非变性原位杂交技术在芝麻染色体识别上的探索与应用.pdfVIP

中国农业科学 2013,46(17):3729-3735 Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2013.17.023 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 （河南省农业科学院/河南省芝麻研究中心，郑州 450002 ） 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) 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 8 -1 -1 最优试验方案。【结果】芝麻ND-FISH 最优方案：制片在浓度100μg·mL RNase A 酶解1 h，探针浓度6 µL （20 ng·µL ）， 杂交4 h，4 × SSC/0.2% Tween 20 溶液洗脱10 min；18 条染色体存在(AC) 杂交信号，其中，14 条染色体上存 8 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) 为探针成功识别栽培芝麻的18 条染色体。 8 关键词：芝麻；寡聚核苷酸；染色体；核型；ND-FISH 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 (Henan Academy of Agricultural Sciences/Henan Sesame Research Center, Zhengzhou 450002) 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·µ