The DOF protein, SAD, interacts with GAMYB in plant nuclei and activates transcription of endosperm-specific genes during barley seed development
The DOF protein, SAD, interacts with GAMYB in plant nuclei and activates transcription of endosperm-specific genes during barley seed development
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DOI：
10.1111/j.1365-313x.2005.02402.x
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发表时间：
2005-06-01
期刊：
影响因子：
7.2
通讯作者：
Carbonero, P
中科院分区：
文献类型：
--
作者：
Diaz, I;Martinez, M;Carbonero, P
DOF蛋白SAD是大麦糊粉细胞种子萌发时的转录激活子，在胚乳发育过程中也发挥着重要的基因调控作用。RT-PCR和原位杂交分析表明，在大麦种子发育早期(开花后10d)，在淀粉胚乳、糊粉细胞、珠心突起、维管组织和幼胚中都有表达。SAD蛋白在细菌中表达，与含有醇溶蛋白盒的寡核苷酸结合，5‘-A/TAAAG-3’序列来自胚乳特异基因Hor2和Itr1的启动子区域，分别编码B-醇溶蛋白和胰蛋白酶抑制剂BTI-CME。SAD与另一种胚乳表达的DOF蛋白BPBF竞争相同的结合部位。在共同轰击的发育胚乳中的瞬时表达实验表明，SAD通过与完整的DOF基序结合来反式激活Hor2和Itr1启动子的转录。当两个DOF因子共同轰击时，观察到Itr1基因的表达存在相加效应。Sad ORF与报告基因绿色荧光蛋白基因的框内融合将荧光表达引导到瞬间转化的洋葱表皮层的细胞核。用双分子荧光复合体(BIFC)方法对洋葱细胞核内的荧光进行可视化，表明SAD与R2R3MYB蛋白GAMYB在体内存在相互作用。DOF蛋白BPBF和GAMYB在植物细胞中的相互作用也已被证实，但通过这种方法无法检测到BPBF和SAD之间的核相互作用。
The DOF protein, SAD, previously shown to be a transcriptional activator in barley aleurone cells upon seed germination, also has an important role in gene regulation during endosperm development. mRNA was detected in early (10 days after flowering) developing barley seeds where it accumulated in the starchy endosperm, aleurone cells, nucellar projection, vascular tissues and the immature embryo, as shown by RT-PCR and in situ hybridization analyses. The SAD protein, expressed in bacteria, binds to oligonucleotides containing the prolamine box, 5'-A/TAAAG-3'sequence, derived from the promoter regions of the endosperm-specific genes Hor2 and Itr1, encoding a B-hordein and trypsin-inhibitor BTI-CMe, respectively. SAD competed for the same binding sites with another endosperm-expressed DOF protein, BPBF. Transient expression experiments in co-bombarded developing endosperms demonstrated that SAD trans-activated transcription from Hor2 and Itr1 promoters through binding to the intact DOF motifs. When the two DOF factors are co-bombarded together an additive effect was observed upon the expression of the Itr1 gene. In-frame fusion of the Sad ORF to the reporter green fluorescent protein gene directs the fluorescence expression to the nucleus in transiently transformed onion epidermal layers. The visualization of fluorescence in the nucleus of onion cells, using the bimolecular fluorescent complex (BiFC) approach, has shown the in vivo interaction between SAD and the R2R3MYB protein GAMYB. The interaction in plant cells has also been documented for the DOF protein BPBF and GAMYB, but nuclear interaction could not be detected between BPBF and SAD by this procedure.