通过全基因组关联分析鉴定茶树中关键的咖啡碱相关上游基因CsAK

doi:10.1016/j.jia.2025.06.026

Journal of Integrative Agriculture ›› 2026, Vol. 25 ›› Issue (4): 1519-1530.DOI: 10.1016/j.jia.2025.06.026

通过全基因组关联分析鉴定茶树中关键的咖啡碱相关上游基因CsAK

收稿日期:2025-01-08 修回日期:2025-06-30 接受日期:2025-05-20 出版日期:2026-04-20 发布日期:2026-03-11

Identification of CsAK as a critical caffeine-related upstream gene in tea accessions through genome-wide association study

Kaixin Rao¹, Yuting Ouyang¹, Yanjun Chen¹, Xiaojing Wang^{1, 2}, Ting Liu³, Qinfei Song¹, Shaojuan Zhang^{1, 2}, Biao Xiong¹, Suzhen Niu^{1, 2#}

¹College of Tea Science/Institute of Agro-Bioengineering, Guizhou University, Guiyang 550025, China
²The Key Laboratory of Plant Resources Conservation and Germplasm Innovation in the Mountainous Region (Ministry of Education), Guiyang 550025, China
³Zhengzhou Foreign Language School, Zhengzhou 450006, China

Received:2025-01-08 Revised:2025-06-30 Accepted:2025-05-20 Online:2026-04-20 Published:2026-03-11
About author:Kaixin Rao, E-mail: 1778424029@qq.com; #Correspondence Suzhen Niu, E-mail: niusuzhen@163.com
Supported by:
This study was supported by the National Natural Science Foundation of China (32060700), the National Guidance Foundation for Local Science and Technology Development of China [(2023) 009], the Guizhou Provincial Basic Research Program (Natural Sciences) - Youth Initiative Project, China([2024] Youth 157), the Guiyang Science and Technology Plan Project (Construction Technology Contract, China [2023] 48-21), the Post-funding Project of the National Key R&D Program of China (2021YFD1100307), the Guizhou Provincial Science and Technology Program, China ([2023] General 481), the Qianxinan Prefecture Science and Technology Program, China (2022-1-52), the Youth Science and Technology Talent Project of Guizhou Provincial Department of Education, China (KY [2022]153), and the Basic Research Project of Guizhou University, China (2023-38).

摘要/Abstract

摘要： 咖啡碱（CAF）作为茶叶主要风味成分，是茶饮料广受欢迎的核心元素之一。作为茶树重要的次级代谢产物，不同茶树种质资源间CAF含量呈现显著差异。然而，CAF生物合成的遗传调控机制尚未明晰。在这项研究中，基于贵州高原359份茶树种质资源开展全基因组关联研究（GWAS）以确定与CAF含量相关的遗传变异。共鉴定出19个显著关联的单核苷酸多态性（SNPs）及CAF生物合成关键基因（CsAK）。亚细胞定位分析表明CsAK-GFP融合蛋白位于细胞膜上。通过反义寡核苷酸（AsODN）靶向沉默茶树芽叶CsAK基因，结果显示AsODN处理的茶树中CsAK基因的表达水平显著下调，且CAF含量降低。在真核细胞中过表达CsAK基因可促进CAF生物合成过程中关键中间产物（L-蛋氨酸）的积累。该研究为后续培育具有高或低CAF水平的优良种质资源的茶树育种计划提供了理论依据。

Abstract:

Caffeine (CAF), a primary flavor component in tea, is one of the main reasons for the popularity of tea beverages. As an important secondary metabolite in tea plants, the CAF content varied greatly among different tea accessions. However, the genetic mechanisms underlying the CAF biosynthesis were still unclear. In this study, we performed a genome-wide association study (GWAS) on 359 tea accessions in the Guizhou Plateau to identify genetic variation associated with CAF content. A total of 19 significant single nucleotide polymorphisms (SNPs) and key gene (CsAK) involved in CAF biosynthesis were identified. Subcellular localization revealed that the CsAK-GFP fusion protein was located on the cell membrane. Antisense oligodeoxynucleotide (AsODN) targeting the CsAK gene to the buds and leaves revealed that the expression levels of the CsAK gene were significantly reduced, and the corresponding CAF content was also decreased in AsODN-treated tea plants. Overexpression of the CsAK gene in eukaryotic cells resulted in the accumulation of key intermediate product (L-methionine) during CAF biosynthesis process. These findings offered a theoretical foundation for future tea breeding programs aimed at cultivating excellent germplasm with high or low levels of CAF.

