水稻染色体双链寡核苷酸荧光原位杂交技术

doi:10.11983/CBB22055

植物学报 ›› 2023, Vol. 58 ›› Issue (3): 433-439.DOI: 10.11983/CBB22055

水稻染色体双链寡核苷酸荧光原位杂交技术

孙尚¹, 胡颖颖¹, 韩阳朔², 薛超², 龚志云¹^,²()

¹扬州大学农学院, 江苏省作物基因组学和分子育种重点实验室/植物功能基因组学教育部重点实验室/江苏省作物遗传生理重点实验室, 扬州 225009
²扬州大学, 江苏省粮食作物现代产业技术协同创新中心, 扬州 225009

收稿日期:2022-03-24 接受日期:2022-08-04 出版日期:2023-05-01 发布日期:2023-05-17
通讯作者: *E-mail: zygong@yzu.edu.cn
基金资助:
国家自然科学基金(31571266)

Double-stranded Labelled Oligo-FISH in Rice Chromosomes

Shang Sun¹, Yingying Hu¹, Yangshuo Han², Chao Xue², Zhiyun Gong¹^,²()

¹Jiangsu Key Laboratory of Crop Genetics and Physiology/Key Laboratory of Plant Functional Genomics of the Ministry of Education/Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Agricultural College of Yangzhou University, Yangzhou 225009, China
²Jiangsu Co-innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, China

Received:2022-03-24 Accepted:2022-08-04 Online:2023-05-01 Published:2023-05-17
Contact: *E-mail: zygong@yzu.edu.cn

摘要/Abstract

摘要： 染色体制备与识别技术是遗传学研究的重要手段, 而寡核苷酸荧光原位杂交(oligo-FISH)是近年来兴起的染色体识别技术。灵活高效的探针是荧光原位杂交过程中的关键因素。传统的单链寡核苷酸探针标记过程复杂, 且获得单个探针的成本较高。在单链寡核苷酸探针的基础上进行改良, 利用靶向全染色体(片段)的特异性引物进行扩增, 将获得产物纯化, 即可得到目的探针, 简化了探针标记过程, 降低了成本, 并提高了标记效率。该文详述了水稻(Oryza sativa)改良后的双链寡核苷酸探针文库的合成及标记方法、有丝分裂时期染色体制片和探针杂交过程。通过设计梯度实验发现水稻中寡核苷酸荧光原位杂交技术染色体和寡核苷酸探针的最佳变性时间与温度分别为85°C 3分钟30秒及90°C 6分钟。该研究在水稻中建立染色体双链寡核苷酸荧光原位杂交技术, 可为多种植物染色体制备与精准识别提供有力的工具。

关键词: 水稻, 寡核苷酸荧光原位杂交技术, 有丝分裂

Abstract: The technique of identification and preparation of chromosome(s) are important tools in genetic research. Oligonucleotide fluorescence in situ hybridization (oligo-FISH) is an emerging chromosome identification technique in recent years. Flexible and efficient probes are the key factors in the process of oligo-FISH. The labelling process of traditional single-stranded oligo probes (ss-oligos) is complicated and the cost of obtaining individual probes is high. By improving the ss-oligo probes labelling process, we obtain the probes by PCR amplification with specific primers targeting the whole chromosome (fragment), which simplifies the probe labelling process, reduces the cost and improves the labelling efficiency. In this study, we describe in detail the synthesis and labelling of a modified double-stranded labelled oligo probes (ds-oligos) library in rice (Oryza sativa), the preparation of mitotic chromosomes and the hybridization process of ds-oligo probes. By designing gradient experiments, the optimal denaturation time and temperature of chromosome and oligo probe in rice were found to be 85°C for 3.5 min and 90°C for 6 min, respectively. This is the first study to establish a chromosomal double-stranded labelled oligo-FISH system in rice, which provides a powerful tool for the preparation and precise identification of chromosomes in a variety of plants.

