应用水稻叶片直接PCR扩增的方法_罗天宽_张小玲_朱世杨_徐谦_裘_省略_清_唐.pdf

分子植物育种，2015 年， 第11 期，第2590-2592页 第13 卷， Molecular Plant Breeding, 2015, Vol.13, No.11, 2590-2592 技术主题 Technology Feature 应用水稻叶片直接PCR 扩增的方法 * 朱世杨 徐谦 裘波音 卢华金 杨文清 唐征 刘庆 荆赞革 吴海涛 罗天宽 张小玲 温州科技职业学院, 浙南作物育种重点实验室, 温州, 325006 * 通讯作者, wzltk@126.com 摘 要 本研究以新鲜和冷冻的水稻幼苗叶片为材料， 探索水稻叶片直接作为模板进行PCR 扩增的方法。结 2 水稻叶片直接作为模板在缓冲液为 果表明，与对照SDS 法提取的DNA 相比，应用新鲜或冷冻的 1~2 mm 10 mmol/L KCl ，2.5 mmol/L MgCl ，25 mmol/L (NH ) SO ，40 mmol/L Tris-HCl (pH 9.0) ，0.8% PCR 增强剂的反 2 4 2 4 应体系中扩增，PCR 扩增效果无明显差异；研究结果表明，该方法省去DNA 体外提取过程，节约成本，操作简 便，可以直接利用水稻叶片作为DNA 模板进行PCR 扩增。 关键词 水稻, PCR, 叶片, 直接扩增, DNA The Method of Direct-PCR Amplification in Rice Leaves (Oryza Sativa L) Luo Tiankuan * Zhang Xiaoling Zhu Shiyang Xu Qian Qiu Boying Lu Huajin Yang Wenqing Tang Zhen Liu Qing Jing Zange Wu Haitao Key Laboratory of Crop Breeding in South Zhejiang, Wenzhou Vocational College of Science and Technology, Wenzhou, 325006 * Corresponding author, wzltk@126.com DOI: 10.13271/j.mpb.013.002590 Abstract In this research fresh and frozen rice seedling leaves were used as experimental materials, the method of direct-PCR amplification in rice leaves was developed and analyzed. The results showed that compared to PCR amplification with template DNA extracted by SDS method, there was no significant difference in the amplification effect by directly using fresh or frozen 1~2 mm2 rice leaf as template in mixed DNA extracting and PCR amplifying buffer of 10 mmol/L KCl, 2.5 mmol/L MgCl , 25mmol/L (NH ) SO , 40 mmol/L Tris-HCl (pH 9.0) 2 4 2 4 and 0.8% PCR enhancer. The results indicated that the rice leaf could be directly used as template for PCR-amplification, which eliminates the process of DNA extraction, saves the cost, and operates