Functional Characterization of Key Genes CsANS and CsLAR Involved in Catechin Biosynthesis in Camellia sinensis

doi:10.16420/j.issn.0513-353x.2023-0247

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (4): 804-814.doi: 10.16420/j.issn.0513-353x.2023-0247

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

Functional Characterization of Key Genes CsANS and CsLAR Involved in Catechin Biosynthesis in Camellia sinensis

ZHANG Zhiling¹, ZHANG Yuanyuan¹, LIN Xiaorong¹, LI Bin¹^,², CHEN Zhongzheng¹^,²^,^*()

¹ College of Food Science，South China Agricultural University，Guangzhou 510642，China
² Guangdong Provincial Key Laboratory of Nutraceuticals and Functional Foods，South China Agricultural University，Guangzhou 510642，China

Received:2023-07-14 Revised:2023-12-05 Online:2024-04-25 Published:2024-04-26
Contact: CHEN Zhongzheng

Abstract

Abstract:

Two key enzyme genes CsANS and CsLAR involved in the catechin synthetic pathway from‘Yinghong 9’were cloned，and successfully identified their functions by prokaryotic expression，subcellular localization and transgenic overexpression analyses. The results showed that the ORFs（open reading frame）of CsANS and CsLAR were 1 068 and 1 029 bp，respectively，which were 99% homologous to the corresponding genes from‘Shuchazao’in TPIA database. Soluble proteins of CsANS and CsLAR can be obtained in prokaryotic induced expression system and can be successfully purified by GST tag. Transient expression of CsANS and CsLAR in Nicotiana benthamiana leaves showed that proteins they encoded were localized in the cytoplasm and nucleus. Furthermore，CsANS and CsLAR were overexpressed in tea calli by Agrobacterium-mediated genetic transformation. The results showed that both of them could significantly promote the biosynthesis of total catechins in tea calli，in which CsANS mainly increased content of EC and EGCG，while CsLAR mainly promoted the accumulation of trans-catechins C，GC，CG and GCG. It is speculated that the function of CsANS is mainly to promote the synthesis of cis-catechins，while CsLAR is more conducive to the synthesize trans-catechins in‘Yinghong 9’.

Key words: Camellia sinensis, catechins, ANS, LAR, overexpression

ZHANG Zhiling, ZHANG Yuanyuan, LIN Xiaorong, LI Bin, CHEN Zhongzheng. Functional Characterization of Key Genes CsANS and CsLAR Involved in Catechin Biosynthesis in Camellia sinensis[J]. Acta Horticulturae Sinica, 2024, 51(4): 804-814.

Figures/Tables 7

Table 1 The primers information for qPCR in this study

引物名称 Primer name	序列（5′-3′） Sequence
GAPDH	F：TTGGCATCGTTGAGGGTCT；R：CAGTGGGAACACGGAAAGC
q-LARa	F：CGCAACCGGGGGCGGAGTCCTCATC；R：GACAACACCATGAATAACTTTAGCG
q-ANS	F：GATGACTACAGTGGCTGCCCCGAGA；R：TCCGAGTCTATGTCTTTCAAGTCAA
q-ANR	F：TCTTATCACTGTCATCCCAACTCT；R：TTTGCATACCTTTCAACCCAT
q-UFGT	F：TTGTTGTTGTGCCTCCCTCA；R：TTGGCAAGCGTTGGATGTT
q-UGT	F：CAAGGCCATGTCAACCCTCTTCTCC；R：GCTCGTTCTCGTCCCAACCATCTTC
q-ECGT	F：GGAATTGGTCCACTGGAGTTCGTAA；R：ATGGGACAATCATGCCTGAATAAGA

Fig. 1 The electrophoresis of PCR amplification CsANS and CsLAR

Fig. 2 SDS-PAGE analysis of pGEX-4T-1-CsANS（A）and pGEX-4T-1-CsLAR（B）recombinant proteins 1：Before induction；2：Before fragmentation；3：After 8 h induction；4：After fragmentation；5：After centrifugation of supernatant；6：After centrifugation of precipitate；7-8：Recovery of protein. The red arrows refer to the bands that recycle the protein.

