Cloning and Expression of CsADH2 in Tea Plant (Camellia sinensis)
-
摘要: 在茶树转录组测序基础上，采用RT-PCR技术，克隆了茶树乙醇脱氢酶基因，命名为CsADH2。生物信息学分析结果表明，该基因全长1 540 bp，其开放阅读框（ORF）长度为1 182 bp，编码393个氨基酸，亚细胞定位预测位于细胞质。编码的氨基酸序列与猕猴桃、向日葵和莴苣的ADH相似性分别达到85%、85%和84%。荧光定量PCR结果表明，CsADH2在茶树各组织中均有表达，在茶树叶片中的表达量最高，在茶树根的表达量最低；在白茶萎凋过程中，CsADH2相对表达量在萎凋2 h时开始上升，萎凋2~8 h相对表达量下降；12 h诱导表达上升至最高，为0 h时的6倍；CsADH2相对表达量在24~48 h处于降低趋势，但仍然显著高于0 h的相对表达量，表明CsADH2可能与白茶加工萎凋过程中脂肪族类香气物质的形成有关。Abstract: Based on the transcriptome database on tea plants (Camellia sinensis), the full-length cDNAs of the alcohol dehydrogenase gene (CsADH2) was cloned from Fudingdabai using RT-PCR. A bioinformatics analysis showed that the cDNA had a full length of 1 540 bp with a 1 182 bp ORF encoding 393 amino acids and located probably in the cytoplasm. The amino acids of CsADH2 was 85% homogenous to those of Actinidia deliciosa and Helianthus annuus L, and 84% to that of Lactuca sativa Linn. The results of qRT-PCR showed that CsADH2 expressed differently in different tissues of a tea plant with the highest in the leaves and the lowest in the roots. During white tea withering, the expression of CsADH2 began to up-regulate in the first 2 h and down-regulated when treated for 2-8 h. The expression peaked at 12th h reaching a level 6 times as high as that at the beginning. The expression was down-regulated in 24-48 h, but still was significantly higher than that of 0 h. It appeared that CsADH2 played an important role in the formation of the aromatic lipids in tea.
-
图 1 茶树CsADH2基因PCR扩增结果
注：M为DNA分子量标准; 1为CsADH2基因PCR产物。
Figure 1. PCR amplification of CsADH2
图 2 茶树CsADH2的cDNA全长序列及推导的氨基酸序列
注：方框内为ATG起始子和TAG终止子。
Figure 2. Nucleotide sequence and deduced amino acid sequence of CsADH2 of tea plant
图 4 CsADH2多序列比对及功能结构域分析
注：CsADH2为茶树；AdADH为绿果猕猴桃(ANA12162.1)；LsADH为莴苣ADH(BAA07911.1)；HaADH为向日葵ADH(XP_022039657.1)。
Figure 4. Multiple sequence comparison and functional domain analysis on CsADH2
图 5 NJ法构建的CsADH2氨基酸序列的系统进化树
Figure 5. Phylogenetic tree of CsADH2 amino acid sequence structured by NJ method
图 6 不同组织、白茶萎凋过程中CsADH2的相对表达量
注：图中小写字母不同表示差异显著(P < 0.05)。
Figure 6. Relative expressions of CsADH2 in different tissues and during white tea withering process
表 1 引物序列
Table 1. Sequences of primers
引物名称 序列(5′-3′) 用途 ADH2-F ATGAGGCAGCTAGCAATGCAATC 验证CsADH全长cDNA序列 ADH2-R CCGATGCTAAGGATTTCATCATT q ADH2-F ATTCAACGCTTCTGTCC 实时荧光定量PCR q ADH2-R GCTCAACTGTGGCTGTC q CsGAPDH-F TTGGCATCGTTGAGGGTCT 茶树内参基因 q CsGAPDH-R CAGTGGGAACACGGAAAGC 
下载: 导出CSV
-