Key words: rice, oligo-FISH, mitotic

孙尚, 胡颖颖, 韩阳朔, 薛超, 龚志云. 水稻染色体双链寡核苷酸荧光原位杂交技术. 植物学报, 2023, 58(3): 433-439.

Shang Sun, Yingying Hu, Yangshuo Han, Chao Xue, Zhiyun Gong. Double-stranded Labelled Oligo-FISH in Rice Chromosomes. Chinese Bulletin of Botany, 2023, 58(3): 433-439.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB22055

https://www.chinbullbotany.com/CN/Y2023/V58/I3/433

图/表 3

图1 在水稻中建立多重PCR扩增的双链寡核苷酸涂染探针蓝色矩形代表染色体; 黄色三角代表着丝粒; 红色矩形代表由TAMRA标记的8号染色体涂染的oligos信号; 绿色矩形代表由FAM标记的10号染色体涂染的oligos信号。F和R分别代表不同染色体区段扩增的特异正向和反向引物。虚线代表筛选出的在两端加接头的特异oligos合集。

Figure 1 Establishment of double-stranded oligo-painting probes based on multiplex PCR in rice The blue rectangles are chromosomes; the small yellow triangles are centromeres; the red rectangles represent TAMRA-labelled chromosome 8 oligo signals; the green rectangles represent FAM-labelled chromosome 10 oligo signals. Specific forward and reverse primers for the amplification of different chromosomal segments represented by F and R, respectively. The dashed line represents the selected specific oligo set with connectors at both ends.

图2 双链寡核苷酸荧光原位杂交技术中水稻染色体变性时间的探索 (A)-(C) 实验过程中水稻染色体变性时间为1分30秒、2分30秒和3分30秒时, 由TAMRA标记的8号全染色体的oligos探针信号的呈现效果; (D)-(F) 实验过程中水稻染色体变性时间为1分30秒、2分30秒和3分30秒时, 由FAM标记的10号全染色体的oligos探针信号的呈现效果。白色箭头示放大后的涂染染色体。染色体由DAPI复染。Bars=5 μm

Figure 2 Exploring the denaturation time of rice chromosomes in double-stranded oligo-FISH (A)-(C) During the experiment, the effects of oligo signals labelled by TAMRA on chromosome 8 while the denaturation time at 1 min 30 s, 2 min 30 s and 3 min 30 s, respectively. (D)-(F) During the experiment, the effects of oligo signals labelled by FAM on chromosome 10 while the denaturation time at 1 min 30 s, 2 min 30 s and 3 min 30 s, respectively. The white arrow points to the painting-chromosomes after magnification. Chromosomes were counterstained with DAPI. Bars=5 μm

图3 双链寡核苷酸涂染荧光原位杂交技术靶向水稻日本晴有丝分裂时期第8号和10号染色体核型图 (A) 有丝分裂间期8号染色体的涂染核型图; (B) 有丝分裂间期10号染色体的涂染核型图; (C) 有丝分裂中期8号染色体的涂染核型图; (D) 有丝分裂中期10号染色体的涂染核型图。红色: TAMRA标记的8号全染色体的oligos探针信号; 绿色: FAM标记的10号全染色体的oligos探针信号。染色体由DAPI复染。Bars= 5 μm

Figure 3 Double-stranded labelled oligo-FISH based on chromosome painting targets the mitosis of chromosome 8 and 10 analysis of Nipponbare (A) Oligo-painting analysis on mitotic interphase chromoso-me 8 of Nipponbare; (B) Oligo-painting analysis on mitotic interphase chromosome 10 of Nipponbare; (C) Oligo-painting analysis on mitotic prometaphase chromosome 8 of Nippon-bare; (D) Oligo-painting analysis on mitotic prometaphase chromosome 10 of Nipponbare. Red: TAMRA-labelled probe signals of whole chromosome 8; Green: FAM-labelled probe signals of whole chromosome 10. Chromosomes were coun-terstained with DAPI. Bars=5 μm

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水稻染色体双链寡核苷酸荧光原位杂交技术

Double-stranded Labelled Oligo-FISH in Rice Chromosomes

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