韭菜S-烃基半胱氨酸亚砜合成与分解代谢关键基因筛选

doi:10.16420/j.issn.0513-353x.2024-0940

摘要/Abstract

摘要：

为了进一步丰富韭菜基因组信息，挖掘韭菜S-烃基半胱氨酸亚砜[S-alk（en）yl-L-cysteine sulfoxides，CSOs]合成代谢相关基因，测定50份韭菜种质资源的S-烯丙基半胱氨酸亚砜（S-allyl-L- cysteine sulfide，ACSO）、S-甲基半胱氨酸亚砜（S-methyl-L-cysteine sulfide，MCSO）和S-丙烯基半胱氨酸亚砜（S-propenyl-L-cysteine sulfide，PeCSO）3种S-烃基半胱氨酸亚砜的含量，筛选出S-烃基半胱氨酸亚砜总含量最高的韭菜品种‘黄格子’和最低的‘哈密钩韭’。通过转录组测序比较两个品种基因表达的差异性，筛选与S-烃基半胱氨酸亚砜合成和代谢相关的差异基因。结果显示，转录组数据共筛选出差异表达基因11 493个，其中上调的有6 203个，下调的有5 290个。GO数据库富集到S-烃基半胱氨酸亚砜生物合成相关的硫代谢通路。KEGG分析共富集到122条通路，其中与S-烃基半胱氨酸亚砜合成代谢相关的有硫代谢、半胱氨酸代谢、谷胱甘肽代谢通路。进一步通过基因注释得到参与CSO合成和代谢的关键基因51个，其中4个含黄素单加氧酶基因FMOs、2个γ-谷氨酰转肽酶基因GGT和7个蒜氨酸酶基因Alliinase均在‘黄格子’中高表达，推测其在调控韭菜S-烃基半胱氨酸亚砜的合成与分解代谢中发挥重要作用。

关键词: 韭菜, 转录组, S-烃基半胱氨酸亚砜, 合成与分解代谢

Abstract:

The aim of this study was to further enrich the genomic information and uncover genes related to the synthesis and catabolism of S-alk（en）yl-L-cysteine sulfoxides（CSOs）. In the study，the content of three types of CSOs，namely S-allyl-L-cysteine sulfide（ACSO），S-methyl-L-cysteine sulfide（MCSO）， and S-propenyl-L-cysteine sulfide（PeCSO），was measured in 50 Chinese chives germplasm resources. Based on these result，two cultivars were selected for further analysis：‘Huanggezi’（HGZ），which exhibited the highest S-alk（en）yl-L-cysteine sulfoxide content，and‘Hami Goujiu’（HMGJ），which had the lowest S-alk（en）yl-L-cysteine sulfoxide content. Then，transcriptome sequencing was performed to compare gene expression differences between the two cultivars，and genes associated with the synthesis and catabolism of S-alk（en）yl-L-cysteine sulfoxides were identified. A total of 11 493 differentially expressed genes（DEGs）were identified，with 6 203 up-regulated，and 5 290 down-regulated. Gene Ontology（GO）enrichment analysis revealed that sulfur metabolic pathways related to S-alk（en）yl-L-cysteine sulfoxide biosynthesis were significantly enriched. Additionally，KEGG pathway analysis identified 122 enriched pathways，including those involved in sulfur metabolism，cysteine metabolism，and glutathione metabolism，which are closely associated with the synthesis and metabolism of S-alk（en）yl-L-cysteine sulfoxides. Through gene annotation，51 key genes involved in CSOs synthesis and catabolism were obtained. Among these，four flavin monooxygenase genes（AtuFMOs），two gamma glutamyl transpeptidase genes（AtuGGT），and seven alliinase genes（AtuALL）were found to be highly expressed in the‘Huanggezi’cultivar. These findings suggest that these genes may play a crucial role in regulating the synthesis and degradation of CSOs in Chinese chive.