Fig. 3 The subcellular localization of CsANS and CsLAR

Fig. 4 Screening and culture of overexpressing transgenic tea callus of CsANS（A）and CsLAR（B）

Fig. 5 Amplification and identification of G418 in overexpressed tea callus

Fig. 6 Detection of gene expression levels and analysis of catechin content and fractions in CsANS and CsLAR overexpression callus（CsANS-OE and CsLAR-OE） *：P < 0.05；**：P < 0.01；ns：P > 0.05.

References 42

[1]	An J P, Qu F J, Yao J F, Wang X N, You C X, Wang X F, Hao Y J. 2017. The bZIP transcription factor MdHY5 regulates anthocyanin accumulation and nitrate assimilation in apple. Horticulture Research, 4:7023.
[2]	Ashihara H, Deng W W, Mullen W, Crozier A. 2010. Distribution and biosynthesis of flavan-3-ols in Camellia sinensis seedlings and expression of genes encoding biosynthetic enzymes. Phytochemistry, 71 (5-6):559-566. doi: 10.1016/j.phytochem.2010.01.010 pmid: 20189205
[3]	Cao H L, Li J M, Ye Y J, Lin H Z, Hao Z L, Ye N X, Yue C. 2020. Integrative transcriptomic and metabolic analyses provide insights into the role of trichomes in tea plant(Camellia sinensis). Biomolecules, 10 (2):311.
[4]	Chen C S, Wei K, Wang L Y, Ruan L, Li H L, Zhou X G, Lin Z H, Shan R Y, Cheng H. 2017. Expression of key structural genes of the phenylpropanoid pathway associated with catechin epimerization in tea cultivars. Frontiers in Plant Science, 8:702. doi: 10.3389/fpls.2017.00702 pmid: 28515736
[5]	Chen S M, Li C H, Zhu X R, Deng Y M, Sun W, Wang L S, Zhang Z. 2012. The identification of flavonoids and the expression of genes of anthocyanin biosynthesis in the chrysanthemum flowers. Biologia Plantarum, 56 (3):458-464.
[6]	Cheng J, Yu K J, Zhang M Y, Shi Y, Duan C Q, Wang J. 2020. The effect of light intensity on the expression of leucoanthocyanidin reductase in grapevine calluses and analysis of its promoter activity. Genes, 11 (10):1156.
[7]	Ferrer J L, Jez J M, Bowman M E, Dixon R A, Noel J P. 1999. Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis. Nature Structural Biology, 6 (8):775-784. doi: 10.1038/11553 pmid: 10426957
[8]	Grzesik M, Naparło K, Bartosz G, Sadowska, Bartosz I. 2018. Antioxidant properties of catechins:comparison with other antioxidants. Food Chemistry, 241:480-492.
[9]	Hao P F, Liu H, Lin B G, Ren Y, Huang L, Jiang L X, Hua S J. 2022. BnaA03.ANS identified by metabolomics and RNA-seq partly played irreplaceable role in pigmentation of red rapeseed(Brassica napus)petal. Frontiers in Plant Science, 13:940765.
[10]	Huang Jiyue, Wang Yufeng, Yang Jinshui. 2010. Over-expression of OsPSK3 increases chlorophyll content of leaves in rice. Hereditas, 32 (12):1281-1289. (in Chinese)
	黄霁月, 王玉锋, 杨金水. 2010. 过表达OsPSK3基因增加水稻叶片叶绿素含量. 遗传, 32 (12):1281-1289.
