Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests, Hainan University, Ministry of Education, Haikou, China.
College of Plant Protection, Hainan University, Haikou, China.
PLoS One. 2020 Jun 1;15(6):e0233911. doi: 10.1371/journal.pone.0233911. eCollection 2020.
Promoters are very important for transcriptional regulation and gene expression, and have become invaluable tools for genetic engineering. Owing to the characteristics of obligate biotrophs, molecular research into obligate biotrophic fungi is seriously lagging behind, and very few of their endogenous promoters have been developed. In this study, a WY7 fragment was predicted in the genome of Oidium heveae Steinmann using PromoterScan. Its promoter function was verified with transient transformations (Agrobacterium tumefaciens-mediated transformation, ATMT) in Nicotiana tabacum cv. Xanthi nc. The analysis of the transcription range showed that WY7 could regulate GUS expression in both monocots (Zea mays Linn and Oryza sativa L. spp. Japonica cv. Nipponbare) and dicots (N. tabacum and Hylocereus undulates Britt). The results of the quantitative detection showed that the GUS transient expression levels when regulated by WY7 was more than 11.7 times that of the CaMV 35S promoter in dicots (N. tabacum) and 5.13 times that of the ACT1 promoter in monocots (O. sativa). GUS staining was not detected in the T1 generation of the WY7-GUS transgenic N. tabacum. This showed that WY7 is an inducible promoter. The cis elements of WY7 were predicted using PlantCARE, and further experiments indicated that WY7 was a low temperature- and salt-inducible promoter. Soluble proteins produced by WY7-hpa1Xoo transgenic tobacco elicited hypersensitive responses (HR) in N. tabacum leaves. N. tabacum transformed with pBI121-WY7-hpa1Xoo exhibited enhanced resistance to the tobacco mosaic virus (TMV). The WY7 promoter has a lot of potential as a tool for plant genetic engineering. Further in-depth studies will help to better understand the transcriptional regulation mechanisms of O. heveae.
启动子对于转录调控和基因表达非常重要，已经成为基因工程的宝贵工具。由于专性生物营养型真菌的特点，对专性生物营养型真菌的分子研究严重滞后，很少开发其内源启动子。本研究利用 PromoterScan 在橡胶疫霉基因组中预测了一个 WY7 片段。通过瞬时转化（农杆菌介导的转化，ATMT）在烟草 cv. Xanthi nc 中验证了其启动子功能。转录范围分析表明，WY7 可以在单子叶植物（玉米和水稻亚种粳稻 cv. Nipponbare）和双子叶植物（烟草和波浪仙人掌）中调节 GUS 的表达。定量检测结果表明，WY7 调控的 GUS 瞬时表达水平在双子叶植物（烟草）中比 CaMV 35S 启动子高 11.7 倍，在单子叶植物（水稻）中比 ACT1 启动子高 5.13 倍。WY7-GUS 转基因烟草 T1 代未检测到 GUS 染色。这表明 WY7 是一种诱导型启动子。利用 PlantCARE 预测了 WY7 的顺式作用元件，进一步的实验表明 WY7 是一种低温和盐诱导型启动子。WY7-hpa1Xoo 转基因烟草产生的可溶性蛋白在烟草叶片中引发了过敏反应（HR）。转化了 pBI121-WY7-hpa1Xoo 的烟草对烟草花叶病毒（TMV）表现出增强的抗性。WY7 启动子作为植物遗传工程的工具具有很大的潜力。进一步的深入研究将有助于更好地理解橡胶疫霉的转录调控机制。
