varieties ,organs ,developmental stages and pH of extraction buffer on its extraction amounts were analyzed and its enzymatic activity for mismatch cleavage was also characterized by using DNA heteroduplexes containing a single mismatch as substrates . Results showed that differences of CEL I amounts extracted among varieties and organs were significant at ５％and １％level , respectively . The cultivar Thailand Huangxinqinshowed the highest extractive amount among three different varieties ( Xuebai,  Wentula and  Thailand Huangxinqin ) compared , while the leaf showed much higher extraction amounts than the stalk and root among three different organs tested . The effect of developmental stages on the CEL I extraction was compared by using the variety  Shanghai Huangxinqin as extractive material and it was higher at reproductive stage than that at vegetative stage . The pH of extraction buffer also showed important effect on the CEL I extraction , and the higher extraction amount was obtained by using the extraction buffer with pH ７.５ than that with pH ５.５ andpH ９.５. Tests on enzymatic activity showed that the mismatch in DNA heteroduplexes could be effectively recognized and cleaved by the CEL I crude extracts .

Published: 25 November 2011
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CUI Hai‐rui
SUN Jie
LI Chun‐nan
CUI Bi‐jun
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CUI Hai‐rui,LI Chun‐nan,SUN Jie,CUI Bi‐jun. Effects of some factors on extraction of celery CELI nuclease. Journal of Zhejiang University (Agriculture and Life Sciences), 2011, 37(6): 591-591.

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http://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2011.06.001     OR     http://www.zjujournals.com/agr/Y2011/V37/I6/591


几种因素对芹菜CEL I核酸酶提取的影响

为有效提取芹菜CELI 核酸酶和进一步利用它建立点突变检测技术,分析了品种、器官、发育时期和提取液pH 值对其提取量的影响,并以含有单个错配的DNA 异质双链为底物鉴定其对错配切割的酶学活性.结果表明:不同芹菜品种和器官间CEL I 提取量的差异分别达到显著和极显著水平,在比较的３个不同品种( 雪白 、文图拉 和 泰国黄心芹 )中,以品种 泰国黄心芹 的CEL I 提取量最高;而在测定的３种不同器官中,叶片的CEL I 提取量远远高于茎秆和根;以 上海黄心芹 为材料比较发育时期对提取量的影响表明,生殖生长期的CEL I 提取量明显高于营养生长期;缓冲液pH 值对CEL I 提取量也有重要影响,pH ７.５ 缓冲液的CEL I 核酸酶提取量高于pH ５.５ 和pH ９.５ ;酶学活性鉴定显示,CEL I 核酸酶粗提物能有效地识别和切割DNA 异质双链中的碱基错配.
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