Isolation of full-length transcript promoter from the Strawberry vein banding virus (SVBV) and expression analysis by protoplasts transient assays and in transgenic plants
Isolation of full-length transcript promoter from the Strawberry vein banding virus (SVBV) and expression analysis by protoplasts transient assays and in transgenic plants
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DOI：
10.1016/j.plantsci.2004.04.011
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发表时间：
2004-09-01
期刊：
影响因子：
5.2
通讯作者：
Maiti, IB
中科院分区：
文献类型：
--
作者：
Pattanaik, S;Dey, N;Maiti, IB
从草莓条脉病毒（Strawberry vein banding virus，SVBV）基因组克隆中克隆了一个全长转录本（FLt）启动子，并对其在原生质体和转基因植株中的活性进行了分析。最大启动子活性所需的5 '-3'-边界通过与P-葡萄糖醛酸酶（GUS）报告基因融合的SVBV启动子的5 '-和Y-末端缺失分析来确定。在瞬时原生质体表达测定中发现371-bp启动子片段（距转录起始位点-352至+19; TSS）足以获得最大启动子活性，并选择该片段用于进一步分析。对SVBV FLt启动子的90-bp序列（TSS坐标为-392至-302）的更精细的缺失分析揭示了该区域中存在负调控元件和正调控元件。在功能获得性实验中，推定的正调控元件与最小启动子的融合显示启动子活性的非常小的增量，这表明各种顺式序列的组合作用参与启动子功能。全长转录启动子的转录起始位点被定位到位于TATA盒下游25-bp的A残基。在原生质体瞬时表达分析中，SVBV FLt启动子在烟草中的活性比玉米高约6倍。GUS活性的定量分析表明，在转基因烟草植株中，根中的平均启动子活性比叶中高约3倍，这种较高的活性是由于根中积累了更多的GUS特异性mRNA。实时荧光定量PCR分析和GUS活性测定结果表明，SVBV FLt启动子在转基因烟草中的相对表达强度大于CaMV 35 S启动子。SVBV FLt启动子是一种组成型强启动子，在植物外源基因表达中具有巨大的应用潜力。(C)2004爱思唯尔爱尔兰有限公司保留所有权利。
A full-length transcript (FLt) promoter was isolated from a genomic clone of Strawberry vein banding virus (SVBV), a double-stranded DNA plant pararetrovirus belonging to the Caulimoviridae family, and its activity was analyzed both in protoplasts and transgenic plants. The 5'-3'-boundaries required for maximal promoter activity were determined by 5'- and Y-end deletion analysis of the SVBV promoter fused to a P-glucuronidase (GUS) reporter gene. A 371-bp promoter fragment (-352 to + 19 from the transcription start site; TSS) was found sufficient for maximal promoter activity in a transient protoplast expression assay, and this was chosen for further analysis. Finer deletion analysis of a 90-bp sequence (coordinates -392 to -302 from TSS) of the SVBV FLt promoter revealed the presence of a negative and a positive regulatory element in this region. In gain-of-function experiment, the fusion of the putative positive regulatory elements with minimal promoter showed very little increment in promoter activity, suggesting that a combinatorial action of various cis-sequences is involved in promoter function. The transcription start site of the full-length transcript promoter was mapped to an A-residue that is located 25-bp downstream of the TATA-box. In protoplast transient expression analysis, the SVBV FLt promoter showed about six-fold higher activity in tobacco compared to maize. A quantitative GUS activity assay showed that in transgenic tobacco plants the average promoter activity was about three-fold higher in roots than in leaves, and this higher activity was due to the accumulation of more GUS specific mRNA in roots. Real-time qRT-PCR analysis and quantitative GUS activity assay showed that the relative strength of the SVBV FLt promoter was greater than the CaMV35S promoter in transgenic tobacco plants. The SVBV FLt promoter is a strong, constitutive promoter and has great application potential in expression of foreign genes in plants. (C) 2004 Elsevier Ireland Ltd. All rights reserved.