Genetic manipulation of plant genes in prokaryotes has been widely used in molecular biology, but the function of a DNA sequence is far from being fully known. Here, we discovered that a plant protein-coding gene containing the CRAL_TRIO domain serves as a promoter in bacteria. We firstly characterized CitPITP1 from Citrus, which contains the CRAL_TRIO domain, and identified a 64-bp sequence (key64) that is critical for prokaryotic promoter activity. In vitro experiments indicated that the bacterial RNA polymerase subunit RpoD specifically binds to key64. We then expanded our research to fungi, plant and animal species to identify key64-like sequences. Five such prokaryotic promoters were isolated from Amborella, Rice, Arabidopsis and Citrus. Two conserved motifs were identified, and mutation analysis indicated that the nucleotides at positions 7, 29 and 30 are crucial for key64-like transcription activity. We detected full-length recombinant CitPITP1 from E. coli, and visualized a CitPITP1-GFP fusion protein in plant cells, supporting the idea that CitPITP1 encodes a protein. However, although exon 4 of CitPITP1 contained key64, it did not demonstrate promoter activity in plants. Our study describes a new basal promoter, provides evidence for neofunction of gene elements across different kingdoms, and provides new knowledge for the modular design of promoters.

原核生物中植物基因的遗传操作已广泛应用于分子生物学，但 DNA 序列的功能远未完全了解。在这里，我们发现包含 CRAL_TRIO 结构域的植物蛋白编码基因在细菌中充当启动子。我们首先表征了来自Citrus的 CitPITP1，它包含 CRAL_TRIO 结构域，并鉴定了一个对原核启动子活性至关重要的 64 bp 序列（key64）。体外实验表明细菌 RNA 聚合酶亚基 RpoD 与 key64 特异性结合。然后，我们将研究扩展到真菌、植物和动物物种，以识别 key64 样序列。从Amborella、Rice、Arabidopsis中分离出五个这样的原核启动子和柑橘。鉴定了两个保守基序，突变分析表明第 7、29 和 30 位的核苷酸对 key64 样转录活性至关重要。我们从大肠杆菌中检测到全长重组 CitPITP1 ，并在植物细胞中观察到 CitPITP1-GFP 融合蛋白，支持 CitPITP1 编码蛋白质的观点。然而，尽管 CitPITP1 的外显子 4 含有 key64，但它在植物中并未表现出启动子活性。我们的研究描述了一种新的基础启动子，为不同界的基因元件的新功能提供了证据，并为启动子的模块化设计提供了新知识。