Key words: tea accessions , caffeine (CAF) , single nucleotide polymorphism (SNP) , genome-wide association study (GWAS) , Guizhou Plateau

Kaixin Rao, Yuting Ouyang, Yanjun Chen, Xiaojing Wang, Ting Liu, Qinfei Song, Shaojuan Zhang, Biao Xiong, Suzhen Niu. 通过全基因组关联分析鉴定茶树中关键的咖啡碱相关上游基因CsAK[J]. Journal of Integrative Agriculture, 2026, 25(4): 1519-1530.

Kaixin Rao, Yuting Ouyang, Yanjun Chen, Xiaojing Wang, Ting Liu, Qinfei Song, Shaojuan Zhang, Biao Xiong, Suzhen Niu. Identification of CsAK as a critical caffeine-related upstream gene in tea accessions through genome-wide association study[J]. Journal of Integrative Agriculture, 2026, 25(4): 1519-1530.

参考文献

Ahammed G J, Li X. 2022. Hormonal regulation of health-promoting compounds in tea (Camellia sinensis L.). Plant Physiology and Biochemistry, 185, 390–400.

Bradbury P J, Zhang Z, Kroon D E, Casstevens T M, Ramdoss Y, Buckler E S. 2007. TASSEL: Software for association mapping of complex traits in diverse samples. Bioinformatics, 23, 2633–2635.

Chen W, Hou L, Zhang Z Y, Pang X M, Li Y Y. 2017. Genetic diversity, population structure, and linkage disequilibrium of a core collection of Ziziphus jujuba assessed with genome-wide SNPs developed by genotyping-by-sequencing and SSR markers. Frontiers in Plant Science, 8, 575.

Chen Y F, Jiang C J, Yin S X, Zhuang J H, Zhao Y, Zhang L J, Jiang X L, Liu Y J, Gao L P, Xia T. 2023. New insights into the function of plant tannase with promiscuous acyltransferase activity. The Plant Journal, 113, 576–594.

Chen Y J, Niu S Z, Deng X Y, Song Q F, He L M, Bai D C, He Y Q. 2023. Genome-wide association study of leaf-related traits in tea plant in Guizhou based on genotyping-by-sequencing. BMC Plant Biology, 23, 196.

Van Dam R M, Hu F B. 2022. Caffeine consumption and cardiovascular health. Nature Reviews Cardiology, 19, 429–430.

Danecek P, Auton A, Abecasis G, Albers C A, Banks E, DePristo M A, Handsaker R E, Lunter G, Marth G T, Sherry S T, McVean G, Durbin R, 1000 Genomes Project Analysis Group. 2011. The variant call format and VCFtools. Bioinformatics, 27, 2156–2158.

Deng X L, Huang D J, Wang Y H, An H W, Bai D C, Wang X J, Niu S Z, Song X M. 2025. Genome-wide association study of salicylic acid provides genetic insights for tea plant selective breeding. Horticulture Research, 12, 362.

Devitt G, Thomas M, Klibanov A M, Pfeiffer T, Bosch V. 2007. Optimized protocol for the large scale production of HIV pseudovirions by transient transfection of HEK293T cells with linear fully deacylated polyethylenimine. Journal of Virological Methods, 146, 298–304.

Fang K X, Xia Z Q, Li H J, Jiang X H, Qin D D, Wang Q H, Wang Q, Pan C D, Li B, Wu H L. 2021. Genome-wide association analysis identified molecular markers associated with important tea flavor-related metabolites. Horticulture Research, 8, 42.