[11]	Jiang W B, Yin Q G, Wu R R, Zheng G S, Liu J Y, Dixon, Richard A, Pang Y Z. 2015. Role of a chalcone isomerase-like protein in flavonoid biosynthesis in Arabidopsis thaliana. Journal of Experimental Botany, 66 (22):7165-7179.
[12]	Kang Xin, Liu Ping, Ma Wenhui, Zhang Yuanyuan, Lin Xiarong, Li Bin, Chen Zhongzheng. 2020. RNAi silencing CsHB1 reduces the accumulation of caffeine in tea callus. Acta Horticulturae Sinica, 47 (12):2373-2384. (in Chinese)
	康馨, 刘平, 马雯慧, 张媛媛, 林晓蓉, 李斌, 陈忠正. 2020. RNAi沉默CsHB1降低茶树叶片愈伤组织咖啡碱积累. 园艺学报, 47 (12):2373-2384.
[13]	Kondou Y, Higuchi M, Matsui M. 2010. High-throughput characterization of plant gene functions by using gain-of-function technology. Annual Review of Plant Biology, 61:373-393. doi: 10.1146/annurev-arplant-042809-112143 pmid: 20192750
[14]	Lei Zhiwei, Li Lulu, Guo Can, Yin Rongxiu. 2019. Advances in catechin glycosylation modifications. Chinese Traditional and Herbal Drugs, 50 (21):5362-5372. (in Chinese)
	雷志伟, 李露露, 郭灿, 尹荣秀. 2019. 儿茶素糖基化修饰研究进展. 中草药, 50 (21):5362-5372.
[15]	Li Daxiang, Xian Shu, Yang Wei, Wan Xiaochun. 2011. Advances in research on the neuroprotective effects of tea. Journal of Tea Science, 31 (2):79-86. (in Chinese)
	李大祥, 鲜殊, 杨卫, 宛晓春. 2011. 茶叶的神经保护作用研究进展. 茶叶科学, 31 (2):79-86.
[16]	Liao L, Vimolmangkang S, Wei G, Zhou H, Korban S S, Han Y. 2015. Molecular characterization of genes encoding leucoanthocyanidin reductase involved in proanthocyanidin biosynthesis in apple. Frontiers in Plant Science, 6:243. doi: 10.3389/fpls.2015.00243 pmid: 25914714
[17]	Molla K A, Karmakar S, Chanda P K, Ghosh S, Sarkar S N, Datta S K, Datta K. 2013. Rice oxalate oxidase gene driven by green tissue-specific promoter increases tolerance to sheath blight pathogen(Rhizoctonia solani)in transgenic rice. Molecular Plant Pathology, 14 (9):910-922.
[18]	Pang Y, Abeysinghe I S B, He J, He X, Huhman D, Mewan K M, Dixon R A. 2013. Functional characterization of proanthocyanidin pathway enzymes from tea and their application for metabolic engineering. Plant Physiology, 161 (3):1103-1116. doi: 10.1104/pp.112.212050 pmid: 23288883
[19]	Prelich G. 2012. Gene overexpression:uses,mechanisms,and interpretation. Genetics, 190 (3):841-854.
[20]	Punyasiri P A N, Abeysinghe I S B, Kumar V, Treutter D, Duy D, Gosch C, Fischer T C. 2004. Flavonoid biosynthesis in the tea plant Camellia sinensis:properties of enzymes of the prominent epicatechin and catechin pathways. Archives of Biochemistry and Biophysics, 431 (1):22-30. doi: 10.1016/j.abb.2004.08.003 pmid: 15464723
[21]	Reddy A M, Reddy V S, Scheffler B E, Wienand U, Reddy A R. 2007. Novel transgenic rice overexpressing anthocyanidin synthase accumulates a mixture of flavonoids leading to an increased antioxidant potential. Metabolic Engineering, 9 (1):95-111. pmid: 17157544
