Ds 因子在烟草染色体插入位点旁邻顺序的克隆

Cloning of the DNA Fragments Flanking Ds Insertion Sites in Tobacco Genome

用ＮｅｓｔｅｄＰＣＲ 方法从含Ｄｓ 因子的转基因烟草ＤＮＡ 中克隆了Ｄｓ 因子在烟草染色体插入位点的９ 个旁邻ＤＮＡ 片段，以这些片段作探针，和野生型烟草的ＤＮＡ 进行Ｓｏｕｔｈｅｒｎ 杂交，以检测这些片段在烟草基因组中的拷贝数，结果表明它们都属于烟草染色体上的低拷贝ＤＮＡ。另外，对这些ＤＮＡ 片段进行核苷酸序列测定，并将它们的顺序与Ｇｅｎｂａｎｋ 数据库中已有的核苷酸序列相比较，其中长度为１２８ 核苷酸的片段１ 和荷兰芹的４ＣＬ２ 基因的一个区段有５７ ．８ ％ 同源性，而另一长度为１６９ 核苷酸的片段３ 和百合中的反转座子的一个区段有６０ ．９ ％ 的同源性。这都表明Ｄｓ 因子在异源植物烟草中插入染色体单拷贝基因的机率很高，这对转座子标签法克隆基因是非常有利的。

Transposon tagging is one of the gene cloning strategies. The selection of a phynotype mutant was possible upon insertional mutagenesis with a transposable element, and subsequently the gene fragment flanking the transposable element could be cloned using the transposable element as a probe, then the mutant and wild type genes could be isolated from genomic library using the flanking DNA fragment as probe. In order to know whether the maize Ds element can transpose within tobacco chromosome, we constructed two element systems Ac/Ds, both elements were cotransformed into tobacco plants. Results of genetic analysis of T 1 and T 2 generations of transformed tobacco plants showed that Ac/Ds could transpose from plasmid DNA into tobacco genomic DNA and were inherited stably. To estimate the proportion of the mutants of Ds inserted into repetitive DNA sequence to those inserted into single copy DNA of tobacco genome, we have cloned and sequenced nine DNA fragments flanking nine different Ds insertion sites in tobacco genome (Fig.2). Then we used these DNA fragments as probes to hybridize with the tobacco wild type genomic DNA digested by Bam HⅠ . Results showed that these nine DNA fragments were low copy number DNA sequences in tobacco genome (Fig.4). Their nucleotide sequences were also determined (Fig.3) and compared with those of registered genes in Genbank data base and it has been shown that a 128 nt fragment in fragment 1 shared 57.8% sequence identity with a region of Parsley 4 coumarate: CoA ligase (4CL 2) gene, and another 169 nt fragment in fragment 3 showed 60.9% sequence identity with a region of retro transposon in Lilium henryi(Fig.5). These results showed that Ds was most frequently inserted and transposed into low copy genes in tobacco genome. This feature is favorable for gene cloning using transposon tagging strategy.