Gao Y N, Han C J, Liu C L, Wang J, Zhao L, Fang L, Min W H. 2019. Enzymatic characterization and molecular mechanism of a novel aspartokinase mutant M372I/T379W from Corynebacterium pekinense. Royal Society of Chemistry Advances, 9, 21344–21354.

Gong A D, Lian S B, Wu N N, Zhou Y J, Zhao S Q, Zhang L M, Cheng L, Yuan H Y. 2020. Integrated transcriptomics and metabolomics analysis of catechins, caffeine and theanine biosynthesis in tea plant (Camellia sinensis) over the course of seasons. BMC Plant Biology, 20, 294.

Grover A, Sharma P C. 2016. Development and use of molecular markers: Past and present. Critical Reviews in Biotechnology, 36, 290–302.

Hacham Y, Matityahu I, Schuster G, Amir R. 2008. Overexpression of mutated forms of aspartate kinase and cystathionine gamma-synthase in tobacco leaves resulted in the high accumulation of methionine and threonine. The Plant Journal, 54, 260–271.

He L M, Luo J, Niu S Z, Bai D C, Chen Y J. 2023. Population structure analysis to explore genetic diversity and geographical distribution characteristics of wild tea plant in Guizhou Plateau. BMC Plant Biology, 23, 255.

Helyar S J, Hemmer-Hansen J, Bekkevold D, Taylor M I, Ogden R, Limborg M T, Cariani A, Maes G E, Diopere E, Carvalho G R, Nielsen E E. 2011. Application of SNPs for population genetics of nonmodel organisms: New opportunities and challenges. Molecular Ecology Resources, 1, 123–136.

Jander G, Joshi V. 2010. Recent progress in deciphering the biosynthesis of aspartate-derived amino acids in plants. Molecular Plant, 3, 54–65.

Jiang P, Ren L J, Zhi L, Yu Z, Lv F X, Xu F L, Peng W, Bai X Y, Cheng K L, Quan L, Zhang X Q, Wang X H, Zhang Y, Yang D, Hu X L, Xiao R P. 2021. Negative regulation of AMPK signaling by high glucose via E3 ubiquitin ligase MG53. Molecular Cell, 81, 629–637.

Jin J Q, Chai Y F, Liu Y F, Zhang J, Yao M Z, Chen L. 2018. Hongyacha, a naturally caffeine-free tea plant from Fujian, China. Journal of Agricultural and Food Chemistry, 66, 11311–11319.

Jin J Q, Yao M Z, Ma C L, Ma J Q, Chen L. 2016. Association mapping of caffeine content with TCS1 in tea plant and its related species. Plant Physiology and Biochemistry, 105, 251–259.

Jin Q F, Wang Z, Sandhu D, Chen L, Shao C Y, Shang F H Z, Xie S Y, Huang F Y, Chen Z Y, Zhang X Q, Hu J Y, Liu G Z, Su Q, Huang M D, Liu Z H, Huang J A, Tian N, Liu S Q. 2023. mRNA-miRNA analyses reveal the involvement of CsbHLH1 and miR1446a in the regulation of caffeine biosynthesis in Camellia sinensis. Horticulture Research, 11, 282.

Kim H, Kang S H, Kim S H, Kim S H, Hwang J, Kim J G, Han K, Kim J B. 2021. Drinking coffee enhances neurocognitive function by reorganizing brain functional connectivity. Scientific Reports, 11, 14381.

Koshiishi C, Kato A, Yama S, Crozier A, Ashihara H. 2001. A new caffeine biosynthetic pathway in tea leaves: Utilisation of adenosine released from the S-adenosyl-L-methionine cycle. FEBS Letters, 499, 50–54.

Kotaka M, Ren J S, Lockyer M, Hawkins A R, Stammers D K. 2006. Structures of R- and T-state Escherichia coli aspartokinase III. Mechanisms of the allosteric transition and inhibition by lysine. Journal of Biological Chemistry, 281, 31544–31552.