Key words: Chinese chive, transcriptome, S-alk（en）yl-L-cysteine sulfoxides, syntheis and catabolism

赵超胜, 陈梦然, 郑永琪, 李菊, 马培芳, 肖雪梅, 郁继华. 韭菜S-烃基半胱氨酸亚砜合成与分解代谢关键基因筛选[J]. 园艺学报, 2025, 52(12): 3257-3270.

ZHAO Chaosheng, CHEN Mengran, ZHENG Yongqi, LI Ju, MA Peifang, XIAO Xuemei, YU Jihua. Screening of Key Genes Involved in the Synthesis and Catabolism of S-Alk（en）yl-L-cysteine Sulfoxide in Chinese Chive[J]. Acta Horticulturae Sinica, 2025, 52(12): 3257-3270.

图/表 6

表1 qRT-PCR所用引物序列

Table 1 Primer sequence for qRT-PCR

基因编号Gene ID	正向引物（5′-3′）Forward primer	反向引物（5′-3′）Reverse primer
Cluster-28231.30244	CCTACAGCCTGCTTACCCAAGTC	TCCTCCACCAAAGCCTTATCAGTAG
Cluster-28231.33055	CCCAGAGACCTTTGCCAAGAACC	ACCACACAGCCTCACCGAATTTG
Cluster-28231.63396	TGTATCCATGAAGCTCATCCCAGTG	TCAACAAGCCTTCCCATAACATTGC
Cluster-28231.76095	ATTTGGCATGGACTCTCGTTGTTTG	CCTGAGGATAATGAGTGGCGACATAG
Cluster-28231.77842	GCTGCACATGACCATCCAAA	ACCACCAGTACTCCTGCAAA
Cluster-28231.78451	GCTGTGAAGTGCATCGAGAGAGG	CGATGGCTAAATGCGTTGGTGTC
Cluster-28231.102399	TGTTTGTGGCGATCAGCAGAATTG	TGGGAAGAGGGAAGCTAGGTTGTC
Cluster-28231.85523	ATCCACAACCAACGCCTACTCAAC	CTTTGGGAACGAGACCTGTGAGC
Cluster-28231.4568	CTCGGCATGGTCACAGATGAAGC	TCCGCCAAGGTGTAGTCGTCTC
Cluster-28231.25081	CGACGCCTTGGAATCCGAGTATC	CCGCCGTCTTGTTTCCTCTGTC
Cluster-28231.116929	CGAGGAAGTCCAAAGTGTTAAGAGTG	ACCAGCTTGTTATGTTCAGGCATTAC

图1 两个品种韭菜差异基因火山图

Fig. 1 Volcanic diagram of differential expressed genes between two cultivars of Chinese chives

图2 两个品种韭菜差异表达基因的GO功能注释

Fig. 2 GO functional annotation of differentially expressed genes between two cultivars of Chinese chives

图3 两个品种韭菜差异基因的KEGG富集图

Fig. 3 KEGG enrichment map of differentially expressed genes between two cultivars of Chinese chives

图4 S-烃基半胱氨酸亚砜合成与分解代谢通路模式图 HMGJ：哈密钩韭；HGZ：黄格子。虚线表示推测过程。下同

Fig. 4 Schematic diagram of S-alk（en）ylcysteine sulfoxide synthesis and catabolism pathway HMGJ：Hami Goujiu；HGZ：Huanggezi。The dashed line represents the inference process. The same below