[22]	Riboni M, Galbiati M, Tonelli C, Conti L. 2013. GIGANTEA enables drought escape response via abscisic acid-dependent activation of the florigens and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1. Plant Physiology, 162 (3):1706-1719.
[23]	Scharbert S, Hofmann T. 2005. Molecular definition of black tea taste by means of quantitative studies,taste reconstitution and omission experiments. Journal of Agricultural and Food Chemistry, 53 (13):5377-5384. doi: 10.1021/jf050294d pmid: 15969522
[24]	Shi Y, Tian S, Hou L, Huang X, Zhang X, Guo H, Yang S. 2012. Ethylene signaling negatively regulates freezing tolerance by repressing expression of CBF and type-A ARR genes in Arabidopsis. The Plant Cell, 24 (6):2578-2595.
[25]	Speeckaert N, El Jaziri M, Baucher M, Behr M. 2022. UGT72,a Major glycosyltransferase family for flavonoid and monolignol homeostasis in plants. Biology, 11 (3):441.
[26]	Su H, Zhang X Y, He Y Q, Li L Y, Wang Y F, Hong G J, Xu P. 2020. Transcriptomic analysis reveals the molecular adaptation of three major secondary metabolic pathways to multiple macronutrient starvation in tea(Camellia sinensis). Genes, 11 (3):241.
[27]	Sun Meilian, Wang Yunsheng, Yang Dongqing, Wei Chaoling, Gao Liping, Xia Tao, Shan Yu, Luo Yang. 2010. Reference genes for real-time fluorescence quantitative PCR in Camellia sinensis. Bulletin of Botany, 45 (5):579-587. (in Chinese)
	孙美莲, 王云生, 杨冬青, 韦朝领, 高丽萍, 夏涛, 单育, 骆洋. 2010. 茶树实时荧光定量PCR分析中内参基因的选择. 植物学报, 45 (5):579-587.
[28]	Tang Xiuhua, Sun Weijiang, Hong Yaoxin, Xie Feng, Chen Jiajia, Chen Zhidan. 2019. Differential expression of genes associated with anthocyanin synthesis in Camellia sinensis varieties with different leaf colors. Journal of Fujian Agriculture and Forestry University(Natural Science Edition), 48 (6):742-745. (in Chinese)
	唐秀华, 孙威江, 洪瑶新, 谢凤, 陈佳佳, 陈志丹. 2019. 不同叶色茶树品种(系)花青素合成相关基因的表达水平. 福建农林大学学报(自然科学版), 48 (6):742-745.
[29]	Wang P Q, Zhang L J, Jiang X L, Dai X L, Xu L J, Li T, Xing D W, Li Y Z, Li M Z, Gao L P. 2018. Evolutionary and functional characterization of leucoanthocyanidin reductases from Camellia sinensis. Planta, 247:139-154.
[30]	Wang Y S, Xu Y J, Gao L P, Yu O, Wang X Z, He X J, Jiang X L, Liu Y J, Xia T. 2014. Functional analysis of flavonoid 3′, 5′-hydroxylase from tea plant(Camellia sinensis):critical role in the accumulation of catechins. BMC Plant Biology, 14:1-14.
[31]	Wei K, Wang L Y, Zhou J, He W, Zeng J M, Jiang Y W, Cheng H. 2011. Catechin contents in tea(Camellia sinensis)as affected by cultivar and environment and their relation to chlorophyll contents. Food Chemistry, 125 (1):44-48.
