Cloning and Functional Analysis of Cuticular Wax Synthesis Gene SlCER1-7 in Tomato

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

Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (7): 1733-1744.doi: 10.16420/j.issn.0513-353x.2023-0624

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Cloning and Functional Analysis of Cuticular Wax Synthesis Gene SlCER1-7 in Tomato

ZHANG Ruiyi¹, FENG Wenjia¹, LI Benben¹, SONG Yiying¹, WEI Mingyue¹, CHAI Guaiqiang¹^,²^,^*(), and HUO Yanbo¹^,²

¹ College of Advanced Agricultural Sciences，Yulin University，Yulin，Shaanxi 719000，China
² Shaanxi Key Laboratory of Ecological Restoration in Shaanbei Mining Area，Yulin，Shaanxi 719000，China

Received:2025-04-01 Revised:2025-05-21 Online:2025-07-23 Published:2025-07-23
Contact: CHAI Guaiqiang

Abstract

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

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.

Figures/Tables 9

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

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

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

Fig. 3 Phylogenetic analysis of SlCER1-7 in tomato

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

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

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

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

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）

References 37

[1]	Ainiwaer · Kadierya, Jia Yunxia, Wang Yan, Zhou Jiyang, Zhong Li. 2024. Ultrastructure and composition analysis of pericarp of six melon varieties with different storage characteristics. Food Science, 45 (17):199-205. (in Chinese)
	佧迪尔亚 · 艾尼瓦尔, 贾云霞, 王艳, 周继阳, 钟俐. 2024. 贮藏特性不同的6个甜瓜品种果皮超微结构与成分分析. 食品科学, 45 (17):199-205. doi: 10.7506/spkx1002-6630-20240109-083
[2]	Al-Abdallat A M, Al-Debei H S, Ayad J Y, Hasan S. 2014. Over-expression of SlSHN1 gene improves drought tolerance by increasing cuticular wax accumulation in tomato. International Journal Molecular Science, 15 (11):19499-19515.
[3]	Chai G Q, Li C L, Xu F, Li Y, Wang Y, Wang Z H. 2018. Three endoplasmic reticulum-associated fatty acyl-coenzyme a reductases were involved in the production of primary alcohols in hexaploid wheat(Triticum aestivum L.). BMC Plant Biology, 18 (1):35.
[4]	Chai Guaiqiang, Wei Mingyue, Feng Wenjia, Li Lin, Duan Yizhong, Fan Xifeng. 2022. Preliminary establishment of genetic transformation system for artificial spike bud blocks in switchgrass. Chinese Journal of Grassland, 44 (7):13-22. (in Chinese)
	柴乖强, 韦明月, 封文佳, 李琳, 段义忠, 范希峰. 2022. 柳枝稷人工穗芽块遗传转化体系的初步建立. 中国草地学报, 44 (7):13-22.
[5]	Chai Y, Li A, Chit Wai S, Song C, Zhao Y, Duan Y, Zhang B, Lin Q. 2020. Cuticular wax composition changes of 10 apple cultivars during postharvest storage. Food Chemistry,324:126903.
[6]	Chu W J, Gao H Y, Chen H J, Fang X J, Zheng Y H. 2018. Effects of cuticular wax on the postharvest quality of blueberry fruit. Food Chemistry,239:68-74.
[7]	Feng Wenjia, Han Ruirui, Li Jiali, Chai Guaiqiang. 2020. Cuticular wax composition determination and expression profiles of fatty alcohols synthesis gene in tomato. Journal of Yulin University, 30 (6):27-31. (in Chinese)
	封文佳, 韩瑞瑞, 李嘉丽, 柴乖强. 2020. 设施番茄表皮蜡质测定及脂肪醇合成基因的表达分析. 榆林学院学报, 30 (6):27-31.
[8]	Kimbara J, Yoshida M, Ito H, Kitagawa M, Takada W, Hayashi K, Shibutani Y, Kusano M, Okazaki Y, Nakabayashi R, Mori T, Saito K, Ariizumi T, Ezura H. 2013. Inhibition of CUTIN DEFICIENT 2 causes defects in cuticle function and structure and metabolite changes in tomato fruit. Plant Cell Physiology, 54 (9):1535-1548.
[9]	Lee S B, Suh M C. 2022. Regulatory mechanisms underlying cuticular wax biosynthesis. Journal of Experimental Botany, 73 (9):2799-2816. doi: 10.1093/jxb/erab509 pmid: 35560199