[1] FUKUDA T, YOKOYAMA J, NAKAMURA T, et al. Molecular phylogeny and evolution of alcohol dehydrogenase (Adh) genes in legumes[J]. BMC Plant Biology, 2005, 5(1):6. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=Open J-Gate000001533589 [2] STROMMER J. The plant ADH gene family[J]. The Plant Journal, 2011, 66(1):128-142. doi: 10.1111/tpj.2011.66.issue-1 [3] KOMATSU S, THIBAUT D, HIRAGE S, et al. Characterization of a novel flooding stress-responsive alcohol dehydrogenase expressed in soybean roots[J]. Plant Molecular Biology, 2011, 77(3):309-322. doi: 10.1007/s11103-011-9812-y [4] 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. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zwslxtx201410006 [5] 张计育, 王刚, 黄胜男, 等.乙醇脱氢酶基因家族在植物抵抗非生物胁迫过程中的作用研究进展[J].中国农学通报, 2015, 31(10):246-250. doi: 10.11924/j.issn.1000-6850.casb14120001 [6] 廖凤玲, 汪志辉, 熊博, 等.爱甘水梨果实发育过程香气形成与代谢关键酶活性的关系[J].华北农学报, 2014, 29(2):152-155. http://d.old.wanfangdata.com.cn/Periodical/hbnxb201402028 [7] 齐红岩, 李岩, 关小川, 等.两个不同类型薄皮甜瓜品种成熟特性、香气成分及其相关酶活性分析[J].中国农业科学, 2011, 44(4):771-780. doi: 10.3864/j.issn.0578-1752.2011.04.015 [8] 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. doi: 10.3969/j.issn.1000-4025.2013.05.002 [9] 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. http://d.old.wanfangdata.com.cn/Periodical/xbzwxb201708001 [10] KENNETH J, LIVAK, THOMAS D, SCHMITTHGE. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-ΔΔ CT Method[J]. Methods, 2001, 25(4):402-408. doi: 10.1006/meth.2001.1262 [11] 郭宇立, 倪逸声.从一级结构预测蛋白质稳定性——Guruprasad, Reedy和Pandit方法[J].生物化学与生物物理进展, 1993(2):95-99. [12] 武雪, 黄晓丽, 王喆之.葡萄乙醇脱氢酶基因Ⅲ的电子克隆及生物信息学分析[J].生物技术通报, 2009(5):71-75. http://d.old.wanfangdata.com.cn/Periodical/swjstb200905018 [13] 苏炜华, 黄宁, 凌辉, 等.甘蔗乙醇脱氢酶基因的克隆与表达[J].应用与环境生物学报, 2017, 23(3):474-481. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=yyyhjswxb201703012 [14] 胡涛.拟南芥乙醇脱氢酶纯化及酶学性质研究[D].杭州: 浙江农林大学, 2015. http://cdmd.cnki.com.cn/Article/CDMD-10341-1015654820.htm [15] 王绍华.香蕉乙醇脱氢酶基因的克隆及其表达分析[D].海口: 海南大学, 2010. http://cdmd.cnki.com.cn/Article/CDMD-10589-2010183584.htm [16] 靳亚忠.甜瓜醇脱氢酶基因家族成员鉴定、分类及表达调控[D].沈阳: .沈阳农业大学, 2014. http://cdmd.cnki.com.cn/Article/CDMD-10157-1014295086.htm [17] 宁文彬, 刘菊华, 贾彩红, 等.香蕉果实采后乙醇脱氢酶活性与乙烯代谢的关系[J].果树学报, 2009, 26(3):386-389. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=gskx200903024 [18] 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. http://cdmd.cnki.com.cn/Article/CDMD-10335-1013187067.htm [19] 叶乃兴.白茶科学·技术与市场[M].北京:中国农业出版社, 2010. [20] 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. http://d.old.wanfangdata.com.cn/Periodical/cytx201504004 -
点击查看大图
计量
- 文章访问数: 1488
- HTML全文浏览量: 259
- PDF下载量: 18
- 被引次数: 0
