苹果细菌人工染色体双色DNA纤维荧光原位杂交体系的建立及应用

doi:10.3864/j.issn.0578-1752.2014.11.014

摘要/Abstract

摘要： 【目的】建立苹果DNA纤维荧光原位杂交（Fiber FISH）技术体系，从而为利用该技术确定苹果基因组DNA序列间的位置关系、构建精细物理图谱等方面的应用奠定基础。【方法】以‘Florina’苹果幼叶为试材，通过液氮研磨，尼龙膜过滤和TritonX-100去除叶绿素等步骤提取细胞核。细胞核经碱裂解，采用盖玻片拉伸方法制备DNA纤维。比较细胞核不同裂解时间和在5种不同包被类型的载玻片上DNA纤维拉伸的效果。用碱裂解方法提取来自苹果‘Florina’自交不亲和S9基因座的4个细菌人工染色体（Bacterium Artificial Chromosome）BAC 34G16、 BAC 45M19、BAC 70J19和BAC 69A4。提取的质粒经PEG纯化，用地高辛或生物素标记探针。探针与DNA纤维制片经过80℃变性、37℃杂交2—3 d和洗片，采用“三明治”方法进行信号放大和检测，在荧光显微镜下观察试验结果。【结果】建立了以苹果幼叶为材料，液氮研磨提取细胞核，碱裂解细胞核和盖玻片拉伸制备DNA纤维的试验方法。提取的细胞核纯净、结构完整，细胞核浓度＞5×103个/μL。试验结果表明，细胞核裂解4 min，在多聚赖氨酸包被的载玻片上制备的DNA纤维平直、伸展均匀，纤维量多、细长，效果好。经原位杂交和信号检测，获得了清晰的、具有Fiber FISH典型特征的“念珠状”杂交信号。对已知大小和位置关系的两个BAC克隆BAC 34G16和BAC 45M19进行杂交信号测量和分析，得出BAC 克隆大小（Y，Kb）与信号长度（X，μm）的相关方程为Y=3.47X（R2=0.9215），其斜率即为该试验技术体系Fiber FISH的分辨率3.47 kb•μm-1。试验对未知大小和位置关系的两个BAC克隆BAC 70J19和BAC 69A4成功地进行了鉴定，它们大小分别为(112.1±18.4)kb和(133.2±16.3 )kb，之间有（90.2±7.3）kb的重叠区域。【结论】建立了以苹果幼叶提取细胞核，制备DNA纤维和原位杂交的方法，获得了高分辨率的苹果DNA纤维原位杂交试验技术体系。

关键词: 苹果 , 细菌人工染色体 , DNA纤维荧光原位杂交

Abstract: 【Objective】The fluorescence in situ hybridization (FISH) on extended DNA fibers is a powerful tool in physical mapping construction, gene site determination in the genome, and so on. However, the technique is less reported in apple for its difficulties in nuclei extraction and fiber extension. To apply it widely in the apple genome research, the double color DNA fiber fluorescence in situ hybridization using apple bacterium artificial chromosomes (BAC) as probes was established in the experiment.【Method】The young leaves from apple ‘Florina’ were well ground in liquid nitrogen. The nuclear suspension was filtrated through nylon mesh of pore size from 161, 100, to 48 μm in turn, and subsequently triton X-100 was added into the suspension for removing chlorophyll. The apple nuclei were broken in STE buffer and DNA fibers were dragged and extended with a clean coverslip. The effects of different nuclear lysis times and five coating-type slides on DNA fibers extension were compared. The bacterium artificial chromosomes BAC 34G16, BAC 45M19, BAC 70J19 and BAC 69A4 from ‘Florina’ self incompatibility S9 locus were extracted by alkaline lysis method. The BAC plasmids were purified by PEG and then labeled with either digoxigenin-11-UTP or biotin-16-UTP using standard nick translation method. The probes and DNA fibers were denatured at 80℃, followed by incubation at 37℃ in a hybridization chamber for 2-3 days. FISH signals were amplified by ‘sandwich’ method and detected under fluorescent microscope.【Result】The experimental system of nuclei extraction from apple young leaves, DNA fiber extension, in situ hybridization and signal amplification was established. The purity and structural integrity of nuclei were obtained and the concentration exceeds 5×103 per microlitre. When the nuclei lyzed in STE buffer for 4 min and fiber extended on the polylysine coating slide, a great many of the ideal DNA fibers exhibiting long, slight, even and straight were obtained. The clear and typical “beads-on-a-string” nature of the DNA fiber FISH signals were observed after in situ hybridization, signal detection and amplification. The two BAC clones 45M19 and 34G16 with the known insert size and site relationship were hybridized on extended DNA fibers to calibrate and standardize the resolution of fiber FISH in apple. The results indicate that a regression equation of Y=3.47X for the best fit to the experimental data, where Y is the molecular probe length in kb and X is the fluorescence signal in μm. The correlation coefficient is R2=0.9215. The experiment also successfully identified two BAC clones 70J19 and 69A4 with unknown insert size and site relationship. The size of BAC70J19 and BAC 69A4 was (112.1±18.4)kb and (133.2±16.3)kb, respectively. There was an overlap region of size (90.2±7.3)kb between the two BAC clones. 【Conclusion】A method of nuclear extraction, DNA fiber extension and in situ hybridization was established using apple young leaves as material and the high resolution of apple fiber FISH was achieved.

Key words: Malus×domestica , bacterium artificial chromosome , DNA fiber fluorescence in situ hybridization

王三红, 章镇, 蔡斌华, 渠慎春. 苹果细菌人工染色体双色DNA纤维荧光原位杂交体系的建立及应用[J]. 中国农业科学, 2014, 47(11): 2205-2213.

WANG San-Hong, ZHANG Zhen, CAI Bin-Hua, QU Shen-Chun. Establishment and Application of Double Color DNA Fiber Fluorescence in situ Hybridization Using Bacterium Artificial Chromosomes in Apple[J]. Scientia Agricultura Sinica, 2014, 47(11): 2205-2213.

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