茶树1-脱氧-D-木酮糖-5-磷酸合成酶基因CsDXS1的克隆与表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2017-0652

摘要/Abstract

摘要： 1-脱氧-D-木酮糖-5-磷酸合成酶（1-deoxy-D-xylulose 5-phosphate synthase,DXS）是MEP 萜类合成途径的第一个关键酶,在植物萜类物质的生物合成过程中发挥重要的作用。以福鼎大白茶为实验材料,在课题组前期转录组数据的基础上,运用 RT-PCR 技术克隆了茶树CsDXS1基因的完整开放阅读框（ORF）。该 ORF长度为 2 154 bp,编码717个氨基酸。生物信息学分析结果表明,茶树CsDXS1蛋白与其他植物的同源蛋白相似性高达85%-90%,属于 DXS基因家族的I类基因,该蛋白是不稳定的亲水蛋白,不存在信号肽和跨膜结构域,有15个可信度高的磷酸化位点,具有典型的转酮醇酶亚家族功能域。利用实时荧光定量 PCR 对 CsDXS1基因在茶树不同组织和不同激素处理下的表达谱进行检测,结果显示：不同组织中,CsDXS1基因的表达水平在第三叶中最高,嫩叶中的表达量显著高于茎和老叶,表达水平从高到低依次为第三叶>第四叶>第二叶>第一叶>嫩茎>老茎>老叶。在不同激素处理中,CsDXS1的表达量受到不同程度的诱导,且表达量峰值的出现时间存在差异。CsDXS1在IAA和ABA激素处理下的表达量峰值最高（4 h）,均为对照的1.5倍,在 MeJA 处理下表达量峰值最低（24 h）,仅为对照的1.1倍。

关键词: 茶树, 1-脱氧-D-木酮糖-5-磷酸合成酶, 萜类化合物, 序列分析, 表达分析

Abstract: 1-deoxy-D-xylulose-5-phosphate synthase（DXS）is the first key enzyme of the MEP terpenoids synthetic pathway,thus it plays an important role in the biosynthesis of plant terpenoid. In this study,the complete ORF of CsDXS1 gene was cloned from Camellia sinensis var. fudingdabaicha based on the previous tea plant transcriptome database,and it’s length was 2 154 bp encoding 717 amino acids. The bioinformatics analysis indicated that CsDXS1 gene belonged to the class I of DXS gene family. The deduced CsDXS1 protein shared high similarity（85%-90%）with other homologous protein,and it was an instable and hydrophilic protein with no signal peptide and transmembrane domain. There were 15 high-probability phosphorylation sites. Furthermore,CsDXS1 had two typical conserved domains of transketolase superfamily. The expression profiles of CsDXS1 gene in different tissues of tea plant or under hormonal treatments were detected using quantitative real-time PCR analysis. Results showed that CsDXS1 exhibited the highest expression in the third leaf,and higher expressions in the young leaves than in the steam and old leaves. The tissue expression profiles showed that the successive order as the third leaf > the fourth leaf > the first leaf > the second leaf > young stem > old stem >old leaf. The CsDXS1 gene expression were induced at varied degrees in response to six hormonal treatments,also the occurrence times of expression peak differed. The expressions of CsDXS1 gene was in the peak（4 h）under IAA and ABA treatments,which was 1.5 times of the control. CsDXS1 gene was at the lowest expression（24 h）under MeJA treatment,which was only 1.1 times of the control.

Key words: Camellia sinensis, DXS,terpenoids, sequence analysis, gene expression

郭亚飞, 王君雅, 郭飞, 倪德江. 茶树1-脱氧-D-木酮糖-5-磷酸合成酶基因CsDXS1的克隆与表达分析[J]. 生物技术通报, 2018, 34(1): 144-152.

GUO Ya-fei, WANG Jun-ya, GUO Fei, NI De-jiang. Cloning and Expression Analysis of CsDXS1 Gene Encoding 1-Deoxy-D-Xylulose-5-Phosphate Synthase in Camellia sinensis[J]. Biotechnology Bulletin, 2018, 34(1): 144-152.

