Characterization and structural features of a chalcone synthase mutation in a white-flowering line of Matthiola incana R. Br. (Brassicaceae)
Characterization and structural features of a chalcone synthase mutation in a white-flowering line of Matthiola incana R. Br. (Brassicaceae)
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DOI：
10.1007/s11103-004-1125-y
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发表时间：
2004-05-01
影响因子：
5.1
通讯作者：
Austin, MB
中科院分区：
文献类型：
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作者：
Hemleben, V;Dressel, A;Austin, MB
对于紫罗兰（菊科），作为一个模型系统来研究花色素苷生物合成的生化和遗传方面，几个几乎同基因的彩色野生型线和白色开花突变株系，每个负责花色素苷生产的基因（基因e，f，和g）的特定缺陷。对于被认为编码查耳酮合酶（CHS; EC 2.3.1.74）的基因f，该查耳酮合酶是类黄酮/花色素苷生物合成途径的关键酶，属于III型聚酮化合物，脱氢酶（PKS）的组，M.与白花CHS突变系18相比，确定了incana系04。chs基因突变的类型被表征为编码进化上保守的精氨酸的三联体AGG中的单核苷酸取代为编码丝氨酸的AGT（R72 S）。北方杂交和RT-PCR结果表明突变基因在花瓣中表达。野生型和突变型CHS cDNA在E.通过Western印迹法和各种起始分子的酶测定验证的Scherichiacoli，揭示了突变蛋白没有可检测的活性，表明严格保守的精氨酸残基对于酶促反应是必需的。这种突变，这是以前没有检测到的诱变筛选，讨论了苜蓿CHS和相关的III型PKS酶的结构和功能信息的光。
For Matthiola incana (Brassicaceae), used as a model system to study biochemical and genetical aspects of anthocyanin biosynthesis, several nearly isogenic colored wild type lines and white-flowering mutant lines are available, each with a specific defect in the genes responsible for anthocyanin production (genes e, f, and g). For gene f supposed to code for chalcone synthase (CHS; EC 2.3.1.74), the key enzyme of the flavonoid/ anthocyanin biosynthesis pathway belonging to the group of type III polyketide, synthases (PKS), the wild type genomic sequence of M. incana line 04 was determined in comparison to the white-flowering CHS mutant line 18. The type of mutation in the chs gene was characterized as a single nucleotide substitution in a triplet AGG coding for an evolutionary conserved arginine into AGT coding for serine (R72S). Northern blots and RT-PCR demonstrated that the mutated gene is expressed in flower petals. Heterologous expression of the wild type and mutated CHS cDNA in E. Scherichia coli, verified by Western blotting and enzyme assays with various starter molecules, revealed that the mutant protein had no detectable activity, indicating that the strictly conserved arginine residue is essential for the enzymatic reaction. This mutation, which previously was not detected by mutagenic screening, is discussed in the light of structural and functional information on alfalfa CHS and related type III PKS enzymes.