[10]	Li T, Sun Y, Liu T, Wu H, An P, Shui Z, Wang J, Zhu Y, Li C, Wang Y, Jetter R, Wang Z. 2019. TaCER1-1A is involved in cuticular wax alkane biosynthesis in hexaploid wheat and responds to plant abiotic stresses. Plant Cell and Environment, 42 (11):3077-3091.
[11]	Li M, Zhang C, Hou L, Liu X R, Zhao H, Pang X, Bo W, Li Y. 2024. Differences in leaf cuticular wax induced by whole-genome duplication in autotetraploid sour jujube. Horticultural Plant Journal, 10 (1):66-76.
[12]	Liu X, Gao S, Liu Y, Cao B, Chen Z, Xu K. 2020. Comparative analysis of the chemical composition and water permeability of the cuticular wax barrier in Welsh onion(Allium fistulosum L.). Protoplasma, 257 (3):833-840.
[13]	Liu Xianfeng, Fu Bojie. 2021. Drought impacts on crop yield:progress,challenges and prospect. Acta Geographica Sinica, 76 (11):2632-2646. (in Chinese) doi: 10.11821/dlxb202111003
	刘宪锋, 傅伯杰. 2021. 干旱对作物产量影响研究进展与展望. 地理学报, 76 (11):2632-2646. doi: 10.11821/dlxb202111003
[14]	Pan Z, Liu M, Zhao H, Tan Z, Liang K, Sun Q, Gong D, He H, Zhou W, Qiu F. 2020. ZmSRL5 is involved in drought tolerance by maintaining cuticular wax structure in maize. Journal of Integrative Plant Biology, 62 (12):1895-1909.
[15]	Sajeevan R S, Nataraja K N, Shivashankara K S, Pallavi N, Gurumurthy D S, Shivanna M B. 2017. Expression of Arabidopsis SHN1 in Indian mulberry(Morus indica L.) increases leaf surface wax content and reduces post-harvest water loss. Frontiers in Plant Science,8:418.
[16]	Samuels L, Kunst L, Jetter R. 2008. Sealing plant surfaces:cuticular wax formation by epidermal cells. Annual Review Plant Biology,59:683-707.
[17]	Shaheenuzzamn M, Shi S, Sohail K, Wu H Q, Liu T, An P, Wang Z, Hasanuzzaman M. 2021. Regulation of cuticular wax biosynthesis in plants under abiotic stress. Plant Biotechnology Reports,15:1-12.
[18]	Sun Y, Hegebarth D, Jetter R. 2021. Acyl-CoA desaturase ADS4. 2 is involved in the formation of characteristic wax alkenes in young Arabidopsis leaves. Plant Physiology, 186 (4):1812-1831.
[19]	Sun Yuyan. 2012. Improvement of drought and salt tolerance using Arabidopsisi BOUNTIFUL and HARDY gene and epicuticular wax synthesis genes in tomato[M. D. Dissertation]. Beijing: Chinese Academy of Agricultural Sciences. (in Chinese)
	孙玉燕. 2012. 利用拟南芥BOUNTIFUL和HARDY基因以及表皮蜡质合成基因提高番茄耐旱和耐盐性[硕士论文]. 北京: 中国农业科学院.
[20]	Tian R, Liu W, Wang Y, Wang W Q. 2024. Cuticular wax in wheat:biosynthesis,genetics and the stress response. Frontiers in Plant Science,15:1498505.
[21]	Usman M, Ahmad N, Raza W, Zhao Z, Abubakar M, Rehman S, Ikram S, Tariq H. 2023. Impact of biochar on the yield and nutritional quality of tomatoes(Solanum lycopersicum)under drought stress. Journal of the Science Food and Agriculture, 103 (7):3479-3488.
[22]	Vogg G, Fischer S, Leide J, Emmanuel E, Jetter R, Levy AA, Riederer M. 2004. Tomato fruit cuticular waxes and their effects on transpiration barrier properties:functional characterization of a mutant deficient in a very-long-chain fatty acid β-ketoacyl-CoA synthase. Journal of Experimental Botany, 55 (401):1401-1410.
[23]	Wang W, Zhang Y, Xu C, Ren J, Liu X, Black K, Gai X, Wang Q, Ren H. 2015. Cucumber ECERIFERUM1(CsCER1),which influences the cuticle properties and drought tolerance of cucumber,plays a key role in VLC alkanes biosynthesis. Plant Molecular Biology, 87 (3):219-233.
[24]	Wang Wenjiao, Xing Junjie, Shen Chengcheng, Li Bin. 2024. Screening and functional analysis of CsCOL5,the upstream regulator of CsCER1,in cucumber pericarp wax synthesis. Acta Horticulturae Sinica, 51 (5):1005-1016. (in Chinese)
	王文娇, 邢军杰, 申成丞, 李斌. 2024. 黄瓜蜡质基因CsCER1调控因子CsCOL5的筛选及其功能分析. 园艺学报, 51 (5):1005-1016.
[25]	Wang Z, Guhling O, Yao R, Li F, Yeats T H, Rose J K, Jetter R. 2011. Two oxidosqualene cyclases responsible for biosynthesis of tomato fruit cuticular triterpenoids. Plant Physiology, 155 (1):540-552. doi: 10.1104/pp.110.162883 pmid: 21059824