参考文献

[1] 张正竹, 施兆鹏, 宛晓春. 萜类物质与茶叶香气（综述）[J]. 安徽农业大学学报, 2000, 27：51-53.
[2] 袁海波, 尹军峰, 叶国柱, 等. 茶叶香型及特征物质研究进展[J]. 中国茶叶, 2009, 31（8）：14-15.
[3] 金蓉, 朱长青, 徐昌杰. 1-脱氧木酮糖-5-磷酸合成酶（DXS）及其编码基因[J]. 细胞生物学杂志, 2007, 29：706-712.
[4] 李莉, 高凌云, 董越, 等. 植物类异戊二烯生物合成相关酶基因研究进展[J]. 浙江师范大学学报：自然科学版, 2008, 31（4）：461-466.
[5] 张冬姚, 孙君, 叶乃兴, 等. 茶树萜烯类香气物质合成相关酶研究进展[J]. 茶叶学报, 2015, 56（2）：68-79.
[6] 赵飞. 茶树品种及加工工艺对茶叶中糖苷类香气前体的影响[D]. 杭州：浙江大学, 2012.
[7] 王道平, 甘秀海, 等. 固相微萃取法与同时蒸馏萃取法提取茶叶香气成分[J]. 西南农业学报, 2013, 26（1）：131-135.
[8] 程波, 杨伟俊, 何江, 等. 10种药用植物萜类合酶（DXS）生物信息学分析[J]. 中国农学通报, 2015, 31（2）：146-152.
[9] Cordoba E, Salmi M, León P. Unravelling the regulatory mechanisms that modulate the MEP pathway in higher plants[J]. J Exp Bot, 2009, 60（10）：2933-2943.
[10] Xiang S, Usunow G, Lange G, et al. Crystal structure of 1-deoxy-D-xylulose 5-phosphate synthase, a crucial enzyme for isoprenoids biosynthesis[J]. J Biol Chem, 2007, 282（4）：2676-2682.
[11] Battilana J, Costantini L, Emanuelli F, et al. The 1-deoxy-D-xylulose 5-phosphate synthase gene co-localizes with a major QTL affecting monoterpene content in grapevine[J]. Theor Appl Genet, 2009, 118：653-669.
[12] Estévez JM, Cantero A, Romero C, et al. Analysis of the expression of CLA1, a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis[J]. Plant physiology, 2000, 124（1）：95-104.
[13] Muñoz-Bertomeu J, Arrillaga I, Ros R, et al. Up-regulation of 1-Deoxy-D-Xylulose-5-Phosphate synthase enhances production of essential oils in transgenic spike lavender[J]. Plant Physiology, 2006, 142：890-900.
[14] Peebles CA, Sander GW, Hughes EH, et al. The expression of 1-deoxy-D-xylulose synthase and geraniol-10-hydroxylase or anthranilate synthase increases terpenoid indole alkaloid accumulation in Catharanthus roseus hairy roots[J]. Metab Eng, 2011, 13：234-240.
[15] Khemvong S, Suvachittanont W. Molecular cloning and expression of a cDNA encoding 1-deoxy-D-xylulose-5-phosphate synthase from oil palm Elaeis guineensis Jacq[J]. Plant Science, 2005, 169（3）：571-578.
[16] Rodríguez-Concepción M, Ahumada I, Diez-Juez E, et al. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase and plastid isoprenoid biosynthesis during tomato fruit ripening[J]. Plant J, 2001, 27 （3）：213-222.
[17] Lois LM, Rodríguez-Concepción M, Gallego F, et al. Carotenoid biosynthesis during tomato fruit development：regulatory role of 1-deoxy-d-xylulose 5-phosphate synthase[J]. The Plant Journal, 2000, 22（6）：503-513.
[18] 刘晶晶, 王富民, 刘国峰, 等. 茶树萜类香气物质代谢谱与相关基因表达谱时空变化的关系[J]. 园艺学报, 2014, 41（10）：2094-2106.
[19] Cordoba E1, Porta H, Arroyo A, et al. Functional characterization of the three genes encoding 1-deoxy-D-xylulose 5-phosphate synthase in maize[J]. J Exp Bot, 2011, 6：2023-2038.
[20] Kim BR, Kim SU, Chang YJ. Differential expression of three 1-deoxy-D-xylulose-5-phosphate synthase genes in rice[J]. Biotechnology Letters, 2005, 27：991-1001.
[21] Florence B, Alain H, Claud S, at el. Dedicated roles of plastid transketolases during the early onset of isoprenoid biogenesis in pepper fruits[J]. Plant Physiology, 1998, 117：1423-1431.
[22] Cordoba E, Porta H, Arroyo A, et al. Functional characterization of the three genes encoding 1-deoxy-D-xylulose 5-phosphate synthase in maize[J]. J Exp Bot, 2011, 62（6）：2023-2038.
[23] 岳川, 曹红利, 曾建明, 等. 茶树中植物激素研究进展[J]. 茶叶科学, 2012, 32（5）：382-392.
[24] 林坤律, 高锦华. 赤霉素对茶树新梢生长, 茶叶品质和产量的影响[J]. 植物生理学通讯, 1981, 3：22-27.
[25] Kim YB, Kim SM, Kang MK, et al. Regulation of resin acid synthesis in Pinus densiflora by differential transcription of genes encoding multiple 1-deoxy-D-xylulose 5-phosphate synthase and 1-hydroxy-2-methyl-2-（E）-butenyl 4-diphosphate reductase genes[J]. Tree Physiology, 2009, 29：737-749.
[26] Cho JY, Mizutani M, Shimizu B, et al. Chemical profiling and gene expression profiling during the manufacturing process of Taiwan oolong tea “oriental beauty”[J]. Biosci Biotechnol Biochem, 2007, 71（6）：1476-1486.
[27] 陈寿松, 林宏政, 孙云, 等. 乌龙茶萜类物质及其代谢调控研究进展[J]. 中国农业科技导报, 2016, 18（5）：72-80.
[28] 彭浩, 宋文路, 王晓强. 水杨酸, 脱落酸对盐胁迫下玉米种子萌发和幼苗生长的影响[J]. 玉米科学, 2016, 6：013.