云南药用野生稻高质量染色体DNA的制备

药用野生稻是我国3种野生稻之一,蕴含丰富的优异基因资源,但由于其CC基因组与栽培稻的AA基因组差异大,远缘杂交不亲合,不易获得后代材料,导致育种中优异基因无法利用。构建大片段基因组文库是研究及利用基因的重要方法,而提取高质量的染色体DNA是建立BIBAC等大片段基因组文库的前提,但按常规方法很难从药用野生稻中提取到Mb级的染色体DNA,需要对提取体系进行优化。本研究对取材与研磨、细胞核的分离与包埋、组蛋白的去除与DNA释放、DNA的酶解与回收等实验步骤进行优化,从药用野生稻中成功提取出1.9Mb的高质量染色体DNA,并对酶解消化体系进行了摸索,制备的染色体DNA可用于构建大片段基因组文库。

Extraction of High-Quality Chromosome DNA in Yunnan Wild Rice Oryza officinalis

Oryza officinalis is one of the three wild species found in China. Although riched in excellent genes, however, due to the large difference between the CC genome of O.officinalis and the AA genome of cultivated rice, distant hybridization is not compatible, and excellent genes are difficult to use. Genomic library construction with large fragment DNA is an important method to study and to use its genes. This kind of genomic library, such as BIBAC library requires a high-quality genomic DNA. But it is not easy to extract chromosome DNA with the size of Mb from O.officinalis by convention methods, so the extraction system need to be optimized. In this paper, the experimental steps such as sampling and grinding, nucleus separation and embedding, histone removal and DNA release, and enzymatic hydrolysis and recovery of DNA were optimized. High quality chromosomal DNA of 1.9Mb was successfully extracted from O.officinalis, and the enzymatic digestion of DNA was also explored. The isolated DNA fragment can used to construct a large-fragment genomic library.