Li F, Deng X Y, Huang Z, Zhao Z F, Li C Y, Song Q F, He Y Q, Niu S N. 2023. Integrated transcriptome and metabolome provide insights into flavonoid biosynthesis in “P113”, a new purple tea of Camellia tachangensis. Beverage Plant Research, 3, 3.

Li G F, Tan M, Ma J J, Cheng F, Li K, Liu X J, Zhao C P, Zhang D, Xing L B, Ren X L, Han M Y, An N. 2021. Molecular mechanism of MdWUS2-MdTCP12 interaction in mediating cytokinin signaling to control axillary bud outgrowth. Journal of Experimental Botany, 72, 4822–4838.

Lu Q, Zhang M C, Niu X J, Wang C H, Xu Q, Feng Y, Wang S, Yuan X P, Yu H Y, Wang Y P, Wei X H. 2016. Uncovering novel loci for mesocotyl elongation and shoot length in indica rice through genome-wide association mapping. Planta, 243, 645–657.

McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo M A. 2010. The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data. Genome Research, 20, 1297–1303.

Mi X Z, Yang C, Qiao D H, Tang M S, Guo Y, Liang S H, Li Y, Chen Z W, Chen J. 2023. De novo full length transcriptome analysis of a naturally caffeine-free tea plant reveals specificity in secondary metabolic regulation. Scientific Reports, 13, 6015.

Misako K, Kouichi M. 2004. Caffeine synthase and related methyltransferases in plants. Frontiers in Bioscience-Landmark, 9, 1833–1342.

Mohanpuria P, Kumar V, Yadav S K. 2010. Tea caffeine: Metabolism, functions, and reduction strategies. Food Science and Biotechnology, 19, 275–287.

Niu S N, Koiwa H, Song Q F, Qiao D H, Chen J, Zhao D G, Chen Z W, Wang Y, Zhang T Y. 2020. Development of core-collections for Guizhou tea genetic resources and GWAS of leaf size using SNP developed by genotyping-by-sequencing. PeerJ, 8, 8572.

Niu S N, Song Q F, Koiwa H, Qiao D H, Zhao D G, Chen Z W, Liu X, Wen X P. 2019. Genetic diversity, linkage disequilibrium, and population structure analysis of the tea plant (Camellia sinensis) from an origin center, Guizhou plateau, using genome-wide SNPs developed by genotyping-by-sequencing. BMC Plant Biology, 19, 328.

Qiao D H, Yang C, Chen J, Guo Y, Li Y, Niu S Z, Cao K M, Chen Z W. 2019. Comprehensive identification of the full-length transcripts and alternative splicing related to the secondary metabolism pathways in the tea plant (Camellia sinensis). Scientific Reports, 9, 2709.

Pandey J, Scheuring D C, Koym J W, Coombs J, Novy R G, Thompson A L, Holm D G, Douches DS, Miller Jr J C, Vales M I. 2021. Genetic diversity and population structure of advanced clones selected over forty years by a potato breeding program in the USA. Scientific Reports, 11, 8344.

Ravanel S, Gakière B, Job D, Douce R. 1998. The specific features of methionine biosynthesis and metabolism in plants. Proceedings of the National Academy of Sciences of the United States of America, 95, 7805–7812.

Reddy V S, Shiva S, Manikantan S, Ramakrishna S. 2024. Pharmacology of caffeine and its effects on the human body. European Journal of Medicinal Chemistry Reports, 10, 100138.

Shi R, Cao Y, Yang T H, Wang Y P, Xiang Y N, Chen F, Zhang W, Zhou X Y, Sun C M, Fu S X, Hu M L, Zhang J F, Wang X D. 2024. Genome-wide association study reveals the genetic basis of crude fiber components in Brassica napus L. shoots at stem elongation stage. Journal of Agricultural and Food Chemistry, 72, 16530–16540.

Srdjenovic B, Djordjevic-Milic V, Grujic N, Injac R, Lepojevic Z. 2008. Simultaneous HPLC determination of caffeine, theobromine, and theophylline in food, drinks, and herbal products. Journal of Chromatographic Science, 46, 144–149.

