五種菸草的核仁形成區

Abstract

螢光原位雜交（fluorescence in situ hybridization, FISH）是能將基因定位在染色體上的一項技術。本實驗以digoxigenin-11-dUTP系統標定水稻的核醣體RNA基因（rDNA），用其做探針，與菸草的根尖細胞做原位雜交；這段rDNA探針包含了17S - 5.8S - 25S基因序列，長3.8 kb。在菸草屬的Petunioides亞屬Alatae節中，N. alata和N. langsdorffii各有九對染色體，前者有三條衛星染色體，後者有二對衛星染色體，原位雜交結果前者有二對rDNA基因座，後者有三對rDNA基因座。N. longiflora和N. plumbaginifolia各有十對染色體，編號30a與30c的N. longiflora植物，原位雜交訊號數和核型觀察具次級收縮的染色體數符合，皆有二對rDNA基因座，但在編號30b的N. longifolra植物，核型觀察只看到三條衛星染色體，原位雜交結果有二對rDNA基因座；N. plumbaginifolia有一對衛星染色體，原位雜交顯示有二對rDNA基因座。N. sylvestris有十二對染色體，原位雜交結果和核型觀察均顯示其有三對rDNA基因座。將野生菸草中期的染色體進行銀染色，以觀察有多少具活性的rDNA基因座，發現銀染的核仁形成區數目皆與由傳統染色法觀察的次級收縮數目相同。此實驗中偵測到N. langsdorffii及N. plumbaginifolia皆有一對不表現的rDNA基因座；N. alata及N. sylvestris各有一對染色體的rDNA基因座具多型性。

The nucleolar organizer regions (NORs) of five Nicotiana species belonging to section Alatae were investigated by conventional cytological technique, fluorescence in situ hybridization (FISH) and silver staining. For FISH, clone pRY18 carrying a 3.8 kb 17S-5.8S-25S repeat of rice ribosomal DNA (rDNA) was labeled with digoxigenin-11-dUTP and used as a probe to hybridize chromosomal DNA in root-tip cells. The results showed that: (1) two pairs of chromosomes in N. alata (2n = 18), N. longiflora (2n = 20) and N. plumbaginifolia (2n = 20), and three pairs of chromosomes in N. langsdorffii (2n = 18) and N. sylvestris (2n = 24) possessed rDNA loci; (2) in N. alata and N. sylvestris the sizes of NORs in one pair of chromosomes were polymorphic. In all species, the number of chromosomes with secondary constrictions determined by Feulgen staining corresponded to the number of AgNORs revealed by silver staining. Comparison of the results from FISH with those of obtained by Feulgen and silver staining indicated that in N. langsdorffii and N. plumbaginifolia rDNA one pair of chromosomes was not functioning.