Abstract

Carotenoids are essential participants in photosynthesis and photo protection as well as growth, development, and stress response in plants. Phytoene desaturase (PDS; EC 1.3.5.5) is an enzyme that catalyzes the first stage of desaturation of 15-cis-phytoene (a precursor to all carotenoids). In this work, we examined for the first time the effect of PDS knockout in the genome of Nicotiana tabacum L. using the CRISPR-Cas9 system on activity of downstream genes involved in biosynthesis of carotenoids. Nine transgenic lines of tobacco were obtained with a mosaic editing of gene NtPDS; three versions of indels (350^351→ins^g#, 350^351→ins^t#, and t351→del(1nt)#) in the exon II resulting in the synthesis of a shortened non-functional version of the protein. The lines were characterized by spotted, green-white pigmentation of the leaves, altered flowering time and morphology as well as a reduced content of carotenoids and chlorophylls in the leaf tissue. A rise in the level of transcripts of phytoene synthase gene NtPSY2 was shown in late-flowering lines as compared to control material. Edited line L29 with the latest flowering showed a considerable increase in the level of transcripts of downstream structural genes of carotenogenesis. A reduction in the carotenoid content in the leaves with mosaic editing was accompanied by a decrease in the level of expression of MADS-box gene NtSEP1, the product of which presumably participates in regulation of transcription of the carotenoid biosynthesis genes. The obtained results may be used for further studies of regulation of biosynthesis of carotenoids and apocarotenoids in Solanaceae.

中文翻译：

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八氢番茄红素去饱和酶基因 NtPDS 嵌合敲除对烟草类胡萝卜素生物合成的影响。

摘要

类胡萝卜素是植物光合作用、光保护以及生长、发育和应激反应的重要参与者。八氢番茄红素去饱和酶（PDS；EC 1.3.5.5）是一种催化 15-顺式八氢番茄红素（所有类胡萝卜素的前体）去饱和第一阶段的酶。在这项工作中，我们首次利用 CRISPR-Cas9 系统检测了烟草基因组中PDS敲除对参与类胡萝卜素生物合成的下游基因活性的影响。通过NtPDS基因嵌合编辑获得了9个烟草转基因品系; 外显子 II 中的三个版本的插入缺失（350^351→ins^g#、350^351→ins^t# 和 t351→del(1nt)#）导致合成缩短的非功能性蛋白质版本。这些品系的特征是叶子出现斑点、绿白色色素沉着、开花时间和形态改变以及叶组织中类胡萝卜素和叶绿素含量降低。与对照材料相比，晚花品系中八氢番茄红素合酶基因NtPSY2的转录物水平有所上升。具有最晚开花的编辑品系L29显示胡萝卜素生成的下游结构基因的转录物水平显着增加。镶嵌编辑后叶片中类胡萝卜素含量的减少伴随着 MADS-box 基因表达水平的降低NtSEP1，其产物可能参与类胡萝卜素生物合成基因的转录调节。所得结果可用于茄科类胡萝卜素和类胡萝卜素生物合成调控的进一步研究。