Yamamoto Masayuki P, Onodera Yasuyuki, Touno Satoru M, Takaiwa Fumio
Transgenic Crop Research and Development Center, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
Plant Physiol. 2006 Aug;141(4):1694-707. doi: 10.1104/pp.106.082826. Epub 2006 Jun 23.
The Dof (DNA binding with one finger) transcriptional activator rice (Oryza sativa) prolamin box binding factor (RPBF), which is involved in gene regulation of rice seed storage proteins, has been isolated from rice cDNA expressed sequence tag clones containing the conserved Dof. RPBF is found as a single gene per haploid genome. Comparison of RPBF genomic and cDNA sequences revealed that the genomic copy is interrupted by one long intron of 1,892 bp in the 5' noncoding region. We demonstrated by transient expression in rice callus protoplasts that the isolated RPBF trans-activated several storage protein genes via an AAAG target sequence located within their promoters, and with methylation interference experiments the additional AAAG-like sequences in promoters of genes expressed in maturing seeds were recognized by the RPBF protein. Binding was sequence specific, since mutation of the AAAG motif or its derivatives decreased both binding and trans-activation by RPBF. Synergism between RPBF and RISBZ1 recognizing the GCN4 motif [TGA(G/C)TCA] was observed in the expression of many storage protein genes. Overexpression of both transcription factors gave rise to much higher levels of expression than the sum of individual activities elicited by either RPBF or RISBZ1 alone. Furthermore, mutation of recognition sites suppressed reciprocal trans-activation ability, indicating that there are mutual interactions between RISBZ1 and RPBF. The RPBF gene is predominantly expressed in maturing endosperm and coordinately expressed with seed storage protein genes, and is involved in the quantitative regulation of genes expressed in the endosperm in cooperation with RISBZ1.
具有单指结构域（DNA binding with one finger，Dof）的转录激活因子水稻（Oryza sativa）醇溶蛋白盒结合因子（RPBF）参与水稻种子贮藏蛋白的基因调控，已从含有保守Dof的水稻cDNA表达序列标签克隆中分离得到。RPBF在单倍体基因组中以单基因形式存在。RPBF基因组序列和cDNA序列的比较显示，基因组拷贝在5′非编码区被一个1892 bp的长内含子中断。我们通过在水稻愈伤组织原生质体中的瞬时表达证明，分离得到的RPBF通过位于其启动子内的AAAG靶序列反式激活多个贮藏蛋白基因，并且通过甲基化干扰实验表明，RPBF蛋白能够识别成熟种子中表达基因启动子中的额外AAAG样序列。结合具有序列特异性，因为AAAG基序或其衍生物的突变会降低RPBF的结合和反式激活能力。在许多贮藏蛋白基因的表达中观察到RPBF与识别GCN4基序[TGA(G/C)TCA]的RISBZ1之间存在协同作用。两种转录因子的过表达所产生的表达水平远高于单独由RPBF或RISBZ1所引发的个体活性之和。此外，识别位点的突变抑制了相互反式激活能力，表明RISBZ1与RPBF之间存在相互作用。RPBF基因主要在成熟胚乳中表达，并与种子贮藏蛋白基因协同表达，且与RISBZ1共同参与胚乳中基因表达的定量调控。
