关键词: 芹菜, 类胡萝卜素裂解双加氧酶基因, 亚细胞定位, 基因表达, 类胡萝卜素

Abstract:

【Objective】 The genotype and relative expression level of carotenoid cleavage dioxygenases gene AgCCD4 in celery with different colors were revealed, and the roles of AgCCD4 gene on carotenoids accumulation in celery tissues were preliminarily analyzed combined with the corresponding carotenoids level, which laid a foundation for further study on the roles of CCD subfamily genes in the pigmentation of different colored celery tissues. 【Method】 The CCD family gene, AgCCD4, was obtained by homology search from genome of celery, and cloned from celery cvs. Jinnan Shiqin, Huangtaiji, Zigan NO.1, and Saixue, respectively. The composition of amino acids, physicochemical properties, genetic relationship, and spatial structure of proteins were analyzed by bioinformatics. The conservative domain and secondary structure were predicted and the tertiary structure model was established. Real-time quantitative PCR (RT-qPCR) was used to detect the expression levels of AgCCD4 in different tissues of celery with different colors. The contents of lutein and β-carotene of leaf blades, petioles, and roots of celery were determined by ultra-performance liquid chromatography (UPLC). The subcellular localization of AgCCD4 protein in tobacco epidermal cells was performed by using Agrobacterium-mediated transient expression system. 【Result】 Sequence analysis results showed that AgCCD4 contained an open reading frame (ORF) with the length of 1 779 bp, encoding 592 amino acids. A total of 18 nucleotides and 9 amino acids sites of AgCCD4s differed in the Saixue and other three celery varieties. The relative molecular weights of AgCCD4 from Saixue and other three celeries were 65.07 and 65.12 kD, and the theoretical pIs were 6.03 and 5.95, respectively. Phylogenetic analysis demonstrated that the CCD4 in celery had the closest genetic relationship with sunflower and lettuce from Compositae family. The secondary structure of AgCCD4 contained multiple α helices and random coils, and the tertiary structure was mainly composed of β strands. Lutein and β-carotene were not detected in celery roots. The contents of lutein and β-carotene in the leaf blades were the highest in Jinnan Shiqin (1 102.58 μg∙g^-1 DW and 241.92 μg∙g^-1 DW, resptectivey), whereas the lowest in Zigan NO.1 (57.12 μg∙g^-1 DW and 45.65 μg∙g^-1 DW, respectively). In petioles, β-carotene was detected only in Huangtaiji, lutein only existed in Jinnan Shiqin and Huangtaiji. The expression level of AgCCD4 was highest in celery leaf blades and lowest in roots, respectively. In leaf blades, the relative expression levels of AgCCD4 in Zigan NO.1 shared similar rhythm change with that Saixue, which were significantly higher than that in others. 【Conclusion】 In this study, AgCCD4 gene was cloned from four celery varieties respectively, while the gene sequence of Saixue was different with other three varieties. AgCCD4 protein contained a RPE65 conserved domain. The expression of AgCCD4 was remarkably varied in different celery tissues and was negatively correlated with the carotenoids content. The content and types of carotenoids affected the plants’ color, and AgCCD4 might regulate the pigmentation of celery tissues by degrading carotenoids.

Key words: Apium graveolens, carotenoid cleavage dioxygenases gene, subcellular localization, gene expression, carotenoids