图5 11个差异表达基因的qRT-PCR验证

Fig. 5 qRT-PCR validation of 11 differentially expressed genes

参考文献 20

[21]	Mehra R, Jasrotia R S, Mahajan A, Sharma D, Iquebal M A, Kaul S, Dhar M K. 2020. Transcriptome analysis of snow mountain garlic for unraveling the organosulfur metabolic pathway. Genomics, 112(1):99-107.
[22]	Novick R M, Elfarra A A. 2008. Purification and characterization of flavin-containing monooxygenase isoform 3 from rat kidney microsomes. Drug Metabolism and Disposition:the Biological Fate of Chemicals, 36(12):2468-2474.
[23]	Ru Hua, Wang Yuxin, Feng Ling, Wang Pingzhi, Song Weitang, Ma Jinhai, Yang Xiaofei. 2022. Analysis of functional bioactive components in Chinese chive seeds. Food Research and Development, 43(5):58-63. (in Chinese)
	茹桦, 王宇欣, 封玲, 王平智, 宋卫堂, 马金海, 杨校飞. 2022. 韭菜籽中功效生物活性成分分析. 食品研究与开发, 43(5):58-63.
[24]	Sun X D, Zhu S Y, Li N Y, Cheng Y, Zhao J, Qiao X G, Lu L, Liu S Q, Wang Y Z, Liu C, Li B P, Guo W, Gao S, Yang Z M, Li F, Zeng Z, Tang Q, Pan Y P, Guan M J, Zhao J, Lu X M, Meng H W, Han Z L, Gao C S, Jiang W K, Zhao X, Tian S L, Su J G, Cheng Z H, Liu T M. 2020. A chromosome-level genome assembly of garlic(Allium sativum)provides insights into genome evolution and allicin biosynthesis. Molecular Plant, 13(9):1328-1339.
[25]	Tian Xiaozhi, Xu Longxiao, Song Jianfei, Xun Mi, Zhang Weiwei, Yang Hongqiang. 2021. Effects of rice husk biochar on enzyme of sulfur assimilation,H₂S content androot architecture of apple trees in compacted soil. Journal of Plant Nutrition and Fertilizers, 27(5):869-877. (in Chinese)
	田孝志, 徐龙晓, 宋建飞, 荀咪, 张玮玮, 杨洪强. 2021. 稻壳炭对紧实土壤中苹果根系硫同化酶活性、H₂S含量及根系构型的影响. 植物营养与肥料学报, 27(5):869-877.
[26]	Wang Yanding, Wang Huan, Li Nana, Wang Lu, Hao Xinyuan, Wang Yuchun, Ding Changqing, Yang Yajun, Wang Xinchao, Qian Wenjun. 2021. Identification and expression analysis of glucose-6-hosphate dehydrogenase gene(CsG6PDHs)in Camellia sinensis. Journal of Tea Science, 41(4):497-510. (in Chinese)
	王彦丁, 王缓, 李娜娜, 王璐, 郝心愿, 王玉春, 丁长庆, 杨亚军, 王新超, 钱文俊. 2021. 茶树中葡萄糖-6-磷酸脱氢酶基因(CsG6PDHs)的克隆与表达分析. 茶叶科学, 41(4):497-510.
[27]	Wu Jiaying, Gao Yuzhong, Wan Wen, Shi Jingjing, Xu Chunyan, Ma Deliang, Su Yiren, Yang Xuqin. 2024. AsNAC570 gene cloning analysis and regulates alliin biosynthesis preliminary exploration. Molecular Plant Breeding, https://kns.cnki.net/kcms/detail/46.1068.S.20220715.1426.004.html. (in Chinese)
	吴家莹, 高玉忠, 宛雯, 师静静, 徐春艳, 马德良, 苏奕人, 杨绪勤. 2024. AsNAC570基因的克隆分析与调控蒜氨酸合成的初步探究. 分子植物育种, https://kns.cnki.net/kcms/detail/46.1068.S.20220715.1426.004.html.
[28]	Wu Wenhui, Xu Yang, Zhang Yadi, Yu Zhengjiao, Pang Yumeng, Qin Lei, Wang Yong. 2024. Cloning and functional analysis of AcFMO from onion during alliine biosynthesis. Chinese Journal of Biotechnology, 40(4):1076-1088. (in Chinese)
	吴文卉, 徐洋, 张雅迪, 于郑娇, 庞雨濛, 秦蕾, 王勇. 2024. 洋葱AcFMO克隆及其在蒜氨酸生物合成过程中的功能分析. 生物工程学报, 40(4):1076-1088.
