以原位雜交技術鑑別菸草體細胞雜種親本的基因組

Abstract

將Nicotiana plumbaginifolia 總基因組 DNA 以 digoxigenin－11－dUTP 標識，與三種菸草體細胞雜種（ N .plumbaginifolia + N. alata、N. plumbaginifolia + N. sylvestris 及N. plumbaginifolia + N .glauca ）根尖染色體做原位雜交，然後以螢光標識的抗體（ fluorescein conjugated anti－digoxigenin antibody）偵測雜交訊息。在波長 450－ 490nm 的藍光激發下，N .plumbaginifolia 染色體被 digoxigenin 標識而發出黃綠色螢光，而另一親本染色體上的 DNA 因與N .plumbaginifolia之 DNA 同源性（ homology ）較低，可於雜交後藉清洗（ washing ) 的嚴謹將探針去除，或雜交時使用阻礙（ blocking ) DNA 而使探針與之無法結合。未被標識的基因組經 propidium iodide 染色會發出紅色螢光。因為基因組原位雜交(genomic in situ hybridization)可以區別雜種菸草兩親本的基因組，所以在遺傳及育種研究上有很大潛力。

Nicotiana plumbaginifolia total genomic DNA was labelled with digoxigenin-11- dUTP and was used as a probe to hybridize chromosomal DNA in root-tip cells of three Nicotiana interspecific somatic hybrids in which N. plumbaginifolia was one parent. The binding of the probe to parental genomes in the hybrids could be differentiated by the addition of blocking DNA (unlabelled DNA from the other species) to the hybridization solution and by stringency washing after hybridization. The hybridization signal was detected immunologically using fluorescein conjugated anti-digoxigenin antibody. When viewed under blue light, the chromosomes of N plumbaginifolia showed yellow-green fluorescence while those of the other species fluoresced red with the counterstain propidium iodide. This technique, commonly known as genomic in situ hybridization, has been demonstrated to have wide application in genetic research and plant breeding, and is being used to investigate various problems encountered in our laboratory.