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

Abstract

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

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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