[29]	Xia S W, Hang L F, Siyad A, Xu X Y, Liu Y J, Yan Q Q, Luo Q Y, Li Y, Lin L J, Li H X, Zhang X A, Huang L K, Ma X, Lai Y S. 2022. Biosynthesis and metabolism of garlic odor compounds in cultivated Chinese chives(Allium tuberosum)and wild Chinese chives(Allium hookeri). International Journal of Molecular Sciences, 23(13):7013.
[30]	Xu Huanhuan, Li Yi, Gao Wei, Wang Yongqin, Liu Lecheng. 2021. Cloning and identification of γ-glutamyl transpeptidase AcGGT gene from onion(Allium cepa). Scientia Agricultura Sinica, 54(19):4169-4178. (in Chinese)
	徐欢欢, 李逸, 高伟, 王永勤, 刘乐承. 2021. 洋葱γ-谷氨酰转肽酶AcGGT的克隆与鉴定. 中国农业科学, 54(19):4169-4178.
[31]	Xu Zhen, Yan Yongzhe, Lu Gang, Jin Qianhuan, Guo Deping. 2007. The biosynthetic pathways of flavor precursors and its control in Alliums. Chinese Journal of Cell Biology,(4):508-512. (in Chinese)
	许真, 严永哲, 卢钢, 金千焕, 郭得平. 2007. 葱属蔬菜植物风味前体物质的合成途径及调节机制. 细胞生物学杂志,(4):508-512.
[32]	Yamazaki Y, Iwasaki K, Mikami M, Yagihashi A. 2011. Distribution of eleven flavor precursors,S-alk(en)yl-L-cysteine derivatives,in seven Allium vegetables. Food Science and Technology Research, 17(1):55-62.
[33]	Yoo K S, Pike L M. 1998. Determination of flavor precursor compound S-alk(en)yl-smallcap L-cysteine sulfoxides by an HPLC method and their distribution in Allium species. Scientia Horticulturae, 75:1-10
[34]	Yoshimoto N, Onuma M, Mizuno S, Sugino Y, Nakabayashi R, Imai S, Tsuneyoshi T, Sumi S, Saito K. 2015a. Identification of a flavin-containing S-oxygenating monooxygenase involved in alliin biosynthesis in garlic. The Plant Journal:For Cell And Molecular Biology, 83(6):941-951.
[35]	Yoshimoto N, Saito K. 2019. S-Alk(en)ylcysteine sulfoxides in the genus Allium:proposed biosynthesis,chemical conversion,and bioactivities. Journal of Experimental Botany, 70(16):4123-4137.
[36]	Yoshimoto N, Yabe A, Sugino Y, Murakami S, Sai-Ngam N, Sumi S, Tsuneyoshi T, Saito K. 2015b. Garlic γ-glutamyl transpeptidases that catalyze deglutamylation of biosynthetic intermediate of Alliin. Front Plant Sci, 5:758
[37]	Zhang Huamin, Chen Jianhua, Yin shouheng, Ma Aichu, Zhang Ming, Xiao Wanlu, Jiao Ziyuan. 2020. Research progress of the formation mechanism of apomictic seed in Chinese chive. Journal of Henan Agricultural Sciences, 49(6):1-7. (in Chinese)
	张华敏, 陈建华, 尹守恒, 马爱锄, 张明, 肖婉露, 焦子源. 2020. 韭菜无融合生殖种子形成机制研究进展. 河南农业科学, 49(6):1-7.
[38]	Zhang Huamin, Ma Qinlin, Yan Fangfang, Li Jijun, Cheng Jianhua, Li Bingbing. 2024. Full-length transcriptome sequencing analysis and mining of sulfur and selenium metabolism related genes in Allium tuberosum. Molecular Plant Breeding, https://link.cnki.net/urlid/46.1068.S.20230919.1218.004. (in Chinese)
	张华敏, 马沁琳, 闫芳芳, 李纪军, 陈建华, 李冰冰. 2024. 韭菜全长转录组测序分析及硫和硒代谢相关基因的挖掘. 分子植物育种, https://link.cnki.net/urlid/46.1068.S.20230919.1218.004.
