German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstr. 7b, D-38124 Braunschweig, Germany.
J Biotechnol. 2010 Nov;150(3):277-87. doi: 10.1016/j.jbiotec.2010.09.934. Epub 2010 Sep 17.
The PR-10a protein (formerly STH-2) is known to be induced by biotic stress in potato. The present study demonstrates that transgenic suspension cells of the potato cultivar Desiree over-expressing the PR-10a protein exhibit significantly increased salt and osmotic tolerance compared to the respective wild type cells. A comparison of the proteome pattern of Solanum tuberosum suspension cultures cv. Desiree before and after the treatment with NaCl or sorbitol under equiosmolar conditions (740mOs/kg) revealed the pathogenesis related protein PR-10a to be one of the predominant differentially expressed proteins in potato cell cultures. The pr-10a mRNA was confirmed to be present by RT-PCR from salt challenged suspension cells and was transcribed into cDNA. For PR-10a over-expression Agrobacterium tumefaciens mediated transformation of the potato cells and a dicistronic vector harboring the cDNA of the pr-10a gene linked to a luciferase gene by an IRES (Internal Ribosome Binding Site) was used. The IRES mediated translation leads to co-expression of PR-10a and luciferase in a fixed ratio. By non-invasive luciferase assay homologous PR-10a over-expressing callus was identified after selection on phosphinothricin supplemented medium. This callus was used for the setup of a transgenic suspension culture. Along with increased salt and osmotic tolerance the transformed culture showed changed proline and glutathione levels under abiotic stress conditions in comparison to the wild type.
PR-10a 蛋白(以前称为 STH-2)已知在马铃薯中受到生物胁迫诱导。本研究表明,与相应的野生型细胞相比,过量表达 PR-10a 蛋白的马铃薯栽培品种 Desiree 的悬浮细胞表现出显著提高的盐和渗透胁迫耐受性。在等渗条件下(740mOs/kg)用 NaCl 或山梨醇处理马铃薯悬浮培养物 cv.Desiree 的蛋白质组图谱比较表明,与发病相关的蛋白 PR-10a 是马铃薯细胞培养物中主要差异表达蛋白之一。通过从盐胁迫悬浮细胞中进行 RT-PCR 确认 pr-10a mRNA 的存在,并将其转录为 cDNA。为了进行 PR-10a 的过表达,使用含有 pr-10a 基因 cDNA 的双顺反子载体通过 IRES(内部核糖体进入位点)与荧光素酶基因连接,通过农杆菌介导转化马铃薯细胞。IRES 介导的翻译导致 PR-10a 和荧光素酶以固定比例共表达。通过非侵入性荧光素酶测定,在含有膦丝菌素的培养基上选择后,鉴定出同源 PR-10a 过表达愈伤组织。该愈伤组织用于建立转基因悬浮培养物。与野生型相比,转化培养物在非生物胁迫条件下显示出增加的盐和渗透胁迫耐受性以及脯氨酸和谷胱甘肽水平的变化。