Zhang Chong, Pan Shufang, Chen Hua, Cai Tiecheng, Zhuang Chunhong, Deng Ye, Zhuang Yuhui, Zeng Yuanhuan, Chen Shunhui, Zhuang Weijian
Fujian Provincial Key Laboratory of Crop Molecular and Cell Biology, Fuzhou, 350002, Fujian, China.
College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, 350002, Fujian, China.
Plant Cell Rep. 2016 Apr;35(4):757-69. doi: 10.1007/s00299-015-1918-2. Epub 2016 Feb 5.
A novel root-specific gene and its upstream promoter were cloned and characterized for potential application in root-specific expression of transgenes. The root is an important plant organ subjected to many biotic and abiotic stresses, such as infection by Ralstonia solanacearum. To isolate tobacco root-specific promoters for genetic applications, microarray screening was performed to identify genes highly and specifically expressed in the root. One root-specific gene encoding an extensin-like protein (NtREL1) was isolated, and its expression pattern was further characterized by both microarray analysis and reverse transcription-polymerase chain reaction. NtREL1 was highly expressed only in roots but not in any other organ. NtREL1 expression was affected by hormone treatment (salicylic acid, abscisic acid, and ethephon) as well as low temperature, drought, and R. solanacearum infection. A full-length 849 bp cDNA containing a 657-nucleotide open reading frame was cloned by Rapid Amplification of cDNA Ends. Subsequently, a fragment of 1,574 bp upstream of NtREL1 was isolated by flanking PCR and named pNtREL1. This promoter fragment contains TATA, GATA, and CAAT-boxes, the basic elements of a promoter, and six root-specific expression elements, namely OSE1, OSE2, ROOTMOTIFTAPOX1, SURECOREATSULTR11, P1BS, and WUSATAg. A construct containing the bacterial uidA reporter gene (β-glucuronidase, GUS) driven by the pNtREL1 promoter was transformed into tobacco plants. GUS staining was only detected in the root, but not in leaves and stems. Additionally, transgenic tobacco plants containing peanut resveratrol synthase gene (AhRS) driven by the pNtREL1 promoter produced resveratrol only in the root. Thus, the pNtREL1 promoter can be used to direct root-specific expression of target genes to protect the root from stress or for biological studies.
克隆并鉴定了一个新的根特异性基因及其上游启动子，以用于转基因根特异性表达的潜在应用。根是植物的一个重要器官，会受到许多生物和非生物胁迫，如青枯雷尔氏菌的感染。为了分离用于基因应用的烟草根特异性启动子，进行了微阵列筛选以鉴定在根中高度特异性表达的基因。分离出一个编码类伸展蛋白的根特异性基因（NtREL1），并通过微阵列分析和逆转录-聚合酶链反应进一步表征其表达模式。NtREL1仅在根中高表达，而在其他任何器官中均不表达。NtREL1的表达受激素处理（水杨酸、脱落酸和乙烯利）以及低温、干旱和青枯雷尔氏菌感染的影响。通过cDNA末端快速扩增克隆了一个全长849 bp的cDNA，其包含一个657个核苷酸的开放阅读框。随后，通过侧翼PCR分离出NtREL1上游1574 bp的片段，并命名为pNtREL1。该启动子片段包含启动子的基本元件TATA、GATA和CAAT框，以及六个根特异性表达元件，即OSE1、OSE2、ROOTMOTIFTAPOX1、SURECOREATSULTR11、P1BS和WUSATAg。将含有由pNtREL1启动子驱动的细菌uidA报告基因（β-葡萄糖醛酸酶，GUS）的构建体转化到烟草植株中。仅在根中检测到GUS染色，而在叶和茎中未检测到。此外，含有由pNtREL1启动子驱动的花生白藜芦醇合酶基因（AhRS）的转基因烟草植株仅在根中产生白藜芦醇。因此，pNtREL1启动子可用于指导靶基因的根特异性表达，以保护根免受胁迫或用于生物学研究。
