多荧光标记引物增强甘蔗染色体寡聚核苷酸探针杂交信号

doi:10.13560/j.cnki.biotech.bull.1985.2022-1085

摘要/Abstract

摘要：

荧光原位杂交技术（fluorescence in situ hybridization, FISH）是植物分子细胞遗传学研究最为重要的手段之一。近些年，基于参考基因组设计的低拷贝寡聚核苷酸探针在FISH中应用得越来越广泛。然而，由于植物基因组中分布大量的重复序列，这使得oligo-FISH的分辨率存在一定局限性。利用包含多个荧光基团的荧光PCR引物，扩增出甘蔗染色体特异oligo探针，并进一步优化甘蔗的荧光原位杂交体系，提高了甘蔗oligo探针识别近缘物种染色体的效率。通过开发多荧光标记的甘蔗oligo探针以及甘蔗荧光杂交体系的优化，有效拓宽荧光信号的最小分辨率，提高信噪比（signal-to-noise ratio, SNR），并成功基于甘蔗oligo探针对高粱1-10号染色体分型。多荧光标记引物增强oligo探针信号的新方法及FISH体系的优化为今后在其他物种中提高oligo-FISH鉴定染色体及捕捉微弱的荧光信号提供了参考。

关键词: 甘蔗, 荧光原位杂交, 寡聚核苷酸探针, 染色体识别, oligo-FISH

Abstract:

Fluorescence in situ hybridization（FISH）is one of the most important methods in plant molecular cytogenetics. In recent years, low copy oligonucleotide probes based on reference genome design have been widely used in FISH. However, the resolution of oligo-FISH is limited due to the large number of repeats distributed in plant genomes. The sugarcane（Sacchanum spp.）chromosome specific oligo probes were amplified by using fluorescent PCR primers containing multiple fluorophore groups, and the sugarcane fluorescence in situ hybridization system was further optimized to improve the efficiency of sugarcane oligo probes in recognizing chromosomes of related species. By developing a new method of multi-fluorescent labeled oligo probes in sugarcane and optimizing the sugarcane fluorescence hybridization system, the minimum resolution of fluorescence signal was effectively broadened，the signal-to-noise ratio（SNR）increased, and sugarcane oligo probes were used to successfully identify sorghum chromosomes 1-10. The new method of multi-fluorescent labeled primers enhanced the signal of oligo probe and the optimization of FISH system provide a reference for improving oligo-FISH of identifying chromosomes and capturing weak fluorescence signals in other species in the future.

Key words: sugarcane, fluorescence in situ hybridization, oligonucleotide probes, chromosome identification, oligo-FISH

李心怡, 姜春秀, 薛丽, 蒋洪涛, 姚伟, 邓祖湖, 张木清, 余凡. 多荧光标记引物增强甘蔗染色体寡聚核苷酸探针杂交信号[J]. 生物技术通报, 2023, 39(5): 103-111.

LI Xin-yi, JIANG Chun-xiu, XUE Li, JIANG Hong-tao, YAO Wei, DENG Zu-hu, ZHANG Mu-qing, YU Fan. Enhancing Hybridization Signal of Sugarcane Chromosome Oligonucleotide Probe via Multiple Fluorescence Labeled Primers[J]. Biotechnology Bulletin, 2023, 39(5): 103-111.

图/表 8

表1 Oligo探针荧光标记引物扩增序列

Table 1 Amplification sequences of oligo probe fluoresc-ence labeled primers

引物名称 Primer name	引物序列 Primer sequence
T7-Cy3	/iCy3dT/ACGACTCACTATAGGGAGA
D9-Cy3	/iCy3dA/GTGTGTACGGACTAATAAT
T7-FAM	/iFAMdT/ACGACTCACTATAGGGAGA
D9-FAM	/iFAMdA/GTGTGTACGGACTAATAAT
T7-Cy3-3	/iCy3dT/ACGAC/iCy3dT/CAC/iCy3dT/ATAGGGAGA
D9-Cy3-3	/iCy3dA/G/iCy3dT/G/iCy3dT/GTACGGACTAATAAT
T7-FAM-3	/iFAMdT/ACGAC/iFAMdT/CAC/iFAMdT/ATAGGGAGA
D9-FAM-3	/iFAMdA/G/iFAMdT/G/iFAMdT/GTACGGACTAATAAT

图1 多荧光标记引物扩增电泳图

Fig. 1 Amplification electrophoretogram of multiple fluo-rescence labeled primers

