以螢光原位雜交定性菸草變異型染色體添加系

Abstract

以基因組原位雜交（genomic in situ hybridization; GISH）技術檢測4個染色體轉座、11個染色體缺失的Nicotiana plumbaginifolia-sylvestris變異型單染色體添加系，發現二基因組間未發生重組，為單純的異源染色體添加系（一條轉座或缺失的N. sylvestris染色體添加至完整N. plumbaginifolia基因組，沒有種間DNA相互插入現象）。以菸草的端粒重複性序列（TTTAGGG）n為探針，與帶缺失染色體的添加系做螢光原位雜交（fluorescence in situ hybridization; FISH），發現所有染色體斷裂處皆有新生的端粒序列。添加的telo 6S及telo 7S各含衛星體及次級收縮，但del 7S則否，以端粒重複性序列及1kb片段的N. plumbaginifolia 25S rDNA序列為探針，與上述三個變異型的染色體添加系做瑩光原位雜交，得知telo 6S及telo 7S含有45S rDNA（18S-5.8S-25S rDNA），而del 7S則否，顯示del 7S除中節處有一個斷點外，在核仁形成區與短臂末端相接處有第二個斷點。未來，可利用放射線處理這些添加系，誘導N. sylvestris染色體斷裂，根據斷點及DNA標誌製作實質圖譜，稱為放射線雜種作圖；另一個用途是利用flow cytometry及染色體顯微切割技術分離添加的N. sylvestris染色體，並構築染色體、臂或特定區域專有的基因組庫。

Genomic in situ hybridization (GISH) was used to identify Nicotiana plumbaginifolia-sylvestris aberrant addition lines (4 lines containing a translocated N. sylvestris chromosome and 11 lines containing a deficient N. sylvestris chromosome). No exchanges of chromosome segments between N. plumbaginifolia and N. sylvestris were observed. This finding indicates that in each addition line a deficient or translocated N. sylvestris chromosome is present in an integral diploid background of N. plumbaginifolia, and no interspecific DNA introgression has occurred. Fluorescence in situ hybridization (FISH) using Nicotiana telomeric repeats (5'-TTTAGGG-3')n as probes indicates that the broken ends of all deficient chromosomes have been healed by telomerase. The added telo 6S and telo 7S chromosomes had satellite regions and secondary constrictions, but del 7S didn't have. The distribution of 45S rDNA (18S-5.8S-25S rDNA) and telomeric repeats on telo 6S, telo 7S and del 7S was studied by FISH. Hybridization signals of 45S rDNA were observed on telo 6S and telo 7S, but not on del 7S. This result suggests that the formation of del 7S is due to occurrence of one break in the centromeric region and another between the short arm and nucleolar organizer region. In the future, we would like to treat these and the normal addition lines produced previously by radiation for the construction of a N. sylvestris physical map based on the occurrence of breakpoints and DNA markers. Such a technique is called radiation hybrid mapping. Another application of addition lines is the isolation of N. sylvestris chromosomes by flow cytometry or microdissection for the construction of the chromosome-, arm- or region-specific library.