| 刺梨及其近缘种PCR实验体系的建立与优化 |
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| 引用本文： | 文晓鹏,邓秀新.刺梨及其近缘种PCR实验体系的建立与优化[J].果树学报,2003,20(1):35-39. |
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| 作者姓名： | 文晓鹏 邓秀新 |
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| 作者单位： | 1. 华中农业大学作物遗传改良国家重点实验室,湖北,武汉,430070;贵州大学农学院,贵州,贵阳,550025
2. 华中农业大学作物遗传改良国家重点实验室,湖北,武汉,430070 |
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| 基金项目： | 国家自然科学基金，贵州省自然科学基金资助项目。 |
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| 摘 要： | 以刺梨及其近缘种月季为试材,进行了RAPD-PCR实验参数的确立和优化试验。结果表明,刺梨及月季25μL反应体系的最优组成为2.5μL10×反应缓冲液,2mmol/LMg2+,0.2mmol/LdNTP,1.6mg/L模板DNA,0.4μmol/L随机引物和1.2UTaqDNA多聚酶。经PCR扩增验证,此反应体系亦适宜于刺梨的部分近缘种,可有效用于RAPD分析;通过将退火温度提高至50℃或采用“Touchdown”扩增程序,并在50μL反应体系中适当增加特异引物对浓度(1.0μmol/L),模板DNA(4.0mg/L)和TaqDNA多聚酶(3.0U/管)的使用量,建立起适合于刺梨特异DNA片段检测及回收的特异PCR扩增实验体系,为刺梨的分子克隆奠定了技术基础。
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| 关 键 词： | 刺梨 近缘种 PCR扩增 实验体系 |
| 文章编号： | 1009-9980-(2003)-01-035-05 |
| 修稿时间： | 2002年5月15日 |
Establishment and Optimization of PCR Technical System in Rosa roxburghii and Its Relatives |
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| Wen Xiaopeng , and Deng Xiuxin.Establishment and Optimization of PCR Technical System in Rosa roxburghii and Its Relatives[J].Journal of Fruit Science,2003,20(1):35-39. |
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| Authors: | Wen Xiaopeng and Deng Xiuxin |
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| Institution: | Wen Xiaopeng 1,2 and Deng Xiuxin 1 |
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| Abstract: | The fruit of Rosa roxburghii Tratt is characterized by the high content of vitamin C among the fruits.To satisfy the increasing molecular studies,the attempts to establish and optimize RAPD-PCR in R.roxburghii Tratt and its relatives,and selective PCR protocols in R.roxburghii Tratt were carried out.The results suggest that,with the25μL cocktail of2.5μL10×reaction buffer,2mmol/L Mg 2+ ,1.6mg /L genomic DNA,0.4μmol/L arbitrary primer,and1.2U Taq DNA polymerase in re-action solution,the profiles are distinct and reproducible in all the tested species.By modifying annealing temperature(50℃)or employing“Touchdown”cycling procedure,as well as using50μL cocktail modified with1.0μmol/L degenerate primer pair,4.0mg /L DNA and3.0U/tube Taq DNA polymerase,the specific band of R.roxburghii Tratt can be distinctly scored for recovery. |
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| Keywords: | Rosa roxburghii Tratt Relatives PCR amplification Experimental system |
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