黄鳝单条染色体的显微分离及特异性检测

MICROISOLATION AND CHARACTERIZATION OF SINGLE CHROMOSOME IN RICE FIELD EELS (MONOPTERUS ALBUS)

建立了显微分离黄鳝单条染色体及检测其特异性的方法。在Olympus倒置显微镜下用毛细玻璃微针手工分离黄鳝减数分裂Ⅰ终变期 3号染色体 ,将其DNA作模板进行DOP PCR扩增后 ,分别以α-3 2 P dCTP和Biotin 11 dUTP标记的单条染色体DNAPCR扩增产物及Biotin 11 dUTP标记的大豆 18SrRNA基因作探针进行Southern杂交、FISH和Dot杂交来检测其特异性。结果表明 :(1)减数分裂Ⅰ终变期染色体标本是进行染色体显微操作的理想材料 ;(2 )DOP PCR扩增产物片段在 2 0 0～ 10 0 0bp之间 ,平均 6 0 0bp左右 ;(3)杂交结果显示 ,本研究所获得的单条染色体是黄鳝 3号染色体 ;(4 )与显微操作仪和微激光分离相比较 ,该方法不需要昂贵仪器 ,在常规实验室即可操作 。

The methods of microisolation and characteristic assay of single chromosome in rice field eels (Monopterus albus) were established. The chromosome 3 of meiosis Ⅰ diakinesis was microisolated manually with a micro-glass needle under the Olympus reverted microscope and its DNA was amplified by DOP-PCR. Several hybridizations were carried out to detect its specialities. 18 S rRNA gene of soybean labeled by Biotin-11-dUTP were hybridized with the metaphase chromosome and the DOP-PCR products dotted on the nylon membrane to map rice field eel rRNA genes.The DOP-PCR products with the positive Dot hybridization results labeled by Biotin-11-dUTP and α-32P-dCTP respectively were hybridized with the genomic DNA digested by different enzymes and the metaphase chromosome to further confirm its specialities. The hybridization results show that: (1) Chromosome specimen of meiosis Ⅰ diakinesis is the ideal material for single chromosome microisolation; (2) The sizes of DOP-PCR products range from 200 bp to 1 000 bp, averagely 600 bp;(3) The single chromosome obtained in the study is rice field eel chromosome 3;(4) Compared with micro-manipulator and micro-laser beam, the methods developed in the study can be more widely used in ordinary laboratory for requiring no expensive instrument.In the paper, we also discuss the future application of microisolation techniques in the study of chromosome evolution, gene mapping and conserved synteny in fish.