Transcriptional and biochemical regulation of a novel Arabidopsis thaliana bifunctional aspartate kinase-homoserine dehydrogenase gene isolated by functional complementation of a yeast hom6 mutant
Transcriptional and biochemical regulation of a novel Arabidopsis thaliana bifunctional aspartate kinase-homoserine dehydrogenase gene isolated by functional complementation of a yeast hom6 mutant
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DOI：
10.1023/a:1021134621488
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发表时间：
2004
影响因子：
5.1
通讯作者：
S. E. Rognes;E. Dewaele;S. F. Aas;M. Jacobs;V. Frankard
中科院分区：
文献类型：
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作者：
S. E. Rognes;E. Dewaele;S. F. Aas;M. Jacobs;V. Frankard
通过高丝氨酸脱氢酶(HSDH)基因(Hom6)突变的酿酒酵母基因(Hom6)的功能互补，克隆了拟南芥的天冬氨酸激酶-高丝氨酸脱氢酶(AK-HSDH)基因。三个分离克隆中的两个还能够补充突变的酵母天冬氨酸激酶(AK)基因(Hom3)。序列分析表明，该基因位于第4染色体上，与之前克隆的AK-HSDH基因不同，对应于一个新的双功能AK-HSDH基因。在hsdh-lesshom6酵母突变体中表达分离的akhor2cdna可赋予细胞苏氨酸和蛋氨酸原生性。无细胞提取物含有苏氨酸敏感的HSDH活性，具有高等株型的反馈特性。相应地，在AK缺陷型酵母突变体中的基因表达导致苏氨酸和蛋氨酸原生营养，并且在无细胞提取物中观察到苏氨酸敏感的AK活性。这些结果证实了akthr2编码苏氨酸敏感的双功能酶。建立了转基因拟南芥植株(在gus报告基因前面含有fakthr2启动子区域的构建)，以比较akthr2基因的表达模式与早先在烟草中描述的fakthr1的表达模式。这两个基因在分生组织细胞、叶片和雄蕊中同时表达。这两个基因之间的主要差异在于akhor2基因在茎、雌蕊和种子形成过程中表达受时间限制或不表达，而akhor1基因在根中表达较少。
An aspartate kinase-homoserine dehydrogenase (AK-HSDH) cDNA ofArabidopsis thalianahas been cloned by functional complementation of aSaccharomyces cerevisiaestrain mutated in its homoserine dehydrogenase (HSDH) gene (hom6). Two of the three isolated clones were also able to complement a mutant yeast aspartate kinase (AK) gene (hom3). Sequence analysis showed that the identified gene (akthr2), located on chromosome 4, is different from the previously clonedA. thalianaAK-HSDH gene (akthr1), and corresponds to a novel bifunctional AK-HSDH gene. Expression of the isolatedakthr2cDNA in a HSDH-lesshom6yeast mutant conferred threonine and methionine prototrophy to the cells. Cell-free extracts contained a threonine-sensitive HSDH activity with feedback properties of higher plant type. Correspondingly, cDNA expression in an AK-deficienthom3yeast mutant resulted in threonine and methionine prototrophy and a threonine-sensitive AK activity was observed in cell-free extracts. These results confirm thatakthr2encodes a threonine-sensitive bifunctional enzyme. TransgenicArabidopsis thalianaplants (containing a construct with the promoter region ofakthr2in front of thegusreporter gene) were generated to compare the expression pattern of theakthr2gene with the pattern ofakthr1earlier described in tobacco. The two genes are simultaneously expressed in meristematic cells, leaves and stamens. The main differences between the two genes concern the time-restricted or absent expression of theakthr2gene in the stem, the gynoecium and during seed formation, whileakthr1is less expressed in roots.