摘要
水稻叶绿体和线粒体基因组较小,且均被测序清楚,可以作为研究细胞质遗传的良好材料,而如何快速有效地分离纯化获得高产量和高质量的细胞质基因组是从DNA水平上研究细胞质遗传变异的前提条件。本研究结合了蔗糖密度梯度离心法-全基因组DNA扩增法(whole genome amplification,WGA),对细胞质基因组的制备进行了改良。改良后的方法仅使用20 g的水稻叶片,即可在2 d时间内得到浓度达300 ng/μL以上,总量达40μg以上的高纯度(OD260/280值为1.8～2.0)、完整的叶绿体DNA(chloroplast DNA,cpDNA)和线粒体DNA(mitochondrial DNA,mtDNA)。该法制备的细胞质基因组可以满足多种细胞质基因组实验的要求,包括Solexa全基因组测序技术的要求。
Due to the small size of rice chloroplast and mitochondria genomes, they are good models for the study on extra-nuclear inheritance. The key step is how to separate and purify the high-quality cytoplasmic genome. In this research, we have developed a new technique based on combination of sucrose step-gradient centrifugation and whole genome amplification （WGA）. Through the developed method, we can get high quality and quantity （ ODa60/2s0 = 1.8 - 2. 0） DNA and mitochondrial DNA with about 300 ng/μL of concentration and 40 μg of grosses from only 20 g rice leaves comparing with traditional methods. The chloroplast DNA and mitochondrial DNA can fulfill the needs of much cytoplasmic genome study, even the needs of Solexa whole genome sequencing （new generation sequencing technology）.
出处
《云南农业大学学报（自然科学版）》
CAS
CSCD
北大核心
2011年第4期437-443,共7页
Journal of Yunnan Agricultural University:Natural Science
基金
国家自然科学基金项目(30360054)
云南省科技厅项目(2005C0011Z)