Stecher G, Tamura K, Kumar S. 2020. Molecular Evolutionary Genetics Analysis (MEGA) for macOS. Molecular Biology and Evolution, 37, 1237–1239.

Suzuki T. 1972. The participation of S-adenosylmethionine in the biosynthesis of caffeine in the tea plant. FEBS Letters, 24, 18–20.

Tang W J, Wu T T, Ye J, Sun J, Jiang Y, Yu J, Tang J P, Chen G M, Wang C M, Wan J M. 2016. SNP-based analysis of genetic diversity reveals important alleles associated with seed size in rice. BMC Plant Biology, 16, 93.

Teng J, Yan C Y, Zeng W, Zhang Y Q, Zeng Z, Huang Y H. 2020. Purification and characterization of theobromine synthase in a Theobromine-enriched wild tea plant (Camellia gymnogyna Chang) from Dayao Mountain, China. Food Chemistry, 311, 125875.

Uffelmann E, Huang Q Q, Munung N S, Vries J D, Okada Y, Martin A R, Martin H C, Lappalainen T, Posthuma D. 2021. Genome-wide association studies. Nature Reviews Methods Primers, 1, 1–21.

Wang J D, Zhang Z L, Gong Z L, Liang Y J, Ai X T, Sang Z W, Guo J P, Li X Y, Zheng J Y. 2022. Analysis of the genetic structure and diversity of upland cotton groups in different planting areas based on SNP markers. Gene, 809, 146042.

Wang R J, Gao X F, Yang J, Kong X R. 2019. Genome-wide association study to identify favorable SNP allelic variations and candidate genes that control the timing of spring bud flush of tea (Camellia sinensis) using SLAF-seq. Journal of Agricultural and Food Chemistry, 67, 10380–10391.

Wang W Y, Xu M Y, Wang G P, Galili G. 2018. New insights into the metabolism of aspartate-family amino acids in plant seeds. Plant Reproduction, 31, 203–211.

Wang Z H, Huang R, Moon D G, Ercisli S, Chen L. 2023. Achievements and prospects of QTL mapping and beneficial genes and alleles mining for important quality and agronomic traits in tea plant (Camellia sinensis). Beverage Plant Research, 3, 22.

Xia E H, Tong W, Hou Y, An Y L, Chen L B, Wu Q, Liu Y L, Yu J, Li F D, Li R P, Li P H, Zhao H J, Ge R H, Huang J, Mallano A L, Zhang Y R, Liu S R, Deng W W, Song C K, Zhang Z L, et al. 2020a. The reference genome of tea plant and resequencing of 81 diverse accessions provide insights into its genome evolution and adaptation. Molecular Plant, 13, 1013–1026.

Xia E H, Tong W, Wu Q, Wei S, Zhao J, Zhang Z Z, Wei C L, Wan X C. 2020b. Tea plant genomics: Achievements, challenges and perspectives. Horticulture Research, 7, 7.

Xia E H, Zhang H B, Sheng J, Li K, Zhang Q J, Kim C, Zhang Y, Liu Y, Zhu T, Li W, Huang H, Tong Y, Nan H, Shi C, Shi C, Jiang J J, Mao S Y, Jiao J Y, Zhang D, Zhao Y, et al. 2017. The tea tree genome provides insights into tea flavor and independent evolution of caffeine biosynthesis. Molecular Plant, 10, 866–877.

Xu J Z, Han M, Ren X D, Zhang W G. 2016. Modification of aspartokinase III and dihydrodipicolinate synthetase increases the production of L-lysine in Escherichia coli. Biochemical Engineering Journal, 114, 79–86.

Yao X Z, Chen H F, Ai A T, Wang F, Lian S S, Tang H, Jiang Y H, Jiao Y J, He Y M, Li T, Lu L T. 2023. The transcription factor CsS40 negatively regulates TCS1 expression and caffeine biosynthesis in connection to leaf senescence in Camellia sinensis. Horticulture Research, 10, 162.

