Department of Genetic Engineering and Graduate School of Biotechnology, College of Life Sciences, Kyung Hee University, Yongin, Republic of Korea.
College of Agriculture and Life Sciences, Chungnam National University, Daejeon, Republic of Korea.
J Exp Bot. 2018 Oct 12;69(21):5105-5116. doi: 10.1093/jxb/ery300.
Carotenoids of staple food crops have a high nutritional value as provitamin A components in the daily diet. To increase the levels of carotenoids, inhibition of carotenoid-cleavage dioxygenases (CCDs), which degrade carotenoids, has been considered as a promising target in crop biotechnology. In this study, suppression of the OsCCD1, OsCCD4a, and OsCCD4b genes using RNAi was verified in transgenic rice plants by quantitative RT-PCR and small RNA detection. Leaf carotenoids were significantly increased overall in OsCCD4a-RNAi lines of the T1 generation, and the highest accumulation of 1.3-fold relative to non-transgenic plants was found in a line of the T2 generation. The effects on seed carotenoids were determined via cross-fertilization between β-carotene-producing transgenic rice and one of two independent homozygous lines of OsCCD1-RNAi, OsCCD4a-RNAi, or OsCCD4b-RNAi. This showed that carotenoids were increased to a maximum of 1.4- and 1.6-fold in OsCCD1-RNAi and OsCCD4a-RNAi, respectively, with a different preference toward α-ring and β-ring carotenoids; levels could not be established in OsCCD4b-RNAi. In addition, the contents of four carotenoids decreased when OsCCD1, OsCCD4a, and OsCCD4b were overexpressed in E. coli strains accumulating phytoene, lycopene, β-carotene, and zeaxanthin. OsCCD1 and OsCCD4a had a similar high carotenoid degrading activity, followed by OsCCD4b without substrate specificity. Overall, our results suggest that suppresing OsCCD4a activity may have potential as a tool for enhancing the carotenoid content of seed endosperms and leaves in rice.
主食作物中的类胡萝卜素作为膳食中的维生素 A 前体成分，具有很高的营养价值。为了增加类胡萝卜素的含量，抑制类胡萝卜素裂解双加氧酶（CCD），即降解类胡萝卜素的酶，已被认为是作物生物技术中的一个有前途的目标。在这项研究中，通过定量 RT-PCR 和小 RNA 检测，证实了 RNAi 抑制 OsCCD1、OsCCD4a 和 OsCCD4b 基因在转基因水稻植株中的作用。T1 代 OsCCD4a-RNAi 系的叶片类胡萝卜素总体显著增加，与非转基因植株相比，最高积累量达到 1.3 倍。通过β-胡萝卜素产生的转基因水稻与 OsCCD1-RNAi、OsCCD4a-RNAi 或 OsCCD4b-RNAi 的两个独立纯合系之一的杂交授粉，确定了对种子类胡萝卜素的影响。结果表明，OsCCD1-RNAi 和 OsCCD4a-RNAi 分别使类胡萝卜素增加到 1.4-和 1.6 倍的最大值，对α-环和β-环类胡萝卜素具有不同的偏好；而在 OsCCD4b-RNAi 中则无法建立。此外，当在积累番茄红素、番茄红素、β-胡萝卜素和玉米黄质的大肠杆菌菌株中过表达 OsCCD1、OsCCD4a 和 OsCCD4b 时，四种类胡萝卜素的含量降低。OsCCD1 和 OsCCD4a 具有相似的高类胡萝卜素降解活性，其次是没有底物特异性的 OsCCD4b。总的来说，我们的结果表明，抑制 OsCCD4a 的活性可能是提高水稻种子胚乳和叶片类胡萝卜素含量的一种潜在工具。
