野生黄瓜5S rDNA-FISH技术体系建立

Establishment of FISH Technique with 5S rDNA as probes in Wild Cucumber

目的通过改进黄瓜中期染色体制片和rDNA-FISH技术的关键因素,对野生种(Cucumis sativus var.hardwickii)PI 183967进行核型分析为继续研究黄瓜各条染色体的准确鉴定和进一步开展细胞学研究奠定基础。方法使用8-羟基喹啉0.002mol·L-1预处理2h,可以得到形态清晰的中期染色体,分裂相最高比例为17.2%。染色体制片以改良酶解空气干燥制片法较好,流程简单,可以获得大量清晰、分散的有丝分裂中期染色体,且背景干净。荧光原位杂交过程中染色体制片采用胃蛋白酶37℃处理30min,70℃变性3min时,探针使用缺口平移法地高辛进行标记,75~100℃变性10min,能获得杂交信号清晰的染色体图像。结果在黄瓜野生种PI 183967中只有一对5SrDNA信号,位于5号染色体短臂上,相对远离着丝点位置。结论野生黄瓜PI 183967的5号染色体可以用5SrDNA鉴定。

Objective Improving the key factor of metaphase chromosome slides and rDNA-FISH,analysis with karyotype and site of5S rDNA signal of Cucumis sativus var.hardwickii PI183967.Methods The pretreatment way of get clear cucumber metaphase chromosomes is bathe in0.002mol/L8-hydroxyquinoline for2h,the highest ratio of mitosis figures is17.2%.The air-drying technique protocol after enzyme digestion of cell wall was a good method for getting clear and well dispersed cucumber chromosome preparation with few debris.With the chromosome preparation being treated at37℃for10min by pepsin,being denatured at70℃for3min,probe DNA labeled by nick translation,probe DNA being denatured at75-100℃for10min,clear hybridization signal in the chromosome was detected.Results PI183967have only one pair of5S rDNA signal,located on chromosome5short arm,far away from the centromere.Conclusion This study will set up the foundation for the related research about cytology in Cucumber.