茶树乙醇脱氢酶基因家族的鉴定及其在白茶萎凋过程的表达分析

doi:10.13305/j.cnki.jts.2021.03.002

摘要/Abstract

摘要： 乙醇脱氢酶（Alcohol dehydrogenase,ADH）作为植物香气物质合成的脂肪酸代谢等途径中关键酶之一,对茶叶芳香物质的形成有着重要作用。从茶树染色体级别的基因组数据库中鉴定到19个CsADH基因家族成员。系统进化树显示,茶树的ADH基因家族成员分成6个亚家族;共线性分析发现,茶树和拟南芥、葡萄与猕猴桃的ADH基因家族之间分别存在2、4、12对共线性关系;茶树ADH基因家族含有1~13个外显子,编码其氨基酸的数目为236~669,分子量为26.15~73.83 kDa,且主要定位于细胞质和叶绿体,仅CsADH1定位于细胞核。此外,对上游启动子区域分析发现了大量与光响应、植物生长发育、胁迫和激素响应密切相关的顺式作用元件。荧光定量检测发现,CsFDH2在萎凋4 h时表达量最高;CsADH4和CsADH10在萎凋32 h时表达量最高,分别是对照的4.11倍和3.54倍;CsADH3在萎凋48 h时达到峰值,略高于萎凋32 h的表达量;CsADH-like1在萎凋40 h时表达量达到最高值;CsADH-like3在萎凋24 h时表达量最高。以上结果为探究萎凋过程中乙醇脱氢酶基因对白茶脂肪族类芳香物质形成的分子机理提供参考。

关键词: 白茶, 萎凋, 香气, ADH基因家族, 表达分析

Abstract: Alcohol dehydrogenase (ADH) plays an important role in the formation of tea aroma as one of the key enzymes in the synthesis of fatty acid metabolism pathway. In this study, 19 CsADH gene family members were identified from the chromosome level genome database of tea plants for the first time. Bioinformatics analysis shows that the members of ADH gene family were divided into six subfamilies. Collinearity analysis shows that there were 2, 4 and 12 pairs of collinearity between ADH gene family of Camellia sinensis and Arabidopsis thaliana, Vitis vinifera and Actinidia chinensis, respectively. The tea ADH gene family contains 1-13 exons, which encode 236-669 amino acids with molecular weight of 26.15-73.83 kDa. It is mainly located in cytoplasm and chloroplast, and only CsADH1is located in nucleus. In addition, a large number of cis-acting elements closely related to light responsive, plant growth, stress and phytohormone responsive were found in the upstream promoter region. Fluorescence quantitative detection shows that the expression of CsFDH2 was the highest at 4 h of withering. The expressions of CsADH4 and CsADH10 were the highest at 32 h of withering, which were 4.11 and 3.54 times that of the control respectively. The expression of CsADH3 reached the peak at 48 h of withering, which was slightly higher than that at 32 h of withering. The expression of CsADH-like1 reached the highest value at 40 h of withering. The highest expression of CsADH-like3 was at 24 h of withering. This study provided a reference for exploring the molecular mechanism of alcohol dehydrogenase genes acting on the formation of aliphatic aromatic substances in the withering process of white tea.

Key words: white tea, withering, aroma, ADH gene family, expression analysis

中图分类号:

S571
Q52

谷梦雅, 王鹏杰, 陈雪津, 郑玉成, 郭永春, 林馨颖, 高婷, 侯炳豪, 叶乃兴. 茶树乙醇脱氢酶基因家族的鉴定及其在白茶萎凋过程的表达分析[J]. 茶叶科学, 2021, 41(3): 302-314. doi: 10.13305/j.cnki.jts.2021.03.002.

GU Mengya, WANG Pengjie, CHEN Xuejin, ZHENG Yucheng, GUO Yongchun, LIN Xinying, GAO Ting, HOU Binghao, YE Naixing. Identification of Alcohol Dehydrogenase Gene Family and Their Expression Analysis in the Withering Process of White Tea[J]. Journal of Tea Science, 2021, 41(3): 302-314. doi: 10.13305/j.cnki.jts.2021.03.002.

