Cloning of a mutable bz2 allele of maize by transposon tagging and differential hybridization.
Cloning of a mutable bz2 allele of maize by transposon tagging and differential hybridization.
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DOI：
10.1093/genetics/117.4.771
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发表时间：
1987-12
期刊：
影响因子：
3.3
通讯作者：
M. McLaughlin;V. Walbot
中科院分区：
文献类型：
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作者：
M. McLaughlin;V. Walbot
利用转座子标签技术和差异杂交技术克隆了玉米紫色色素花青素合成基因Bronze 2（Bz 2）的全序列。首先，从Mutator品系中回收突变的bz 2等位基因（bz 2-mu 1）。该突变被认为是由转座因子Mutator（Mu）的插入引起的，该转座因子Mutator在Mutator品系中以10-60个拷贝存在。使用从bz 2-穆尔分离的DNA制备文库，并选择一小部分Mu特异性候选克隆。由于对不同组织中花青素苷合成的遗传要求有很多了解，我们能够根据与从不同bz 2突变体中分离的RNA的杂交来鉴定bz 2克隆。只有一个克隆与紫色植株（Bz 2，B，P1）的壳RNA杂交，而与绿色植株（an-bz 2 -6923，B，P1）的壳RNA不杂交。通过克隆在含有来自其他bz 2突变体的DNA的印迹上的杂交模式提供进一步的确认。我们提出了一个限制性内切酶图谱的克隆，表明1.4 kbp的穆插入的位置和方向。我们确定的转录区域，转录的方向和位置的1.4 kbp的穆插入在一个独立的分离的突变体，bz 2-mu 2。
Sequences of Bronze2 (Bz2), a maize gene which is required for the synthesis of the purple pigment anthocyanin, have been cloned by combining the techniques of transposon tagging and differential hybridization. First, a mutable bz2 allele (bz2-mu1) was recovered from a Mutator line. The mutation was assumed to result from an insertion of the transposable element Mutator (Mu), which is found in 10-60 copies in Mutator lines. A library was prepared using DNA isolated from bz2-mul, and a small bank of Mu-specific candidate clones was selected. Because much is known about the genetic requirements for the synthesis of anthocyanin in different tissues, we were able to identify the bz2 clone based on its hybridization to RNA isolated from different bz2 mutants. Only one clone hybridized to RNA isolated from the husks of purple (Bz2, B, Pl) plants but not to RNA isolated from the husks of green (an-bz2-6923, b, pl) plants. Further confirmation was provided by the hybridization pattern of the clone on blots containing DNA from other bz2 mutants. We present a restriction map of the clone, indicating the location and orientation of the 1.4-kbp Mu insert. We identify the transcribed region, the direction of transcription and the location of the 1.4-kbp Mu insert in an independently isolated mutant, bz2-mu2.