番茄表皮蜡质合成相关基因SlCER1-7的克隆与功能验证

doi:10.16420/j.issn.0513-353x.2023-0624

摘要/Abstract

摘要： 利用生物信息分析和表达分析、转基因等方法研究了番茄果实表皮蜡质合成相关基因SlCER1-7的功能。结果表明，SlCER1-7序列全长为2 078 bp，开放阅读框为1 884 bp，编码627个氨基酸；SlCER1-7为亲水性蛋白；同源比对和系统进化分析表明SlCER1-7与茄子CER1的亲缘关系最近。SlCER1-7在番茄成熟叶、绿果皮、黄果皮和红果皮中均有较高的表达水平，而在幼叶中的表达量较低。在番茄中过表达SlCER1-7，果实总蜡质含量增高，并以直链C33烷烃和支链iso-C32烷烃增加最明显；保水性测定表明SlCER1-7转基因番茄果实的保水性增强。

关键词: 番茄, 表皮蜡质, CER1, 基因克隆, 保水性

Abstract:

The bioinformatics，expression analysis and transgenic technology were used to reveal the biological function of SlCER1-7，the results showed that SlCER1-7 had a total length of 2 078 bp and contained a complete open reading frame of 1 884 bp，encoding 627 amino acids. Bioinformatics analysis showed that SlCER1-7 was a hydrophilic protein. Homology alignment and phylogenetic analysis showed that SlCER1-7 was closely related to CER1 in eggplant. Meanwhile，SLCER1-7 was mainly expressed in mature leaves，green pericarp，yellow pericarp and red pericarp，but not in young leaves. Finally，the overexpression transgenic lines were obtained by Agrobacterium-mediated transformation，the results showed that the total wax content of transgenic tomato fruits were significantly increased，especially the straight chain C33 alkanes and the branched chain C32 alkanes，and the water-holding capacity of transgenic fruits were enhanced.

Key words: tomato, cuticular wax, CER1, gene cloning, water retention

张瑞益, 封文佳, 李奔奔, 宋怡颖, 韦明月, 柴乖强, 霍彦波. 番茄表皮蜡质合成相关基因SlCER1-7的克隆与功能验证[J]. 园艺学报, 2025, 52(7): 1733-1744.

ZHANG Ruiyi, FENG Wenjia, LI Benben, SONG Yiying, WEI Mingyue, CHAI Guaiqiang, and HUO Yanbo. Cloning and Functional Analysis of Cuticular Wax Synthesis Gene SlCER1-7 in Tomato[J]. Acta Horticulturae Sinica, 2025, 52(7): 1733-1744.

图/表 9

表1 基因表达分析所使用的引物

Table 1 List of primers used for expression analysis

用途 Usage	引物名称 Primer name	引物序列（5′-3′） Primer sequence
PCR扩增目标基因Gene amplification	SlCER1-7-PCR	F：GCTTAAGGATGGCTTCTAAAC；R：CTGGAGCATATTGAAGGATGA
转基因植株检测 Molecular identification of transgenic plants	RT-SlCER1-7	F：CTTGAAGAAGGTGGATGAACTC；R：CATGGTCTCATCACCATTACC
潮霉素抗性基因Hygromycin marker gene	Hpt	F：CGAGTACTTCTACACAGCCAT；R：ACAGCGTCTCCGACCTGATGC
内参基因Internal control gene	SlACTIN	F：CCATCCTCCGTCTTGACCTT；R：ACTTGCCCATCGGGTAATTC
荧光定量PCR Quantitative real-time PCR	Q-PCR-SlCER1-7	F：ATCCATTATGGAGGCAGATAAC；R：GCAATGGAGTAAGCAACTTTGGAC

图1 番茄SlCER1-7（Solyc01g088400.3）的表达分析 A：SlCRE1相关基因表达热图；B：SlCER1-7在番茄不同组织中的表达。使用Duncan’s法分析差异显著性，不同小写字母表示在不同的组织中差异显著（P < 0.05）。绿果皮、黄果皮和红果皮分别指：未成熟果实（绿色）、刚转色成熟果实（橙黄色）和完全成熟果实（红色）的果皮。下同

Fig. 1 Expression analysis of SlCER1-7 in tomato A：Heat map of SlCER1 genes in tomato；B：Expression analysis of SlCER1-7 gene in different tissues of tomato. Significance of differences was analyzed using the Duncan’s method. Different lowercase letters represent significance difference among the the different tissues（P < 0.05）. Green pericarp，yellow pericarp and red pericarp refer to the pericarp of immature fruits（green），just-turned-ripe fruits（orange-yellow）and fully ripe fruits（red），respectively. The same below

图2 番茄SlCER1-7的扩增（A）和阳性单克隆鉴定（B） Marker：DL 2000；1 ~ 12：12个阳性单克隆

Fig. 2 Cloning of SlCER1-7 in tomato（A）and the positive clone identification（B） Marker：DL 2000；1-12：12 positive clone strains

图3 番茄SlCER1-7的系统进化分析

Fig. 3 Phylogenetic analysis of SlCER1-7 in tomato

图4 SlCER1-7转基因番茄的分子鉴定 A：潮霉素标记基因的PCR扩增；B：SlCER1-7的PCR扩增；C：转基因株系中SlCER1-7的表达。+：阳性对照；-：阴性对照；WT：野生型；1 ~ 10、T5-2、T7-6、T9-1、T10-8：转基因再生植株。下同

Fig. 4 Molecular characterization of transgenic SlCER1-7 gene plants in tomato seedlings A：PCR analysis of Hygromycin marker gene from transgenic seedlings；B：PCR analysis of SlCER1-7 from transgenic seedlings； C：SlCER1-7 expression in transgenic lines. +：Positive control；-：Negative control；WT：Wild type；1-10，T5-2，T7-6，T9-1 and T10-8：Transgenic plants. The same below

图5 转SlCER1-7番茄果实表皮蜡质总量（A）及其成分（B）

Fig. 5 Analysis of total wax content（A）and cuticular wax components（B）in SlCER1-7 transgenic tomato

图6 转SlCER1-7番茄果实表皮蜡质成分烷烃的链长分布

Fig. 6 Chain length distribution of alkanes in tomato lines expressing SlCER1-7

图7 转SlCER1-7基因番茄株系T5-2和T7-6果实表皮蜡质晶体结构分析

Fig. 7 The epidermal wax crystal structure of the tomato fruit in SlCER1-7 transgenic lines T5-2 and T7-6

图8 SlCER1-7转基因番茄果实失水处理后表型（A）和失水率（B） t-Test，*表示野生型与转基因株系间差异显著（P < 0.05）

Fig. 8 Phenotype（A）and water loss rate（B）of transgenic SlCER1-7 tomato fruits after water loss treatment t-Test，* indicates significant difference among the wildtype plants and transgenic lines（P < 0.05）

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