中国农业科学 ›› 2013, Vol. 46 ›› Issue (17): 3729-3735.doi: 10.3864/j.issn.0578-1752.2013.17.023

• 研究简报 • 上一篇    下一篇

非变性原位杂交技术在芝麻染色体识别上的探索与应用

 刘艳阳, 崔承齐, 梅鸿献, 武轲, 郑永战   

  1. 河南省农业科学院/河南省芝麻研究中心,郑州 450002
  • 收稿日期:2013-04-10 出版日期:2013-09-01 发布日期:2013-07-05
  • 通讯作者: 通信作者郑永战,Tel:0371-65851559;E-mail:sesame168@163.com
  • 作者简介:刘艳阳,E-mail:liuyanyang001@163.com
  • 基金资助:

    国家现代农业产业技术体系建设专项资金(CARS-15)

Exploration and Application of Non-Denaturing FISH on Chromosomes Research in Sesame (Sesamum indicum L.)

 LIU  Yan-Yang, CUI  Cheng-Qi, MEI  Hong-Xian, WU  Ke, ZHENG  Yong-Zhan   

  1. Henan Academy of Agricultural Sciences/Henan Sesame Research Center, Zhengzhou 450002
  • Received:2013-04-10 Online:2013-09-01 Published:2013-07-05

摘要: 【目的】研究和确定芝麻ND-FISH最优试验条件,并对其在芝麻染色体识别中的可行性进行探索。【方法】以寡聚核苷酸(AC)8为探针,研究不同洗脱强度、酶解时间和杂交时间对ND-FISH的影响,确定芝麻ND-FISH最优试验方案。【结果】芝麻ND-FISH最优方案:制片在浓度100 μg•mL-1 RNase A酶解1 h,探针浓度6 µL(20 ng•µL-1),杂交4 h,4 × SSC/0.2% Tween 20溶液洗脱10 min;18条染色体存在(AC)8杂交信号,其中,14条染色体上存在较强杂交信号,4条染色体上有较弱杂交信号,结合染色体长度及臂比,18条染色体可有效识别。【结论】利用ND-FISH技术、以(AC)8为探针成功识别栽培芝麻的18条染色体。

关键词: 芝麻 , 寡聚核苷酸 , 染色体 , 核型 , ND-FISH

Abstract: 【Objective】The objective of this paper is to study the optimal experimental conditions for non-denaturing FISH (ND-FISH), and to explore its application for chromosome recognition in sesame. 【Method】 Using oligomeric nucleotide (AC)8 as probe, the effects of different elution strengths, digestions and hybriding times on the ND-FISH were analyzed to optimize the protocol. 【Result】The optimal experimental conditions for ND-FISH were as follows: cells on slides were treated with DNase-free RNase A at a concentration of 100 μg•mL-1 for 1 h, 6 µL of the probe (20 ng•µL-1), hybridization for 4 h, elution with the 4×SSC/0.2% Tween 20 optimal solution for 10 min. Using (AC)8 as a probe, mitotic metaphase chromosomes of Yuzhi 11 were analyzed by ND-FISH. Nine pairs of the chromosomes of Yuzhi 11(seven pairs of chromosomes had strong signals, and two pairs of chromosomes had weak signals) could be distinguished based on the hybridization signals combining with the length and arm ratio of chromosomes. 【Conclusion】ND-FISH technology with (AC)8 as a probe was used to successfully identify 18 of 26 chromosomes of cultivated sesame for the first time.

Key words: sesame , oligonucleotide , chromosome , karyotype , ND-FISH