Cloning and Expression of CsADH2 in Tea Plant (Camellia sinensis)
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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.
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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 
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