Division of Plant and Microbial Biotechnology, Institute of Life Sciences, NALCO Nagar Road, NALCO Square, Chandrasekharpur, Bhubaneswar, Odisha, 751023, India.
Planta. 2021 May 15;253(6):121. doi: 10.1007/s00425-021-03624-1.
We analyzed the synthetic full-length transcript promoter of Blueberry red ringspot virus (BRRV) and developed two chimeric promoters (MBR3 and FBR3). Transcriptional activities of these chimeric promoters were found equivalent to that of the CaMV35S promoter. Chimeric promoters driven plant-derived PaDef protein showed high antimicrobial activities against several pathogens. Blueberry red ringspot virus (BRRV) is a pararetrovirus under the genus, Soymovirus belongs to the Caulimoviridae family. We have made a synthetic version of the BRRV-Flt promoter and analyzed its activity in detail. A 372 bp promoter fragment BR3 (- 212 to + 160) showed the strongest transcriptional activity compared with other fragments in both transient and transgenic assays; its activity was found near equivalent to that of the CaMV35S promoter. We constructed two chimeric promoters; MBR3 and FBR3 by fusing the UASs (Upstream activation sequences) of Mirabilis mosaic virus (MUAS; - 297 to - 38; 335 bp) and Figwort mosaic virus (FUAS; - 249 to - 54; 303 bp) respectively to the core promoter domain of BR3 (BR3; - 212 to + 160; 372 bp). The activities of MBR3 and FBR3 promoters were found equivalent to that of the activity of the CaMV35S promoter and approximately 4.0 (four) times stronger than that of the CaMV35S promoter. Histochemical and fluorometric GUS assays confirmed the above observation. The transcriptional efficacies of these recombinant promoters were tested by evaluating the antibacterial and antifungal activities of recombinant plant-derived antimicrobial peptide Persea americana var. drymifolia defensin (PaDef) driven under these promoters. Bioassays showed promising antifungal activities of the plant made PaDef against Alternaria alternata and antibacterial property against Gram-positive (S. aureus and R. fascians) and Gram-negative bacteria (E. coli and P. aeruginosa). Based upon the above results, MBR3 and FBR3 could be useful promoters for plant genetic engineering and can become useful substitutes for the widely used CaMV35S promoter in plant biology.
我们分析了蓝莓红叶环斑病毒（BRRV）的全长转录启动子，并开发了两个嵌合启动子（MBR3 和 FBR3）。这些嵌合启动子的转录活性与 CaMV35S 启动子相当。由嵌合启动子驱动的植物衍生的 PaDef 蛋白对几种病原体表现出高抗菌活性。蓝莓红叶环斑病毒（BRRV）是一种拟反转录病毒，属于 Soymovirus 属，属于 Caulimoviridae 科。我们已经合成了 BRRV-Flt 启动子，并对其活性进行了详细分析。与瞬时和转基因测定中的其他片段相比，372 bp 的启动子片段 BR3（-212 至+160）表现出最强的转录活性；其活性与 CaMV35S 启动子相当。我们构建了两个嵌合启动子；MBR3 和 FBR3 通过分别融合 Mirabilis mosaic virus（MUAS；-297 至-38；335 bp）和 figwort mosaic virus（FUAS；-249 至-54；303 bp）的 UASs（上游激活序列）到 BR3 的核心启动子区域（BR3；-212 至+160；372 bp）。MBR3 和 FBR3 启动子的活性与 CaMV35S 启动子的活性相当，大约是 CaMV35S 启动子的 4.0 倍。组织化学和荧光 GUS 测定证实了上述观察结果。通过评估这些重组启动子驱动的重组植物衍生抗菌肽 Persea americana var. drymifolia defensin（PaDef）对细菌和真菌的抗菌和抗真菌活性，测试了这些重组启动子的转录效率。生物测定显示，植物产生的 PaDef 对Alternaria alternata 具有良好的抗真菌活性，对革兰氏阳性（金黄色葡萄球菌和 R. fascians）和革兰氏阴性菌（大肠杆菌和铜绿假单胞菌）具有抗菌活性。基于上述结果，MBR3 和 FBR3 可作为植物基因工程的有用启动子，并可成为植物生物学中广泛使用的 CaMV35S 启动子的有用替代品。