[32]	Wu Z J, Li X H, Liu Z W, Xu Z S, Zhuang J. 2014. De novo assembly and transcriptome characterization:novel insights into catechins biosynthesis in Camellia sinensis. BMC Plant Biology, 14 (1):1-16.
[33]	Xie D Y, Sharma S B, Paiva N L, Ferreira D, Dixon R A. 2003. Role of anthocyanidin reductase,encoded by BANYULS in plant flavonoid biosynthesis. Science, 299 (5605):396-399.
[34]	Zhang H L, Zhao X J, Zhang J P, Yang B, Yu Y H, Liu T F, Nie B H, Song B T. 2020. Functional analysis of an anthocyanin synthase gene StANS in potato. Scientia Horticulturae, 272:109569.
[35]	Zhang J, Han Z Y, Tian J, Zhang X, Song T T, Yao Y C. 2015. The expression level of anthocyanidin synthase determines the anthocyanin content of crabapple(Malus)petals. Acta Physiologiae Plantarum, 37 (6):1-10.
[36]	Zhang Xue. 2018. Metabolic mechanism of tea catechins and its subcellular localization in key enzymes of biosynthesis[M. D. Dissertation]. Guangzhou: South China Agricultural University. (in Chinese)
	张雪. 2018. 茶叶差异儿茶素代谢机理及其生物合成关键酶亚细胞定位[硕士论文]. 广州: 华南农业大学.
[37]	Zhang Y Z, Wei K, Li H L, Wang L Y, Ruan L, Pang D D, Cheng H. 2018. Identification of key genes involved in catechin metabolism in tea seedlings based on transcriptomic and HPLC analysis. Plant Physiology and Biochemistry, 133:107-115. doi: S0981-9428(18)30475-3 pmid: 30399544
[38]	Zhao L, Chen C S, Wang Y, Shen J Z, Ding Z T. 2019. Conserved microRNA act boldly during sprout development and quality formation in Pingyang Tezaocha(Camellia sinensis). Frontiers in Genetics, 10:237. doi: 10.3389/fgene.2019.00237 pmid: 31001312
[39]	Zhao Lei. 2013. Transcription factors analysis in flavonoid biosynthesis pathway and gene function of ANR in tea plant Camellia sinensis(L.) O. Kuntze[Ph. D. Dissertation]. Hefei: Anhui Agricultural University. (in Chinese)
	赵磊. 2013. 茶树类黄酮合成转录因子筛选及ANR基因功能验证[博士论文]. 合肥: 安徽农业大学.
[40]	Zhao Z C, Hu G B, Hu F C, Wang H C, Yang Z Y, Lai B. 2012. The UDP glucose:flavonoid-3-O-glucosyltransferase(UFGT)gene regulates anthocyanin biosynthesis in litchi(Litchi chinesis Sonn.) during fruit coloration. Molecular Biology Reports, 39:6409-6415.
[41]	Zheng C, Ma J Q, Chen J D, Ma C L, Chen W, Yao M Z, Chen L. 2019. Gene coexpression networks reveal key drivers of flavonoid variation in eleven tea cultivars(Camellia sinensis). Journal of Agricultural and Food Chemistry, 67 (35):9967-9978. doi: 10.1021/acs.jafc.9b04422 pmid: 31403784
[42]	Zou Rui, Wang Qingfen, Yang Ziyun, Wu Tian. 2021. Cloning and expression analysis of anthocyanin synthase(ANS)gene in leaves of Zijuan tea. Genomics and Applied Biology, 40 (3):1284-1288. (in Chinese)
	邹瑞, 王青芬, 杨自云, 吴田. 2021. 紫娟茶叶片中花青素合成酶基因的克隆及表达分析. 基因组学与应用生物学, 40 (3):1284-1288.

