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作物杂志,2020, 第5期: 103–109 doi: 10.16035/j.issn.1001-7283.2020.05.016

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

云南药用野生稻高质量染色体DNA的制备

李娥贤(), 殷富有, 张敦宇, 陈越, 余腾琼, 雷涌涛, 肖素勤, 程在全, 柯学()   

  1. 云南省农业科学院生物技术与种质资源研究所/农业农村部西南作物基因资源与种质创制重点实验室/云南省稻种材料工程技术研究中心,650205,云南昆明
  • 收稿日期:2020-03-05 修回日期:2020-05-16 出版日期:2020-10-15 发布日期:2020-10-12
  • 通讯作者: 柯学
  • 作者简介:李娥贤,从事野生稻发掘利用研究,E-mail： xiaogaogao4850@126.com
  • 基金资助:
    云南省科技计划(2018ZG005);云南省科技计划(2018FB035);云南省科技计划(2018FG001-068);国家自然科学基金(31460054)

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

Li Exian(), Yin Fuyou, Zhang Dunyu, Chen Yue, Yu Tengqiong, Lei Yongtao, Xiao Suqin, Cheng Zaiquan, Ke Xue()   

  1. Institute of Biotechnology and Germplasm Resources, Yunnan Academy of Agricultural Sciences/Key Laboratory of Southwestern Crop Gene Resources and Germplasm Innovation, Ministry of Agriculture and Rural Affairs/Rice Materials Engineering Technology Research Center of Yunnan Province, Kunming 650205, Yunnan, China
  • Received:2020-03-05 Revised:2020-05-16 Online:2020-10-15 Published:2020-10-12
  • Contact: Ke Xue

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摘要：

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。

关键词: 药用野生稻, 染色体DNA, DNA制备

Abstract:

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.

Key words: Oryza officinalis, Chromosome DNA, DNA extraction

图1

药用野生稻叶片细胞核分离 A.药用野生稻;B.叶片匀浆液;C.细胞核离心;D.细胞核离心和清洗;E.细胞核

图2

药用野生稻叶片细胞核的低熔点琼脂糖包埋 A.核重悬;B.琼脂糖在冰上凝固;C.包埋块凝固于医用注射器中;D.包埋块切成圆片;E.嵌入式块清洗;F.包埋块洗涤

图3

不同浓度BamH I处理8min对药用野生稻基因组DNA消化的影响 M.酵母染色体PFG marker;Non.未经BamH I消化。下同

图4

0.6U BamH I处理不同时间对药用野生稻基因组DNA消化的影响

图5

药用野生稻染色体DNA未经及经BamHⅠ部分消化 A.最适酶解条件,B和C.酶解消化后目标DNA区域的切取

图6

BIBAC质粒及药用野生稻染色体大片段回收 A. BIBAC质粒及酶解,B.染色体大片段回收;M. λDNA/HindⅢ marker (A)和D2000 marker (B)

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[1] 肖放华, 彭绍裘, 李东汉. 水稻稻瘟病抗性鉴定及其利用[J]. 作物杂志, 1987, (1): 22–23
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