参考文献

[1] 郭永春, 王鹏杰, 陈笛, 等. 茶树SRO基因家族的鉴定及表达分析[J]. 茶叶科学, 2019, 39(4): 392-402.
Guo Y C, Wang P J, Chen D, et al.Genome-wide identification and expression analysis of SRO gene family in Camellia sinensis[J]. Journal of Tea Science, 2019, 39(4): 392-402.
[2] 叶乃兴. 白茶:科学·技术与市场[M]. 北京: 中国农业出版社, 2010: 136-137.
Ye N X.The science technology and market of white tea [M]. Beijing: China Agriculture Press, 2010: 136-137.
[3] Zhao F, Qiu X H, Ye N X, et al.Hydrophilic interaction liquid chromatography coupled with quadrupole-orbitrap ultra high resolution mass spectrometry to quantitate nucleobases, nucleosides, and nucleotides during white tea withering process[J]. Food Chemistry, 2018, 266: 343-349.
[4] Fu X M, Chen Y Y, Mei X, et al.Regulation of formation of volatile compounds of tea (Camellia sinensis) leaves by single light wavelength[J]. Scientific Reports, 2015, 5: 16858. doi: 10.1038/srep16858.
[5] 陈雪津, 王鹏杰, 林馨颖, 等. 白茶萎凋过程萜烯类合成相关基因的鉴定和表达分析[J]. 茶叶科学, 2020, 40(3): 363-374.
Chen X J, Wang P J, Lin X Y, et al.Identification and expression analysis of terpene synthesis related genes during the withering of white tea[J]. Journal of Tea Science, 2020, 40(3): 363-374.
[6] 王鹏杰, 陈丹, 俞滢, 等. 茶树单萜合成酶CsTPS基因的克隆及表达分析[J]. 西北植物学报, 2017, 37(8): 1465-1473.
Wang P J, Chen D, Yu Y, et al.Cloning and expression of monoterpene synthase gene CsTPS in tea plant (Camellia sinensis)[J]. Acta Botanica Boreali-Occidentalia Sinica, 2017, 37(8): 1465-1473.
[7] Chase T.Alcohol dehydrogenases: identification and names for gene families[J]. Plant Molecular Biology Reporter, 1999, 17(4): 333-350.
[8] Hedlund J, Jornvall H, Persson B.Subdivision of the MDR superfamily of medium-chain dehydrogenases/reductases through iterative hidden Markov model refinement[J]. BMC Bioinformatics, 2010, 11: 534. doi: 10.1186/1471-2105-11-534.
[9] Jörnvall H, Hedlund J, Bergman T, et al.Superfamilies SDR and MDR: from early ancestry to present forms. Emergence of three lines, a Zn-metalloenzyme, and distinct variabilities[J]. Biochemical and Biophysical Research Communications, 2010, 396(1): 125-130.
[10] Hageman R H, Flesher D.The effect of an anaerobic environment on the activity of alcohol dehydrogenase and other enzymes of corn seedings[J]. Arch Biochem Biophys, 1960, 87: 203-209.
[11] Jin Y Z, Zhang C, Liu W, et al.The alcohol dehydrogenase gene family in melon (Cucumis melo L.): bioinformatic analysis and expression patterns[J]. Frontiers in Plant Science, 2016, 7: 670. doi: 10.3389/fpls.2016.00670.
[12] Zeng W W, Qiao X, Li Q H, et al.Genome-wide identification and comparative analysis of the ADH gene family in Chinese white pear (Pyrus bretschneideri) and other Rosaceae species[J]. Genomics, 2020, 112(5): 3484-3496.
[13] Tesniere C, Verries C.Molecular cloning and expression of cDNAs encoding alcohol dehydrogenases from Vitis vinifera L. during berry development[J]. Plant Science, 2000, 157(1): 77-88.
[14] Su W H, Ren Y J, Wang D J, et al.The alcohol dehydrogenase gene family in sugarcane and its involvement in cold stress regulation[J]. BMC Genomics, 2020, 21(1): 521. doi: 10.1186/s12864-020-06929-9.
[15] Singh R K, Sane V A, Misra A, et al.Differential expression of the mango alcohol dehydrogenase gene family during ripening[J]. Phytochemistry, 2010, 71(13): 1485-1494.
[16] Garabagi F, Duns G, Strommer J.Selective recruitment of Adh genes for distinct enzymatic functions in Petunia hybrida[J]. Plant Molecular Biology, 2005, 58(2): 283-294.
