烟草18S rDNA染色体原位杂交技术优化研究

Optimization of in situ hybridization of tobacco 18S rDNA chromosome

荧光原位杂交(Fluorescence in situ hybridization,FISH)在细胞遗传学中有较多应用,其中重复序列常被作为探针来研究物种进化、分类。但现常用的探针序列均较长(200 bp-500 bp),杂交耗时较多。本研究以烟草(Nictiana tabacum)为材料,克隆并标记18S rDNA保守序列(254 bp),以其为探针对烟草染色体进行FISH,经18 h杂交获得了清晰的信号。在此基础上,利用荧光素直接标记引物序列(寡聚核苷酸),经较短时间(2 h)的杂交,也获得了清晰的信号。以寡聚核苷酸为探针对云烟87四倍体、N. tabacum-N. plumbaginifolia五倍体、烟草六倍体、云烟87八倍体以及N. plumbaginifolia的染色体进行杂交,均获得了良好效果。且杂交液可简化配制,洗脱过程也可简化。18S rDNA寡聚核苷酸探针与5S rDNA寡聚核苷酸探针结合,实现了N. plumbaginifolia的18S rDNA和5S rDNA双色FISH。所以,该寡聚核苷酸序列可以应用于烟草属植物18S rDNA(45S rDNA)位点的分析,且较大程度缩短了操作时间并简化了操作过程。

Fluorescence in situ hybridization (FISH) has been widely used in cytogenetics research, and repeative sequences are often used as probes in researches on evolution and taxonomy of species. However, the commonly used probe sequences are as long as 200 bp-500 bp, which take long time for hybridization. In this study, tobacco (Nictiana tabacum) was used as material, and 18S rDNA conserved sequence (254 bp) was cloned and labeled. FISH was performed on tobacco chromosomes using this sequence as a probe, and the clear signal was detected after 18 h hybridization. On this basis, the primer sequence (oligonucleotide) was directly labeled with fluorescein, and after a short time (2 h) of hybridization on tobacco chromosomes, the clear signal was also detected. Hybridization of the chromosomes of Yunyan87 tetraploid, N. tabacum-N. plumbaginifolia pentaploid, tobacco hexaploid, Yunyan87 octoploid, and N. plumbaginifolia was performed by using the oligonucleotide as the probe, and good results were obtained. The hybridization solution simplifies the preparation and the elution process can be simplified. Through combining the 18S rDNA oligonucleotide probe with the 5S rDNA oligonucleotide probe, dual-color FISH of 18S rDNA and 5S rDNA of N. plumbaginifolia was realized. Therefore, the oligonucleotide sequence can be applied to chromosome site analysis of Nicotiana 18S rDNA(45S rDNA), with the operation time greatly shortened and the operation process simplified.