检索
E-mail
RSS
高级检索 图表检索

当期目录 我要投稿 过刊浏览
高级检索 图表检索

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

RichHTML

4

PDF (PC)

86

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)

[1] 程在全, 黄兴奇, 钱君, 等. 珍稀濒危植物——云南药用野生稻自然生态群的新发现及其特性. 云南植物研究, 2004,26(3):267-274.
[2] 杨雅云, 张敦宇, 陈玲, 等. 云南药用野生稻对四种水稻主要病害的抗性鉴定. 植物病理学报, 2019,49(1):101-112.
[3] 阿新祥, 吴成军, 鄢波, 等. 云南药用野生稻核基因组细菌人工染色体(BAC)文库的构建与分析. 植物生理学通讯, 2005,41(1):70-72.
[4] 苏龙, 徐志健, 乔卫华 .等. 广西药用野生稻遗传多样性分析及SSR引物数量对遗传多样性结果的影响研究. 植物遗传资源学报, 2017,18(4):603-610.
[5] 段峰森, 刘虹, 陈雁, 等. 利用C基因组DNA和C0t-1DNA对药用野生稻、大颖野生稻基因组的比较分析. 湖北农业科学, 2011,50(12):2548-2552.
[6] 齐兰, 王效宁, 张吉贞, 等. 利用SRAP标记研究海南野生稻的遗传多样性与遗传分化. 植物遗传资源学报, 2013,14(3):402-406.
[7] 吴绮, 覃瑞, 李刚, 等. 利用药用野生稻C基因组C0t-1 DNA比较分析宽叶野生稻CCDD基因组. 植物遗传资源学报, 2010,11(5):589-592.
[8] 刘如亮, 刘虹, 陈雁, 等. 药用野生稻(CC)–栽培稻(AA)异源单体附加系MAAL8的遗传分析. 安徽农业科学, 2011,39(17):10139-10142.
[9] 柯杰, 祝贺, 覃瑞, 等. 药用野生稻–栽培稻渗入系后代卷叶表型的遗传分析. 安徽农业科学, 2016,44(3):119-122.
[10] 李培纲, 李红梅, 张帅, 等. 药用野生稻ADF基因的克隆及其抗逆性分析. 华北农学报, 2017,32(6):37-44.
[11] 侯思名, 刘凌云, 殷旭东, 等. 药用野生稻BIBAC文库构建过程中质粒提取方法改进. 西南农业学报, 2014,27(1):1-5.
[12] 张大燕, 李红梅, 李培纲, 等. 药用野生稻OoLEA1基因的克隆和生物信息学分析. 分子植物育种, 2017,15(5):1597-1602.
[13] 汪暖, 陈志雄, 刘蕊, 等. 药用野生稻TAC克隆转化籼稻的体系初探. 植物生理学通讯, 2010,46(3):217-222.
[14] 秦宗燕, 刘石娟, 包颖. 药用野生稻叶中花色苷合成基因的序列特点与表达差异. 植物生理学报, 2016,52(8):1248-1254.
[15] 张欢欢, 刘蕊, 郭海滨, 等. 药用野生稻有利基因发掘与利用研究进展. 中国农学通报 2009,25(19):42-45.
[16] 侯思名, 张薇, 白现广, 等. 野生稻细胞核DNA提取的研究. 西南农业学报, 2009,22(5):1215-1218.
[17] 侯思名, 张薇, 翟书华, 等. 云南药用野生稻BIBAC文库的构建及分析. 湖南农业大学学报, 2011,37(1):17-21.
[18] 刘艳平, 程在全, 殷富有, 等. 云南药用野生稻BIBAC文库混合克隆池制备及筛选. 生物技术通讯, 2012(2):117-120.
[19] 王琳, 戴陆园, 吴丽华, 等. 云南三种野生稻原生境植物种群的调查及比较分析. 中国水稻科学, 2006,20(1):47-52.
[20] 康公平, 徐国云, 陈志, 等. 茶陵普通野生稻光合特性研究. 作物学报, 2007,33(9):1558-1562.
[21] 徐玲玲, 陈善娜, 程在全, 等. 云南野生稻离子吸收效率及其动力学特征研究. 植物生理学通讯, 2006,42(3):406-410.
[22] 蒋春苗, 黄兴奇, 李定琴, 等. 云南野生稻叶茎根组织结构特性的比较研究. 西北植物学报, 2012,32(1):99-105.
[23] Hamilton C M, Frary A, Lewis C, et al. Stable transfer of intact high molecular weight DNA into plant chromosomes. Proceedings of the National Academy of Sciences of the United States of America, 1996,93(18):9975-9979.
[24] Hamilton C M. A binary-BAC system for plant transformation with high-molecular-weight DNA. Gene, 1997,200(2):107-116.
doi: 10.1016/S0378-1119(97)00388-0
[25] Hamilton C M, Frary A, Xu Y M, et al. Construction of tomato genomic DNA libraries in a binary-BAC (BIBAC) vector. Plant Journal, 1999,18(2):223-229.
doi: 10.1046/j.1365-313X.1999.00433.x