[17] Peters J S, Frenkel C.Relationship between alcohol dehydrogenase activity and low-temperature in two maize genotypes, Silverado F₁ and Adh1-Adh2- doubly null[J]. Plant Physiology and Biochemistry, 2004, 42(10): 841-846.
[18] 刘威, 陈昊, 靳亚忠, 等. 高等植物醇脱氢酶及其基因家族研究进展[J]. 植物生理学报, 2014, 50(10): 1479-1493.
Liu W, Chen H, Jin Y Z, et al.Advances in alcohol dehydrogenase enzymes and their gene families in higher plants[J]. Plant Physiology Journal, 2014, 50(10): 1479-1493.
[19] 辛肇军, 孙晓玲, 陈宗懋. 茶树醇脱氢酶基因的表达特征及番茄遗传转化分析[J]. 西北植物学报, 2013, 33(5): 864-871.
Xin Z J, Sun X L, Chen Z M.Expression character of tea alcohol dehydrogenase gene and tomato transformation[J]. Acta Botanica Boreali-Occidentalia Sinica, 2013, 33(5): 864-871.
[20] 彭海娇. 茶蚜降低茶树对茶尺蠖幼虫防御反应的研究[D]. 长春: 东北师范大学, 2015.
Peng H J.The research on tea aphid infestation reduce the defensive response of tea plant to tea geometrid larvae [D]. Changchun: Northeast Normal University, 2015.
[21] 高晨, 郑玉成, 周珍, 等. 茶树乙醇脱氢酶基因CsADH2的克隆与表达分析[J]. 福建农业学报, 2018, 33(12): 1257-1263.
Gao C, Zheng Y C, Zhou Z, et al.Cloning and expression of CsADH2 in tea plant (Camellia sinensis)[J]. Fujian Journal of Agricultural Science, 2018, 33(12): 1257-1263.
[22] Zhou Z W, Wu Q Y, Yao Z L, et al.Dynamics of ADH and related genes responsible for the transformation of C₆-aldehydes to C₆-alcohols during the postharvest process of oolong tea[J]. Food Science & Nutrition, 2019, 8(1): 104-113.
[23] 王鹏杰, 郑玉成, 林浥, 等. 茶树GRF基因家族的全基因组鉴定及表达分析[J]. 西北植物学报, 2019, 39(3): 413-421.
Wang P J, Zheng Y C, Lin Y, et al.Genome-wide identifiction and expression analysis of GRF gene family in Camellia sinensis[J]. Acta Botanica Boreali-Occidentalia Sinica, 2019, 39(3): 413-421.
[24] Strommer J.The plant ADH gene family[J]. The Plant Journal, 2011, 66(1): 128-142.
[25] Perry D J, Furnier G R.Pinus banksiana has at least seven expressed alcohol dehydrogenase genes in two linked groups[J]. PNAS, 1996, 93(23): 13020-13023.
[26] Xu G X, Guo C C, Shan H Y, et al.Divergence of duplicate genes in exon-intron structure[J]. PNAS, 2012, 109(4): 1187-1192.
[27] Manríquez D, El-Sharkawy I, Flores F B, et al.Two highly divergent alcohol dehydrogenases of melon exhibit fruit ripening-specific expression and distinct biochemical characteristics[J]. Plant Molecular Biology, 2006, 61(4): 675-685.
[28] Speirs J, Lee E, Holt K, et al.Genetic manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the balance of some flavor aldehydes and alcohols[J]. Plant Physiol, 1998, 117(3): 1047-1058.
[29] 滑金杰, 袁海波, 江用文, 等. 萎凋过程鲜叶理化特性变化及其调控技术研究进展[J]. 茶叶科学, 2013, 33(5): 465-472.
Hua J J, Yuan H B, Jiang Y W, et al.A review on the regulation technique of withering process and the change in physical and chemical properties of leaves[J]. Journal of Tea Science, 2013, 33(5): 465-472.
[30] 俞少娟, 李鑫磊, 王婷婷, 等. 白茶香气及萎凋工艺对其形成影响的研究进展[J]. 茶叶通讯, 2015, 42(4): 14-18.
Yu S J, Li X L, Wang T T, et al.Research progress on white tea flavor and its withering processing[J]. Journal of Tea Communication, 2015, 42(4): 14-18.
[31] 陈维, 马成英, 王雯雯, 等. 萎凋时间对“英红九号”白茶香气的影响[J]. 食品科学, 2017, 38(18): 138-143.
Chen W, Ma C Y, Wang W W, et al.Effects of withering duration on the aroma profile of Yinghong No. 9 white tea[J]. Food Science, 2017, 38(18): 138-143.
[32] 李凤娟. 白茶的滋味、香气和加工工艺研究[D]. 杭州: 浙江大学, 2012.
Li F J.Studies on the taste, aroma and processing of white tea [D]. Hangzhou: Zhejiang University, 2012.