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基于菜豆荚斑驳病毒的载体开发,用于大豆中稳定的蛋白质表达和序列特异性病毒诱导的基因沉默

Development of Bean pod mottle virus-based vectors for stable protein expression and sequence-specific virus-induced gene silencing in soybean.

作者信息

Zhang Chunquan, Ghabrial Said A

机构信息

Department of Plant Pathology, 201F Plant Science Building, University of Kentucky, 1405 Veterans Drive, Lexington, KY 40546-0312, USA.

出版信息

Virology. 2006 Jan 20;344(2):401-11. doi: 10.1016/j.virol.2005.08.046. Epub 2005 Oct 14.

Abstract

Plant virus-based vectors provide valuable tools for expression of foreign proteins in plants and for gene function studies. None of the presently available virus vectors is suitable for use in soybean. In the present study, we produced Bean pod mottle virus (BPMV)-based vectors that are appropriate for gene expression and virus-induced gene silencing (VIGS) in soybean. The genes of interest were inserted into the RNA2-encoded polyprotein open reading frame between the movement protein (MP) and the large coat protein (L-CP) coding regions. Additional proteinase cleavage sites were created to flank the foreign protein by duplicating the MP/L-CP cleavage site. To minimize the chances of homologous recombination and thus insert instability, we took advantage of the genetic code degeneracy and altered the nucleotide sequence of the duplicated regions without affecting amino acid sequences. The recombinant BPMV constructs were stable following several serial passages in soybean and relatively high levels of protein expression were attained. Successful expression of several proteins with different biological activities was demonstrated from the BPMV vector. These included the reporter proteins GFP and DsRed, phosphinothricin acetyltransferase (encoded by the herbicide resistance bar gene), and the RNA silencing suppressors encoded by Tomato bushy stunt virus, Turnip crinkle virus, Tobacco etch virus, and Soybean mosaic virus. The possible use of BPMV as a VIGS vector to study gene function in soybean was also demonstrated with the phytoene desaturase gene. Our results suggest that the BPMV-based vectors are suitable for expression of foreign proteins in soybean and for functional genomics applications.

摘要

基于植物病毒的载体为在植物中表达外源蛋白和进行基因功能研究提供了有价值的工具。目前可用的病毒载体均不适用于大豆。在本研究中,我们构建了基于菜豆荚斑驳病毒(BPMV)的载体,该载体适用于大豆中的基因表达和病毒诱导的基因沉默(VIGS)。将感兴趣的基因插入RNA2编码的多聚蛋白开放阅读框中,位于运动蛋白(MP)和大外壳蛋白(L-CP)编码区域之间。通过复制MP/L-CP切割位点,在外源蛋白两侧创建了额外的蛋白酶切割位点。为了尽量减少同源重组的可能性,从而降低插入片段的不稳定性,我们利用遗传密码简并性,在不影响氨基酸序列的情况下改变了重复区域的核苷酸序列。重组BPMV构建体在大豆中连续传代几次后仍保持稳定,并实现了较高水平的蛋白表达。从BPMV载体成功表达了几种具有不同生物学活性的蛋白。这些蛋白包括报告蛋白绿色荧光蛋白(GFP)和红色荧光蛋白(DsRed)、膦丝菌素乙酰转移酶(由抗除草剂bar基因编码),以及由番茄丛矮病毒、芜菁皱缩病毒、烟草蚀纹病毒和大豆花叶病毒编码的RNA沉默抑制因子。利用八氢番茄红素去饱和酶基因,还证明了BPMV作为VIGS载体用于研究大豆基因功能的可能性。我们的结果表明,基于BPMV的载体适用于在大豆中表达外源蛋白和用于功能基因组学应用。

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