A specific member of the Cab multigene family can be efficiently targeted and disrupted in the moss Physcomitrella patens
A specific member of the Cab multigene family can be efficiently targeted and disrupted in the moss Physcomitrella patens
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DOI：
10.1007/s004380050945
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发表时间：
1999-02-01
期刊：
影响因子：
--
通讯作者：
Zryd, JP
中科院分区：
文献类型：
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作者：
Hofmann, AH;Codón, AC;Zryd, JP
对同源重组后基因破坏引起的表型变化的分析为基因功能的研究提供了强大的技术。迄今为止，该技术很难应用于植物，因为用含有与基因组序列同源的 DNA 的构建体转化后基因破坏的频率较低（0.01 至 0.1%）。最近的研究表明，使用未知功能的基因组序列可以在小立碗藓中实现高基因破坏率（高达 90%）。我们使用该系统利用 Cab 多基因家族的成员来检查立碗藓中基因破坏的特异性。我们使用了先前表征的 Cab 基因 ZLAB1，并分离了 Cab 基因家族的其他 13 个密切相关成员的片段。在 199 bp 的延伸测序中，Cab 家族的 13 个新成员与 ZLAB1 的 DNA 序列平均有 8.5% 的差异。我们观察到 304 个沉默取代和 16 个导致蛋白质氨基酸序列变化的取代。我们将ZLAB1编码区的1029 bp（包括与13个新Cab基因具有高同源性的199 bp中的177 bp）克隆到含有可选择的潮霉素抗性标记的载体中，并使用该构建体转化P. patens。在测试的九个稳定转化体中的三个中，该构建体已插入并破坏了 ZLAB1 基因。没有与破坏相关的可辨别的表型。因此，我们已经证明，基因破坏在 P. patens 中是可重复的，并且对序列同源性的要求似乎是严格的，因此允许首次在陆地植物中分析基因家族个体成员的作用。
The analysis of phenotypic change resulting from gene disruption following homologous recombination provides a powerful technique for the study of gene function. This technique has so far been difficult to apply to plants because the frequency of gene disruption following transformation with constructs containing DNA homologous to genomic sequences is low (0.01 to 0.1%). It has recently been shown that high rates of gene disruption (up to 90%) can be achieved in the moss Physcomitrella patens using genomic sequences of unknown function. We have used this system to examine the specificity of gene disruption in Physcomitrella using a member of the Cab multigene family. We have employed the previously characterised Cab gene ZLAB1 and have isolated segments of 13 other closely related members of the Cab gene family. In the 199-bp stretch sequenced, the 13 new members of the Cab family show an average of 8.5% divergence from the DNA sequence of ZLAB1. We observed 304 silent substitutions and 16 substitutions that lead to a change in the amino acid sequence of the protein. We cloned 1029 bp of the coding region of ZLAB1 (including 177 of the 199 bp with high homology to the 13 new Cab genes) into a vector containing a selectable hygromycin resistance marker, and used this construct to transform P. patens. In three of nine stable transformants tested, the construct had inserted in, and disrupted, the ZLAB1 gene. There was no discernible phenotype associated with the disruption. We have therefore shown that gene disruption is reproducible in P. patens and that the requirement for sequence homology appears to be stringent, therefore allowing the role of individual members of a gene family to be analysed in land plants for the first time.