RNAi-mediated Suppression of Three Rice Carotenoid Cleavage Dioxygenase Genes, OsCCD1, 4a, and 4b, Increases Carotenoid Content of Rice

First author: Mi Ran Ko; Affiliations: Kyung Hee University (庆熙大学): Yongin, Republic of Korea

Corresponding author: Sun-Hwa Ha

Carotenoids of staple food (主食) crops have high nutritional value as provitamin A (维生素原A) components in the daily diet. To increase the levels of carotenoids, inhibition of carotenoid cleavage dioxygenases (CCDs; 类胡萝卜素裂解双加氧酶), which degrade carotenoids, has been considered as a promising target in crop biotechnology. Suppression of the OsCCD1, OsCCD4a, and OsCCD4b genes using RNA interference (Ri) was verified in transgenic rice plants by quantitative RT-PCR and small RNA detection. Leaf carotenoids were statistically increased overall in OsCCD4a_Ri lines of the T1 generation, and the highest accumulation of 1.3-fold was found in the OsCCD4a_Ri 7 line of the T2 generation relative to non-transgenic plants. The effects on seed carotenoids were elucidated via cross-fertilization (异花受粉) between β-carotene-producing transgenic rice and one of two independent homozygous lines of OsCCD1_Ri, OsCCD4a_Ri or OsCCD4b_Ri. As a result, seed carotenoids were increased to a maximum of 1.4- and 1.6-fold in OsCCD1_Ri and OsCCD4a_Ri, respectively, with a different preference toward α-ring carotenoids and β-ring carotenoids; levels were not established in OsCCD4b_Ri. In addition, the levels of four carotenoids decreased when OsCCD1, OsCCD4a, and OsCCD4b were overexpressed in E. coli strains accumulating phytoene (八氢番茄红素), lycopene (番茄红素), β-carotene (β-胡萝卜素), and zeaxanthin (玉米黄素). OsCCD1 and OsCCD4a had a similar high carotenoid degrading activity, followed by OsCCD4b without substrate specificity. Collectively, the blocking of OsCCD4a activity might have potential as a practical tool for enhancing the carotenoid level of the carotenoid-accumulating seed endosperms (胚乳) as well as leaves of rice plants.

在日常饮食中，主食作物的类胡萝卜素具有与维生素原A一样高的营养价值。为了增加类胡萝卜素的水平，抑制类能够降解累胡萝卜素的胡萝卜素裂解双加氧酶CCD在作物生物技术中可以作为潜在的技术手段。作者通过实时定量PCR和小RNA检测对RNA干扰OsCCD1，OsCCD4a和OsCCD4a基因的转基因水稻植株进行了功能验证。结果显示在所有OsCCD4a_Ri株系T1代的叶片中，类胡萝卜素的含量显著提高；T2代的OsCCD4a_Ri 7株系与非转基因植株叶片类胡萝卜素含量差异达到最大，为1.3倍。作者进一步通过将产β-胡萝卜素的转基因水稻与OsCCD1_Ri、OsCCD4a_Ri或OsCCD4a_Ri独立纯合株系进行异花受粉来研究对于种子类胡萝卜素的影响。结果显示在OsCCD1_Ri和OsCCD4a_Ri株系中，种子的类胡萝卜素含量分别提高了1.4和1.6倍，且分别优先增加α-ring和β-ring类胡萝卜素，而叶片的类胡萝卜素含量在OsCCD4b_Ri中没有差异。另外，在大肠杆菌的OsCCD1、OsCCD4a和OsCCD4b过表达菌株中八氢番茄红素、番茄红素、β-胡萝卜素及玉米黄素四种类胡萝卜素的含量有所降低。OsCCD1和OsCCD4a具有相似的类胡萝卜素降解活性，OsCCD4b紧随其后，并且没有底物特异性。综上，阻断OsCCD4a基因的活性可能对于增强水稻种子胚乳及植株叶片中类胡萝卜素的积累具有潜在的实际应用价值。

doi: https://doi.org/10.1093/jxb/ery300

Journal: Journal of Experimental Botany

Published date: 14 August, 2018

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