[26]	Wei Mingyue, Guo Xiaowen, Qiu Jiaxin, Song Yiying, Chai Guaiqiang, Duan Yizhong. 2023. Preliminary functional analysis of β- ketoacyl-CoA synthase CsKCS11gene from Cucumis sativus L. China Cucurbits and Vegetables, 36 (10):23-31. (in Chinese)
	韦明月, 郭孝文, 邱嘉鑫, 宋怡颖, 柴乖强, 段义忠. 2023. 黄瓜β-酮脂酰辅酶A合成酶基因CsKCS11的功能初步分析. 中国瓜菜, 36 (10):23-31.
[27]	Wu H, Liu L, Chen Y, Liu T, Jiang Q, Wei Z, Li C, Wang Z. 2022. Tomato SlCER1-1catalyzes the synthesis of wax alkanes which increases the drought tolerance and fruit storability. Horticulture Research, 11 (9):uhac004.
[28]	Wu Hongqi, Luo Wenqiao, Zhao Shuai, Wang Cong, Ni Zhen, Zhu Hao, Niu Yanlu, Wang Yong, Wang Zhonghua, Quan Li. 2017. Effect of drought stress on wax accumulation in leaves of tomato. Journal of Northwest A & F University(Nat. Sci. Ed.), 45 (7):73-80. (in Chinese)
	吴洪启, 罗文巧, 赵帅, 王聪, 倪震, 朱浩, 牛艳璐, 汪勇, 王中华, 权力. 2017. 干旱胁迫对番茄叶片蜡质积累的影响. 西北农林科技大学学报(自然科学版), 45 (7):73-80.
[29]	Xiong C, Xie Q M, Yang Q H, Sun P Y, Gao S H, Li H X, Zhang J H, Wang T T, Ye Z B, Yang C X. 2020. WOOLLY,interacting with MYB transcription factor MYB31,regulates cuticular wax biosynthesis by modulating CER6 expression in tomato. The Plant Journal, 103 (1):323-337.
[30]	Xu Chengxiang, Zheng Fuqing, Ma Yanping, Zhang Shaoping, Chen Xiaoting, Ye Simin. 2021. Effect of storage temperature on peel wax content and chemical composition of citrus cultivars with different storability. Food Science, 42 (13):223-232. (in Chinese) doi: 10.7506/spkx1002-6630-20190617-167
	徐呈祥, 郑福庆, 马艳萍, 张少平, 陈小婷, 叶思敏. 2021. 贮藏温度对耐贮性不同的柑橘品种果皮蜡质含量及其化学组成的影响. 食品科学, 42 (13):223-232.
[31]	Yang H B, Zhu Z F, Zhang M F, Li X, Xu R W, Zhu F, Xu J, Deng X X, Cheng Y J. 2022. QTL analysis reveals reduction of fruit water loss by NAC 042 through regulation of cuticular wax synthesis in citrus fruit. Horticultural Plant Journal, 8 (6):737-746.
[32]	Zeng Qiong, Wu Qi, Wang Yuechen, Liu Dechun, Liu Shanbei, Liu Chuanfu, Liu Yong. 2013. A study on fruit storability of glossy mutant of“newhall”navel orange. Acta Agriculturae Universitatis Jiangxiensis, 35 (3):525-529. (in Chinese)
	曾琼, 吴启, 王玥辰, 刘德春, 刘山蓓, 刘传福, 刘勇. 2013. 纽荷尔脐橙果皮光泽型突变体贮藏性研究. 江西农业大学学报, 35 (3):525-529.
[33]	Zhang Y L, You C X, Li Y Y, Hao Y J. 2020. Advances in biosynthesis,regulation,and function of apple cuticular wax. Frontiers in Plant Science,11:1165.
[34]	Zhou L, Ni E, Yang J, Zhou H, Liang H, Li J, Jiang D, Wang Z, Liu Z, Zhuang C. 2013. Rice OsGL1- 6 is involved in leaf cuticular wax accumulation and drought resistance. PLoS ONE, 8 (5):e65139.
[35]	Zhu X, Xiong L. 2013. Putative megaenzyme DWA1 plays essential roles in drought resistance by regulating stress-induced wax deposition in rice. Proceedings of the National Academy of Sciences of the United States of America, 110 (44):17790-17795.
[36]	Wei Shuwei, Wang Shaomin, Wang Hongwei, Ran Kun, Dong Ran, Dong Xiaochang, Zhang Yong. 2021. Effects of Calcium treatment on microstructure,waxy components and aroma emission of‘Nanguoli’pear. Acta Horticulturae Sinica, 48 (6):1197-1207. (in Chinese) doi: 10.16420/j.issn.0513-353x.2020-0413
	魏树伟, 王少敏, 王宏伟, 冉昆, 董冉, 董肖昌, 张勇. 2021. 钙处理对‘南果梨’果皮微观结构、蜡质成分及香气释放的影响. 园艺学报, 48 (6):1197-1207. doi: 10.16420/j.issn.0513-353x.2020-0413
[37]	Wei Xiaoyu, Wang Yuejin. 2022. Correlation between anatomical structure and resistance to powdery mildew in Chinese wild Vitis species. Acta Horticulturae Sinica, 49 (6):1200-1212. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0367
	魏晓羽, 王跃进. 2022. 中国野生葡萄果皮解剖结构与白粉病抗性的相关性研究. 园艺学报, 49 (6):1200-1212. doi: 10.16420/j.issn.0513-353x.2021-0367

Cloning and Functional Analysis of Cuticular Wax Synthesis Gene SlCER1-7 in Tomato

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