摘要
目的:为了提高SSR分子标记技术在水稻遗传育种中的研究效率,介绍一种快速的适于SSR-PCR扩增的水稻幼苗单株DNA提取法及PAGE银染法。方法:在常温下加入少量SDS提取液将单株叶片迅速捣碎,再加入300μLSDS提取液,65℃水浴10min后离心,取上清用乙醇沉淀后离心,超净工作台吹干后用100μLTE回溶即可。改进的PAGE银染法只需染色、冲洗、显影等3步,用乙醇和硝酸银作为染色液,去离子水快速冲洗1次后置显影液中即可显影。结果:本法可快速提取DNA,不须液氮研磨、氯仿抽提,提取的DNA经0.8%琼脂糖凝胶电泳检测表明质量较好,且扩增结果稳定可靠,满足了SSR-PCR的需要。改良的银染法与常规银染法的检出结果相同,但快速方便,整个过程只需10min,且背景较浅,灵敏度高。结论:快速提取法及PAGE银染改良法结合SSR分子标记技术,可有效地用于杂种后代的遗传连锁分析和分子标记辅助育种时的单株检测。
Objective： In order to improving the efficiency in the research of the rice genetics breeding using SSR marker, the methods of DNA rapid extraction from single rice seedling and rapid sliver staining after PAGE were developed. Methods： 10 mg fresh single riee seedling within a 1.5 mL EP tube was grounded rapidly with 100 μL extraction buffer at room temperature. Immediately after grounding 300 μL extraction buffer was added to the tube. Then the extracts was incubated in a water bath at 65℃ for 10 min. After centrifugation, precipitation with pure ethanol, dry under a weak vacuum, dissolved by TE, it could be applied to PCR. The improved method of PAGE sliver staining needed three steps which meant staining, rinsing and developing. Only ethanol and silver nitrate solution were used in staining. After rinsing gels with deionized water, it could transfer into 500 mL developer solution to develop. Results： Results revealed that the method need no liquid nitrogen, no octanol-chloroform, the main band of DNA extracted by the rapid method was clear and slightly degraded, indicating that it could be used to SSR-PCR analysis. The simple and rapid method of PAGE sliver staining was taken only about 10 min to staint the gels, and the background of gels was bright, high sensibility. Conclusion： With adoption of SSR marker techniques, the rapid method of DNA extraction and PAGE sliver staining could be effectively applied to molecular marker detection of F2 segregant population and supplementary screen of breed materials.
出处
《生物技术通讯》
CAS
2006年第2期213-215,共3页
Letters in Biotechnology
基金
辽宁省博士启动基金(辽宁省科委2001102061)