Yoshida H, Okada S, Wang F M, Shiota S, Mori M, Kawamura M, Zhao X, Wang Y Q, Nishigaki N, Kobayashi A, Miura K, Yoshida S, Ikegami M, Ito A, Huang L T, Caroline Hsing Y I, Yamagata Y, Morinaka Y, Yamasaki M, Kotake T, et al. 2023. Integrated genome-wide differentiation and association analyses identify causal genes underlying breeding-selected grain quality traits in japonica rice. Molecular Plant, 16, 1460–1477.

Yu S W, Li P H, Zhao X C, Tan M M, Ahmad M Z, Xu Y J, Tadege M, Zhao J. 2021. CsTCPs regulate shoot tip development and catechin biosynthesis in tea plant (Camellia sinensis). Horticulture Research, 8, 104.

Zhang C, Dong S S, Xu J Y, He W M, Yang T L. 2019. PopLDdecay: A fast and effective tool for linkage disequilibrium decay analysis based on variant call format files. Bioinformatics, 35, 1786–1788.

Zhang S R, Jin J Q, Chen J D, Ercisli S, Chen L. 2022. Purine alkaloids in tea plants: Component, biosynthetic mechanism and genetic variation. Beverage Plant Research, 2, 13.

Zhang W Y, Zhang Y J, Qiu H J, Guo Y F, Wan H L, Zhang X L, Scossa F, Alseekh S, Zhang Q H, Wang P, Xu L, Schmidt M H-W, Jia X X, Li D L, Zhu A T, Guo F, Chen W, Ni D J, Usadel B, Fernie A R, Wen W W. 2020. Genome assembly of wild tea tree DASZ reveals pedigree and selection history of tea varieties. Nature Communications, 11, 3719.

Zhang Y R, Fu J M, Zhou Q Y, Li F D, Shen Y H, Ye Z L, Tang D K, Chi N, Li L Q, Ma S Y, Inayat M A, Guo T Y, Zhao J, Li P H. 2022. Metabolite profiling and transcriptome analysis revealed the conserved transcriptional regulation mechanism of caffeine biosynthesis in tea and coffee plants. Journal of Agricultural and Food Chemistry, 70, 3239–3251.

Zhao M Y, Jin J Y, Wang J M, Gao T, Luo Y, Jing T T, Hu Y T, Pan Y T, Lu M Q, Schwab W, Song C K. 2022. Eugenol functions as a signal mediating cold and drought tolerance via UGT71A59-mediated glucosylation in tea plants. The Plant Journal, 109, 1489–1506.

Zhao Z F, Song Q F, Bai D C, Niu S Z, He Y Q, Qiao D H, Chen Z W, Li C Y, Luo J, Li F. 2022. Population structure analysis to explore genetic diversity and geographical distribution characteristics of cultivated-type tea plant in Guizhou Plateau. BMC Plant Biology, 22, 55.

Zheng Y X, Yuan F, Huang Y Q, Zhao Y F, Jia X Y, Zhu L Y, Guo J J. 2021. Genome-wide association studies of grain quality traits in maize. Scientific Reports, 11, 9797.

Zhou M Z, Yan C Y, Zeng Z, Luo L, Zeng W, Huang Y H. 2020. N-Methyltransferases of caffeine biosynthetic pathway in plants. Journal of Agricultural and Food Chemistry, 68, 15359–15372.

Zou J N, Yu H, Yu Q, Jin X J, Cao L, Wang M Y, Wang M X, Ren C Y, Zhang Y X. 2021. Physiological and UPLC-MS/MS widely targeted metabolites mechanisms of alleviation of drought stress-induced soybean growth inhibition by melatonin. Industrial Crops and Products, 163, 113323.

通过全基因组关联分析鉴定茶树中关键的咖啡碱相关上游基因CsAK

Identification of CsAK as a critical caffeine-related upstream gene in tea accessions through genome-wide association study

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 0

编辑推荐

Metrics