[39]	Zhang Huamin, Zhang Xinling, Yin Shouheng, Wang Liya, Li Bingbing. 2023. Research progress on metabolic pathway of S-alk(en)ylcysteine sulfoxides in Allium. Guihaia, 43(2):221-233. (in Chinese)
[1]	Cui Lina, Jing Wenjuan, Huang Hongyong, Wang Lushi, Tang Hui. 2007. The comparison of fresh garlic enzyme deactivated method. Food Research and Development,(4):11-13. (in Chinese)
	崔利娜, 景文娟, 黄洪勇, 王鲁石, 唐辉. 2007. 鲜蒜灭酶方法的比较. 食品研究与开发,(4):11-13.
[2]	Dong Yuhui, Zhang Jingsheng, Zhao Guanghua. 2008. Improvement techniques for isolation and purification of 1-PeCSO. Science and Technology of Food Industry,(9):169-171. (in Chinese)
	董瑜慧, 张京生, 赵广华. 2008. 洋葱中天然活性成分丙烯基半胱氨酸亚砜分离纯化方法的改进. 食品工业科技,(9):169-171.
[3]	Fritsch R M, Keusgen M. 2006. Occurrence and taxonomic significance of cysteine sulphoxides in the genus Allium L.(Alliaceae). Phytochemistry, 67(11):1127-1135.
[4]	Gurpal S, Helen L, Kenny A, Stephanie L, Rakesh M, Om Parkash D. 2023. Overexpression of gamma-glutamyl cyclotransferase 2;1(CsGGCT2;1)reduces arsenic toxicity and accumulation in Camelina sativa(L.). Plant Cell Reports, 43(1):14.
[5]	Han Beibei, Wang Baoju, Tong Jing, Liu Mingchi, Wu Zhanhui, Meng Yanling, Liu Ning. 2022. Effects of amino acid treatments on growth,quality,and yield of hydroponic Chinese chive. China Vegetables,(5):74-80. (in Chinese)
	韩贝贝, 王宝驹, 佟静, 刘明池, 武占会, 孟艳玲, 刘宁. 2022. 氨基酸处理对水培韭菜产量、品质及风味的影响. 中国蔬菜,(5):74-80.
[6]	Han Qing. 2020. Study on the flavor substance of garlic cysteine derivative in maillard reaction[M. D. Dissertation]. Shenyang: Shenyang Agricultural University. (in Chinese)
	韩晴. 2020. 大蒜半胱氨酸衍生物参与形成美拉德风味物质机制初探[硕士论文]. 沈阳: 沈阳农业大学.
[7]	Han Yunyun, Yuan Xuewen, Li Yifang, Deng Bing. 2022. Cloning and expression analysis of Lentinus edodes GPX gene during postharvest storage. Storage and Process, 22(4):1-8. (in Chinese)
	韩云云, 袁学文, 栗艺芳, 邓冰. 2022. 香菇GPX基因克隆及其在采后贮藏中的表达分析. 保鲜与加工, 22(4):1-8.
[8]	Havey M, King J J, Bradeen J M, Bark O, Sato Y, Gökçe A F. 2001. A low-density genetic map of onion(Allium cepa L.)and its use for marker-facilitated selection of maintainer lines. Acta Horticulturae, 555:87-89.
[9]	Havey M J, Ahn Y K. 2016. Single nucleotide polymorphisms and indel markers from the transcriptome of garlic. Journal of the American Society for Horticultural Science, 141(1):62-65.
[10]	Jakse J, Meyer J D F, Suzuki G, McCallum J, Cheung F, Town C D, Havey M J. 2008. Pilot sequencing of onion genomic DNA reveals fragments of transposable elements,low gene densities,and significant gene enrichment after methyl filtration. Molecular Genetics and Genomics:MGG, 280(4):287-292.
[11]	Jones M G, Hughes J, Tregova A, Milne J, Tomsett A B, Collin H A. 2004. Biosynthesis of the flavour precursors of onion and garlic. Journal of Experimental Botany, 55(404):1903-1918.
