类胡萝卜素裂解双加氧酶基因AgCCD4调控芹菜不同组织的着色

doi:10.3864/j.issn.0578-1752.2021.15.012

Abstract

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

WANG Hao,YIN Lian,LIU JieXia,JIA LiLi,DING Xu,SHEN Di,FENG Kai,XU ZhiSheng,XIONG AiSheng. The Carotenoid Cleavage Dioxygenases Gene AgCCD4 Regulates the Pigmentation of Celery Tissues with Different Colors[J].Scientia Agricultura Sinica, 2021, 54(15): 3279-3294.

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顺式元件 cis-element	序列 Sequence	元件数量 Number of cis-element	功能 Function
ACE	CTAACGTATT	1	光调控元件 Cis-acting element involved in light responsiveness
ARE	AAACCA	1	厌氧诱导必需顺式作用元件 Cis-acting regulatory element essential for the anaerobic induction
Box 4	ATTAAT	2	光应答部分保守DNA模块 Part of a conserved DNA module involved in light responsiveness
CAAT-box	CAAT/CCAAT/CAAAT	42	启动子和增强子区域的共同顺式作用元件 Common cis-acting element in promoter and enhancer regions
ERE	ATTTTAAA	6	乙烯应答元件 Ethylene responsive element
F-box	CTATTCTCATT	1	赤霉素响应元件 Gibberellin response element
GT1-motif	GGTTAA	1	光响应要素 Light responsive element
RY-element	CATGCATG	1	顺式作用的调控要素涉及种子特异性调控 Cis-acting regulatory element involved in seed-specific regulation
STRE	AGGGG	3	渗透压胁迫应答元件 Osmotic stress response element
TC-rich repeats	GTTTTCTTAC	1	顺式作用元素参与防御和应激反应 Cis-acting element involved in defense and stress responsiveness
TCT-motif	TCTTAC	3	光响应元件的一部分Part of a light responsive element
WUN-motif	AAATTACT	1	创伤诱导响应元件 Trauma induced response element

植物 Plant	氨基酸数Number of amino acid	理论分子质量 Relative molecular mass (kD)	等电点 pI	氨基酸比例 Ratio of amino acid (%)				总平均疏水性Grand average of hydrophobicity
植物 Plant	氨基酸数Number of amino acid	理论分子质量 Relative molecular mass (kD)	等电点 pI	脂肪族Aliphatic	芳香族Aromatic	酸性Positive	碱性Negative	总平均疏水性Grand average of hydrophobicity
芹菜 Apium graveolens
赛雪 Saixue	592	65.07	6.03	19	10	12	11	-0.269
津南实芹 Jinnan Shiqin	592	65.12	5.95	19	10	12	11	-0.297
胡萝卜 Daucus carota	588	64.68	6.21	19	10	12	11	-0.284
哥伦比亚锦葵 Herrania umbratica	606	66.72	6.55	21	9	12	11	-0.224
矮牵牛 Petunia hybrida	603	66.01	6.25	20	10	12	11	-0.223
烟草 Nicotiana tabacum	601	65.99	7.16	20	10	13	11	-0.218
枸杞 Lycium chinense	599	65.71	6.34	20	9	13	11	-0.252
欧洲甜樱桃 Prunus avium	597	65.70	6.21	20	10	12	11	-0.238
银白杨 Populus alba	611	66.64	6.26	21	9	12	11	-0.189
黄连木 Pistacia vera	616	67.69	6.44	21	9	12	10	-0.176
甘薯 Ipomoea batatas	594	67.66	5.76	21	9	12	11	-0.171
中华辣椒 Capsicum chinense	603	66.04	6.34	21	9	12	11	-0.248
杨梅 Morella rubra	623	68.40	6.72	20	9	13	11	-0.259
向日葵 Helianthus annuus	592	64.80	5.70	20	10	12	12	-0.155
莴苣 Lactuca sativa	592	65.36	6.05	19	10	13	11	-0.263
山杜鹃 Rhododendron kaempferi	600	65.65	6.17	20	10	12	11	-0.187
克莱门柚 Citrus clementina	603	66.45	6.87	21	9	13	11	-0.236
芜菁 Brassica rapa	595	65.60	6.19	21	9	13	12	-0.230
盐芥 Eutrema salsugineum	602	66.41	7.02	21	8	14	11	-0.255
蓖麻 Ricinus communis	618	68.07	6.85	21	9	12	11	-0.133

The Carotenoid Cleavage Dioxygenases Gene AgCCD4 Regulates the Pigmentation of Celery Tissues with Different Colors

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