茶儿茶素合成关键酶基因CsANS和CsLAR的功能鉴定

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

摘要/Abstract

摘要：

以‘英红9号’茶树[Camellia sinensis（L.）O. Kuntze]为材料，克隆了儿茶素合成途径的两个关键酶基因CsANS和CsLAR，并利用原核表达、亚细胞定位以及转基因表达分析对其功能进行鉴定。结果表明，CsANS和CsLAR开放阅读框（open reading frame，ORF）分别长1 068和1 029 bp，与TPIA数据库中来源于‘舒茶早’的对应基因同源性均高达99%。CsANS和CsLAR可溶性蛋白均能在原核表达系统诱导获得，并经GST标签成功纯化。本氏烟草叶片的瞬时表达结果显示，CsANS和CsLAR编码的蛋白定位于细胞质和细胞核。利用农杆菌介导的遗传转化方法在茶愈伤组织中超表达CsANS和CsLAR，结果显示其均能显著促进总儿茶素的合成，其中CsANS主要促进顺式儿茶素（EC和EGCG）含量的增加，而CsLAR主要促进反式儿茶素（C、GC、CG和GCG）的积累。推测在‘英红9号’茶树中CsANS的功能主要是促进顺式儿茶素的合成，而CsLAR更利于反式儿茶素的合成。

关键词: 茶树, 儿茶素, ANS, LAR, 过表达

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

张芷苓, 张媛媛, 林晓蓉, 李斌, 陈忠正. 茶儿茶素合成关键酶基因CsANS和CsLAR的功能鉴定[J]. 园艺学报, 2024, 51(4): 804-814.

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.

图/表 7

表1 本研究中的qPCR引物信息

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

图1 茶树CsANS和CsLAR基因PCR扩增电泳图

Fig. 1 The electrophoresis of PCR amplification CsANS and CsLAR

图2 pGEX-4T-1-CsANS（A）和pGEX-4T-1-CsLAR（B）重组蛋白的SDS-PAGE分析 1：诱导前；2：破碎前；3：诱导8 h后；4：破碎后；5：离心后上清液；6：离心后沉淀；7 ~ 8：回收蛋白。红色箭头指的是回收蛋白的条带。

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.

图3 CsANS和CsLAR的亚细胞定位

Fig. 3 The subcellular localization of CsANS and CsLAR

图4 CsANS（A）和CsLAR（B）过表达转基因茶愈伤组织的筛选培养

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

图5 过表达愈伤组织中G418基因的扩增鉴定

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

图6 茶树CsANS和CsLAR过表达愈伤组织（CsANS-OE和CsLAR-OE）中基因表达水平和儿茶素组分含量

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.

参考文献 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.