Down-regulation of β-carotene hydroxylase increases β-carotene and total carotenoids enhancing salt stress tolerance in transgenic cultured cells of sweetpotato
Down-regulation of β-carotene hydroxylase increases β-carotene and total carotenoids enhancing salt stress tolerance in transgenic cultured cells of sweetpotato
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DOI：
10.1016/j.phytochem.2011.11.003
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发表时间：
2012-02-01
期刊：
影响因子：
3.8
通讯作者：
Kwak, Sang-Soo
中科院分区：
文献类型：
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作者：
Kim, Sun Ha;Ahn, Young Ock;Kwak, Sang-Soo
甘薯（Ipomoea batatas Lam.）是一种重要的工业作物和食物来源，含有有用的成分，包括类胡萝卜素等抗氧化剂。 β-胡萝卜素羟化酶 (CHY-β) 是类胡萝卜素生物合成 β-β-分支中的关键调节酶，它催化羟基化为 β-胡萝卜素生成 p-隐黄质和 β-隐黄质生成玉米黄质。为了通过抑制β-胡萝卜素的进一步羟基化来增加甘薯的β-胡萝卜素含量，评估了沉默类胡萝卜素生物合成途径中CHY-β的效果。从橙肉甘薯（cv. Shinhwangmi）的贮藏根中克隆了编码 CHY-β 的部分 cDNA，以生成 RNA 干扰-IbCHY-β 构建体。将该构建体引入白肉甘薯（cv. Yulmi）的培养细胞中。逆转录聚合酶链反应分析证实在转基因培养细胞中成功抑制了 IbCHY-β 基因表达。八氢番茄红素合酶和番茄红素β-环化酶的表达水平增加，而其他基因的表达没有显示出可检测到的变化。 IbCHY-beta 基因表达的下调改变了非转基因（NT）和转基因细胞之间类胡萝卜素的组成和水平。在转基因株系#7中，总类胡萝卜素含量达到最大值117μg/g干重，其中β-胡萝卜素含量为34.43μg/g干重。此外，IOCHY-β沉默的愈伤组织显示出β-隐黄质和玉米黄质含量升高以及P450基因的高转录水平。转基因细胞中的2,2-二苯基-1-三硝基苯肼自由基清除活性(DPPH)是NT细胞中的两倍多。 RNA-IbCHY-β愈伤组织增加了脱落酸（ABA）含量，同时伴随着对盐胁迫的耐受性增强。此外，通过3,3'-二氨基联苯胺（DAB）染色测量，转基因培养细胞在盐胁迫下活性氧的产生显着减少。综上所述，目前的结果表明，IbCHY-β的下调增加了转基因植物细胞中β-胡萝卜素的含量和总类胡萝卜素的含量，并增强了它们的抗氧化能力。 (C) 2011 Elsevier Ltd. 保留所有权利。
Sweetpotato (Ipomoea batatas Lam.) is an important industrial crop and source of food that contains useful components, including antioxidants such as carotenoids. beta-Carotene hydroxylase (CHY-beta) is a key regulatory enzyme in the beta-beta-branch of carotenoid biosynthesis and it catalyzes hydroxylation into both beta-carotene to p-cryptoxanthin and beta-cryptoxanthin to zeaxanthin. To increase the beta-carotene content of sweetpotato through the inhibition of further hydroxylation of beta-carotene, the effects of silencing CHY-beta in the carotenoid biosynthetic pathway were evaluated. A partial cDNA encoding CHY-beta was cloned from the storage roots of orange-fleshed sweetpotato (cv. Shinhwangmi) to generate an RNA interference-IbCHY-beta construct. This construct was introduced into cultured cells of white-fleshed sweetpotato (cv. Yulmi). Reverse transcription-polymerase chain reaction analysis confirmed the successful suppression of IbCHY-beta gene expression in transgenic cultured cells. The expression level of phytoene synthase and lycopene p-cyclase increased, whereas the expression of other genes showed no detectable change. Down-regulation of IbCHY-beta gene expression changed the composition and levels of carotenoids between non-transgenic (NT) and transgenic cells. In transgenic line #7, the total carotenoid content reached a maximum of 117 mu g/g dry weight, of which beta-carotene measured 34.43 mu g/g dry weight. In addition, IOCHY-beta-silenced calli showed elevated p-cryptoxanthin and zeaxanthin contents as well as high transcript level P450 gene. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH) in transgenic cells was more than twice that in NT cells. RNA-IbCHY-beta calli increased abscisic acid (ABA) content, which was accompanied by enhanced tolerance to salt stress. In addition, the production of reactive oxygen species measured by 3,3'-diaminobenzidine (DAB) staining was significantly decreased in transgenic cultured cells under salt stress. Taken together, the present results indicate that down-regulation of IbCHY-beta increased beta-carotene contents and total carotenoids in transgenic plant cells and enhanced their antioxidant capacity. (C) 2011 Elsevier Ltd. All rights reserved.