[26] Chang Y L, Chuang H W, Meksem K, et al. Characterization of a plant-transformation-ready large-insert BIBAC library of Arabidopsis and bombardment transformation of a large-insert BIBAC of the library into tobacco. Genome, 2011,54(6):437-447.
doi: 10.1139/G11-011
[27] Wang W Q, Wu Y R, Li Y, et al. A large insert Thellungiella halophila BIBAC library for genomics and identification of stress tolerance genes. Plant Molecular Biology, 2010,72(91):93-99.
[28] Fu Y H, Scheuring C F, Wang H Y, et al. Construction and characterization of a BIBAC library of Jatropha curcas L.and identification of BIBAC clones containing genes associated with fatty acid metabolism. Molecular Breeding, 2011,28(4):559-567.
doi: 10.1007/s11032-010-9505-2
[29] Lee M, Zhang Y, Zhang M P, et al. Construction of a plant-transformation-competent BIBAC library and genome sequence analysis of polyploid upland cotton (Gossypium hirsutum L.). BMC Genomics, 2013,14:208-221.
doi: 10.1186/1471-2164-14-208 pmid: 23537070
[30] 成志伟, 曹筑荣, 陈良碧, 等. 东乡野生稻双元细菌人工染色体（BIBAC）文库的构建. 生物技术通报, 2006(zl):272-275,282.
[31] 何瑞锋. 药用野生稻基因组文库构建与大片段DNA转化. 武汉:武汉大学, 2003.
[32] Thomas G, Joseph L, Varghese G, et al. Discrimination between Oryza malampuzhaensis Krish. et Chand. and Oryza officinalis Wall ex Watt based on RAPD markers and morphological traits. Euphytica, 2001,122(1):181-189.
doi: 10.1023/A:1012602516236
[33] Naredo M E B, Mercado S M Q, Banaticla-Hilario M C N, et al. Genetic diversity patterns in ex situ collections of Oryza officinalis Wall. ex G. Watt revealed by morphological and microsatellite markers. Genetic Resources and Crop Evolution, 2017,64(4):733-744.
doi: 10.1007/s10722-016-0396-x
[34] 闫影, 王春连, 刘丕庆, 等. 野生稻抗病虫基因的挖掘和利用. 作物杂志, 2011(4):15-19.
[35] 陈明亮, 陈大洲, 胡兰香, 等. 东乡野生稻耐冷分子机制研究进展. 作物杂志, 2016(4):15-19.
[36] Tang H W, Zheng X M, Li C L, et al. Multi-step formation, evolution, and functionalization of new cytoplasmic male sterility genes in the plant mitochondrial genomes. Cell Research, 2017,27:130-146.
doi: 10.1038/cr.2016.115 pmid: 27725674
[37] Mickelbart M V, Hasegawa P M, Bailey-Serres J. Genetic mechanisms of abiotic stress tolerance that translate to crop yield stability. Nature Reviews Genetics, 2015,16(4):237-251.
doi: 10.1038/nrg3901 pmid: 25752530
[38] 冯锐, 郭辉, 秦学毅, 等. 利用广西普通野生稻创新选育抗白叶枯病优质水稻三系不育系. 作物杂志, 2014(4):64-67.
[39] Zou X H, Du Y S, Tang L, et al. Multiple origins of BBCC allopolyploid species in the rice genus (Oryza). Scientific Reports, 2015,5:1-8.
[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
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!