Analysis of cis-sequence of subgenomic transcript promoter from the Figwort mosaic virus and comparison of promoter activity with the cauliflower mosaic virus promoters in monocot and dicot cells
Analysis of cis-sequence of subgenomic transcript promoter from the Figwort mosaic virus and comparison of promoter activity with the cauliflower mosaic virus promoters in monocot and dicot cells
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DOI：
10.1016/s0166-0934(02)00146-5
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发表时间：
2002-12-01
期刊：
影响因子：
5
通讯作者：
Maiti, IB
中科院分区：
文献类型：
--
作者：
Bhattacharyya, S;Dey, N;Maiti, IB
从玄参花叶病毒（FMV）基因组克隆中分离亚基因组转录物（Sgt）启动子。将FMV Sgt启动子与异源编码序列连接以形成嵌合基因构建体。在瞬时原生质体表达实验和转基因植物中，通过对与β-葡萄糖醛酸酶报告基因偶联的FMV Sgt启动子片段的5 ′，3 ′-末端缺失和内部缺失分析，确定了在植物中最佳表达的顺式序列的最大活性和参与所需的5 ′-3 ′-边界。一个301 bp的FMV Sgt启动子片段（从转录起始位点开始的-270到+31序列; TSS）提供了最大的启动子活性。FMV Sgt启动子的TSS通过引物延伸分析使用来自为FMV Sgt启动子：uidA融合基因开发的转基因植物的总RNA来确定。位于TATA盒上游距离TSS-70至-100处的激活剂结构域是启动子活性绝对需要的，并且其功能相对于TATA盒是关键的位置依赖性的。发现两个序列基序AGATTTTAAT（坐标-100至-91）和GTAAGCGC（坐标-80至-73）对于启动子活性是必需的。FMV Sgt启动子在单子叶植物细胞中活性较低;与玉米细胞相比，FMV Sgt启动子在烟草细胞中的表达水平高约27.5倍。FMV Sgt启动子与花椰菜花叶病毒（CaMV）35 S启动子的比较表达分析表明，FMV Sgt启动子比CaMV 35 S启动子强约2倍。FMV Sgt启动子为组成型启动子，在幼苗中的表达量为根>叶>茎。(C)2002 Elsevier Science B. V.保留所有权利。
A sub-genomic transcript (Sgt) promoter was isolated from the Figwort mosaic virus (FMV) genomic clone. The FMV Sgt promoter was linked to heterologous coding sequences to form a chimeric gene construct. The 5'-3'-boundaries required for maximal activity and involvement of cis-sequences for optimal expression in plants were defined by 5'-, 3'-end deletion and internal deletion analysis of FMV Sgt promoter fragments coupled with a beta-glucuronidase reporter gene in both transient protoplast expression experiments and in transgenic plants. A 301 bp FMV Sgt promoter fragment (sequence -270 to +31 from the transcription start site; TSS) provided maximum promoter activity. The TSS of the FMV Sgt promoter was determined by primer extension analysis using total RNA from transgenic plants developed for FMV Sgt promoter: uidA fusion gene. An activator domain located upstream of the TATA box at -70 to -100 from TSS is absolutely required for promoter activity and its function is critically position-dependent with respect to TATA box. Two sequence motifs AGATTTTAAT (coordinates -100 to -91) and GTAAGCGC (coordinates -80 to -73) were found to be essential for promoter activity. The FMV Sgt promoter is less active in monocot cells; FMV Sgt promoter expression level was about 27.5-fold higher in tobacco cells compared to that in maize cells. Comparative expression analysis of FMV Sgt promoter with cauliflower mosaic virus (CaMV) 35S promoter showed that the FMV Sgt promoter is about 2-fold stronger than the CaMV 35S promoter. The FMV Sgt promoter is a constitutive promoter; expression level in seedlings was in the order: root > leaf > stem. (C) 2002 Elsevier Science B.V. All rights reserved.