Functional Characterization of Key Genes CsANS and CsLAR Involved in Catechin Biosynthesis in Camellia sinensis

RichHTML

PDF

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 7

References 42

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	LIU Gangyun, DUAN Shixiang, XU Nana, GUO Yaomiao, DOU Junling, YANG Sen, NIU Huanhuan, LIU Dongming, YANG Luming, HU Jianbin, and ZHU Huayu. Molecular Markers Assisted Construction of Cmerecta Near-Isogenic Line of Melon Dwarfing Gene [J]. Acta Horticulturae Sinica, 2024, 51(9): 2048-2062.
[2]	ZHENG Qianming, WANG Xiaoke, WANG Honglin, ZHOU Jia, XIE Pu, and MA Yuhua. The Transport Activity Detection of Pitaya Sugar Transporters HpSWEET1a and HpSWEET4a by pH Indicator Method [J]. Acta Horticulturae Sinica, 2024, 51(9): 2195-2206.
[3]	QIU Hui, ZHU Dejuan, ZHANG Yongle, GAO Yujie, LI Liu, WANG Guoping, HONG Ni. Interaction Between the Coat Protein of Apple Chlorotic Leaf Spot Virus and Two E3 Ubiquitin Ligases of Pear and Their Subcellular Localization [J]. Acta Horticulturae Sinica, 2024, 51(8): 1715-1727.
[4]	KUANG Meimei, LI Li, MA Jianwei, LIU Yuan, JIANG Hongfei, LEI Rui, MAN Yuping, WANG Yifan, HUANG Bo, WANG Yanchang, LIU Shibiao. Screening of Kiwifruit Canker Resistance-Related Genes Based on the Transcriptome Sequencing Analysis of Hybrids from Actinidia chinensis [J]. Acta Horticulturae Sinica, 2024, 51(8): 1743-1757.
[5]	HAN Ying, DUAN Ying, NIU Yijie, LI Yansu, HE Chaoxing, SUN Mintao, WANG Jun, LI Qiang, CHEN Shuangchen, YAN Yan. Transcription Metabolic Mechanism of Humic Acid Biodegradable Plastic Film to Improving the Fruit Quality of Tomato in the Greenhouse [J]. Acta Horticulturae Sinica, 2024, 51(8): 1758-1772.
[6]	DUAN Minjie, LI Yifei, WANG Chunping, YANG Xiaomiao, HUANG Renzhong, HUANG Qizhong, ZHANG Shicai. Integrated Transcriptomic and Targeted Metabolomic Analysis Reveals Regulation of Carotenoid Accumulation During Pepper Fruit Development [J]. Acta Horticulturae Sinica, 2024, 51(8): 1773-1791.
[7]	ZHAO Jingyi, WU Xiaoxu, HU Yunjie, GAI Shuting, ZHU Zhihao, QIN Lei, WANG Yong. Molecular Cloning and Functional Analysis of AcGAI in Onion Flowering Regulation [J]. Acta Horticulturae Sinica, 2024, 51(8): 1792-1802.
[8]	YANG Jinghui, XU Yuan, XIAO Ting, CHU Shupin, LIU Jixiang, YAO Kebing. Resistance of Colletotrichum gloeosporioides Species Complexes from Grape to Azoxystrobin [J]. Acta Horticulturae Sinica, 2024, 51(8): 1906-1912.
[9]	ZHANG Lihua, XU Yu, Zheng Litong, Wang Changzhi, ZHU Lingcheng, MA Baiquan, and LI Mingjun, . The Relevance Research Between Acid Transporters and Fruit Acidity [J]. Acta Horticulturae Sinica, 2024, 51(7): 1474-1488.
[10]	HUANG Yiqi, ZHU Yuqin, DU Zhaoxuan, XU Feng, CHEN Xiaoyi, YANG Guoshun, and XU Yanshuai, . Progress on Bud Sport of Tree Fruit [J]. Acta Horticulturae Sinica, 2024, 51(7): 1547-1564.
[11]	ZHAO Zeyang, ZHOU Yuqing, LIN Deshu, and REN Huibo, . Advances in Morphogenesis of Petal Conical Epidermal Cells [J]. Acta Horticulturae Sinica, 2024, 51(7): 1695-1706.
[12]	SUN Zhichao, GUO Xinmiao, WANG Hui, XIE Yan, GAO Yanxia, LI Jisheng, GAO Yujun. Mechanism of miR408-MnBBP Module Regulating Anthocyanin Biosynthesis in Mulberry [J]. Acta Horticulturae Sinica, 2024, 51(6): 1216-1226.
[13]	QIN Yu, GUO Rongkun, NONG Huilan, WANG Yan, CUI Kai, DONG Ningguang. Using the Fluorescent Labeled SSR Markers to Establish Molecular Identity of Hawthorn [J]. Acta Horticulturae Sinica, 2024, 51(6): 1227-1240.
[14]	LI Xiang, ZHONG Shiwei, WANG Yiguang, DENG Jinping, FANG Qiu, ZHAO Hongbo. Functional Analysis of OfbHLH89 Gene in Regulating Flower Opening in Osmanthus fragrans [J]. Acta Horticulturae Sinica, 2024, 51(6): 1311-1320.
[15]	DENG Shufang, LIU Qian, LIU Ling, CHEN Ou, WANG Wenjun, ZENG Kaifang, DENG Lili. Cloning of Mandarin Fruit CcHY5 and Its Function in Fruit Coloration [J]. Acta Horticulturae Sinica, 2024, 51(5): 939-955.