CHARACTERIZATION OF CIS-ACTING ELEMENTS IN LIGHT REGULATION OF THE NUCLEAR GENE ENCODING THE A-SUBUNIT OF CHLOROPLAST ISOZYMES OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM ARABIDOPSIS-THALIANA
CHARACTERIZATION OF CIS-ACTING ELEMENTS IN LIGHT REGULATION OF THE NUCLEAR GENE ENCODING THE A-SUBUNIT OF CHLOROPLAST ISOZYMES OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM ARABIDOPSIS-THALIANA
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DOI：
10.1128/mcb.14.4.2525
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发表时间：
1994-04-01
影响因子：
5.3
通讯作者：
SHIH, MC
中科院分区：
文献类型：
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作者：
CONLEY, TR;PARK, SC;SHIH, MC
我们已经鉴定了编码拟南芥叶绿体甘油醛-3-磷酸脱氢酶A亚单位的核基因(GAPA)的光调控顺式作用元件。我们的结果表明，以大肠杆菌的β-葡萄糖醛酸酶基因为报告基因，GAPA基因的1.1kb启动子片段足以在转基因烟草中提供光诱导和器官特异性。缺失分析表明，GAPA基因的-359~-110bp区域是光反应所必需的。在这个区域内有三个拷贝的十聚体重复序列(称为Gap box)，其共同序列为5‘-CAAATGAA(A/G)A-3’，这在其他光调控基因的启动子区域中尚未被鉴定。在所有三个Gap盒都存在的情况下，从GAPA启动子缺失这些元件的一个拷贝的缺失(To-247)使光诱导减少两到三倍于启动子缺失(To-359)，而所有三个Gap盒(To-110)的缺失完全取消光诱导。以烟草核为核蛋白来源的凝胶迁移率改变实验表明，包含这些重复序列的GAPA启动子片段与核提取物中的一个因子强烈结合，这种结合可以被只由Gap盒的单体或二聚体组成的合成竞争对手取消。此外，Gap box的三聚体、二聚体和单体显示出结合活性，并且像真正的GAPA启动子衍生的探针一样，显示出与Gap box拷贝数相关的结合活性。这些结果有力地表明，这些重复序列在拟南芥GAPA基因的光调控中起着重要作用。
We have characterized cis-acting elements involved in light regulation of the nuclear gene (GapA) encoding the A subunit of chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in Arabidopsis thaliana. Our results show that a 1.1-kb promoter fragment of the GapA gene is sufficient to confer light inducibility and organ specificity in transgenic Nicotiana tabacum (tobacco) plants, using the beta-glucuronidase gene of Escherichia coli as the reporter gene. Deletion analysis indicates that the -359 to -110 bp region of the GapA gene is necessary for light responsiveness. Within this region there are three copies of a decamer repeat (termed the Gap box) having the consensus sequence 5'-CAAATGAA(A/G)A-3', which has not been characterized in the promoter regions of other light-regulated genes. A deletion (to -247) producing loss of one copy of these elements from the GapA promoter reduces light induction by two- to threefold compared with a promoter deletion (to -359) with all three Gap boxes present, while deletion of all three Gap boxes (to -110) abolishes light induction completely. Gel mobility shift experiments using tobacco nuclei as the source of nuclear proteins show that GapA promoter fragments that contain these repeats bind strongly to a factor in the nuclear extract and that binding can be abolished by synthetic competitors consisting only of a monomer or dimer of the Gap box. Furthermore, a trimer, dimer, and monomer of the Gap box show binding activity and, like the authentic GapA promoter-derived probes, show binding activities that are correlated with Gap box copy number. These results strongly suggest that these repeats play important roles in light regulation of the GapA gene of A. thaliana.