[12]	Krause R J, Glocke S C, Elfarra A A. 2002. Sulfoxides as urinary metabolites of S-allyl-L-cysteine in rats:evidence for the involvement of flavin-containing monooxygenases. Drug Metabolism and Disposition:the Biological Fate of Chemicals, 30(10):1137-1142.
[13]	Krest I, Glodek J, Keusgen M. 2000. Cysteine sulfoxides and alliinase activity of some Allium species. Journal of Agricultural and Food Chemistry, 48(8):3753-3760.
[14]	Li Guanggui, Lin Guodu, Hu Yuqian, Zhang Huaishan, Bai Jing, An Huaming, Lu Min. 2020. Polymorphism of GGP and DHAR gene sequences and their associations with vitamin C content in fruits of wild Rosa roxburghii. Jiangsu Agricultural Sciences, 48(8):63-69. (in Chinese)
	李广贵, 林国都, 胡玉乾, 张怀山, 白静, 安华明, 鲁敏. 2020. 刺梨GGP和DHAR基因序列多态性及其与果实维生素C含量的关联分析. 江苏农业科学, 48(8):63-69.
[15]	Liang Zhile, Shang Kehan, Wang Lihui, Zhou Jin, Wang Guanglong, Xiong Aisheng. 2019. Cloning and expression analysis of the AsGST gene in garlic exposed to salinity stress. Journal of Nuclear Agricultural Sciences, 33(6):1088-1095. (in Chinese)
	梁志乐, 尚珂含, 王立辉, 周瑾, 王广龙, 熊爱生. 2019. 大蒜谷胱甘肽硫转移酶基因AsGST的克隆及其对盐胁迫的响应. 核农学报, 33(6):1088-1095.
[16]	Liu Jiantao, Zhao Li, Su Wei, Wang Shan, Zhang Weimin. 2006. Research progress of bioactive constituent and molecular biology in Allium tuberosum. Food Science and Technology,(8):67-70. (in Chinese)
	刘建涛, 赵利, 苏伟, 王杉, 张伟民. 2006. 韭菜中生物活性成分及其分子生物学的研究进展. 食品科技,(8):67-70.
[17]	Liu N, Tong J, Hu M M, Ji Y H, Wang B J, Liang H, Liu M C, Wu Z H. 2021. Transcriptome landscapes of multiple tissues highlight the genes involved in the flavor metabolic pathway in Chinese chive(Allium tuberosum). Genomics, 113(4):2145-2157.
[39]	张华敏, 张新岭, 尹守恒, 王利亚, 李冰冰. 2023. 葱属植物S-烷(烯)基半胱氨酸亚砜代谢途径研究进展. 广西植物, 43(2):221-233.
[40]	Zhang Ming, Lü Aiqin, Chen Zhongfu, Li Jijun, Ma Peifang, Li Xixiang, Wang Haiping. 2016. The current situation and strategies of chive germplasm development research in China. Journal of Plant Genetic Resources, 17(3):503-506,516. (in Chinese)
	张明, 吕爱琴, 陈中府, 李纪军, 马培芳, 李锡香, 王海平. 2016. 我国韭菜资源研究现状和种质创新研究建议. 植物遗传资源学报, 17(3):503-506,516.
[18]	Liu Pingxiang, Qiu Jing, Wong Rui, Xu Yanyang, Qian Yongzhong. 2021. Advances in the analysis of garlic functional components. Quality and Safety of Agro-Products,(2):67-73. (in Chinese)
	刘平香, 邱静, 翁瑞, 许彦阳, 钱永忠. 2021. 大蒜中主要功效成分分析研究进展. 农产品质量与安全,(2):67-73.
[19]	Martin M N, Saladores P H, Lambert E, Hudson A O, Leustek T. 2007. Localization of members of the γ-glutamyl transpeptidase family identifies sites of glutathione and glutathione S-conjugate hydrolysis. Plant Physiology, 144(4):1715-1732.
[20]	Matsuse K, Abdelrahman M, Ariyanti N A, Tsuji F, Hirata S, Nakajima Tetsuya, Sato M, Hirai Y M, Manochai B, Shigyo M. 2022. Targeted metabolome profiling of indonesian shallots and Japanese long-day/short-day bulb onions. Metabolites, 12(12):1260-1260.