图2 荧光原位杂交优化前后的流程对比图蓝色区域：优化前杂交步骤；红色区域：优化后杂交步骤；黄色标记：改良步骤；虚线部分：预杂交步骤区域

Fig. 2 Comparison of flow before and after optimization of FISH Blue area: Pre-optimization hybridization step; red area: post-optimization hybridization step; yellow marker: improvement step; dashed part: pre-hybridization step area

图3 基于杂交体系优化下不同曝光梯度Cy3与FAM的信号捕获 a:未优化杂交体系下，FAM探针在不同曝光时长的成像；b:优化杂交体系下，FAM探针在不同曝光时长的成像；c:未优化杂交体系下，Cy3探针在不同曝光时长的成像；d:优化杂交体系下，Cy3探针在不同曝光时长的成像；e：优化体系前后荧光密度对比图，红色背景为Cy3标记，绿色背景为FAM标记；IntDen为灰度值；标尺为10 μm

Fig. 3 Signal capture of Cy3 and FAM with different exposure gradients based on hybridization system optimization a: Imaging of the FAM probe at different exposure duration in the unoptimized hybridization system; b: imaging of the FAM probe at different exposure duration in the optimized hybridization system; c: imaging of the Cy3 probe at different exposure duration in the unoptimized hybridization system; d: imaging of the Cy3 probe at different exposure duration in the optimized hybridization system; e: comparison of fluorescence density before and after optimization system, the red background is marked with Cy3, the green background is marked with FAM.IntDen: Intensity Image. bar=10 μm

图4 基于不同荧光基团探针杂交不同曝光梯度Cy3与FAM的信号捕获 a：单荧光修饰下，FAM探针在不同曝光时长的成像；b：3个荧光修饰下，FAM探针在不同曝光时长的成像；c：未优化杂交体系下，Cy3探针在不同曝光时长的成像；d：3个荧光修饰下，Cy3探针在不同曝光时长的成像；e：不同荧光修饰数量下的荧光密度对比图，红色背景为Cy3标记，绿色背景为FAM标记；IntDen为灰度值；标尺为10 μm。

Fig. 4 Signal capture of Cy3 and FAM with different exposure gradients based on different fluorophore group probes a: Imaging of FAM probe at different exposure duration under single fluorescence modification; b: imaging of FAM probe at different exposure duration under three fluorescence modifications; c: imaging of Cy3 probe at different exposure duration under unoptimized hybridization system; d: imaging of Cy3 probe at different exposure duration under three fluorescence modifications; e: comparison graph of fluorescence density under different number of fluorescence modifications, red background for Cy3 marker, green background is FAM marker; IntDen is gray scale value; scale bar is 10 μm

表2 热带种oligos探针序列比对高粱基因组数量统计

Table 2 Number statistics of sorghum genome aligned by S. officinarum oligos probes

oligo探针 oligo probes	高粱染色体 Sorghum chromosome
oligo探针 oligo probes	1	2	3	4	5	6	7	8	9	10
Chr1	18 400	63	120	79	51	84	68	46	55	66
Chr2	73	12 522	55	65	29	55	30	80	34	42
Chr3	45	46	15 771	61	54	47	41	30	125	41
Chr4	28	47	48	11 448	46	42	29	28	18	65
Chr5	27	34	26	107	3 680	29	16	53	25	44
Chr6	85	20	70	93	48	7 959	32	39	41	47
Chr7	75	81	54	33	40	36	7 760	30	39	95
Chr8	29	34	21	65	143	25	25	5 218	24	40
Chr9	28	39	43	15	25	33	15	26	8 592	57
Chr10	86	112	47	28	46	21	33	60	46	9 573

图5 热带种Oligo序列在高粱染色体上的分布 a：Oligo探针分布热图；b：oligo探针密度统计图

Fig. 5 S. officinarum Oligos sequences distribute on the chromosome of sorghum a: The hot map of Oligo probe distribution. b: The statistics of oligo probe density

图6 Oligo探针定位于高粱染色体 a: Chr1, Chr2探针在高粱染色体的定位；b: Chr3, Chr4探针在高粱染色体的定位; c: Chr5, Chr6探针在高粱染色体的定位; d: Chr7, Chr8探针在高粱染色体的定位; e: Chr9, Chr10探针在高粱染色体的定位；Cy3为红色，FAM为绿色；标尺为10 μm

Fig. 6 Oligo probe located on sorghum chromosome a: Probe Chr1 and Chr2 located on sorghum chromosome; b: probe Chr3 and Chr4 located on sorghum chromosome; c: probe Chr5 and Chr6 located on sorghum chromosome; d: probe Chr7 and Chr8 located on sorghum chromosome; e: probe Chr9 and Chr10 located on sorghum chromosome; Cy3 is red, FAM is green; bar=10 μm

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