茶树CsIDM的鉴定、表达分析及互作验证

doi:10.16420/j.issn.0513-353x.2021-1218

摘要/Abstract

摘要：

以不同茶树（Camellia sinensis）基因组为参考，通过序列同源性分析，在‘龙井43’中鉴定并克隆到3个CsIDM基因。CsIDM1属于组蛋白乙酰转移酶家族，具有典型的HAT保守结构域，定位于细胞核中。CsIDM2、CsIDM3均属于热激蛋白家族，具有典型的ACD保守结构域，均定位于细胞核、细胞膜及细胞质中。CsIDM启动子区域含有多种响应环境信号的顺式作用元件，主要为光响应、植物激素响应、胁迫响应和植物生长发育相关。CsIDM在花蕾、腋芽、茎中表达量较高，在盛花组织中较低。在干旱、高温及生物胁迫下，CsIDM均出现不同程度的差异表达。茶树越冬芽休眠形成与解除过程中，CsIDM1的表达丰度在休眠形成、深休眠和休眠解除3个阶段存在高—低—高的表达模式。通过双分子荧光互补试验证实，CsIDM2和CsIDM3之间可形成异源二聚体;CsIDM2与CsMBD5、CsIDM3与CsMBD16存在相互作用。综上所述，CsIDM在茶树的胁迫应答和芽休眠解除中发挥作用，可能通过形成蛋白复合体参与甲基化调控。

关键词: 茶树, 去甲基化, IDM复合物, 亚细胞定位, 表达分析, BiFC互作

Abstract:

In this study，using different tea plant genomes as a reference，three CsIDM genes were identified and cloned from Camellia sinensis‘Longjing 43’. CsIDM1，belongs to the histone acetyltransferase family，has a typical HAT conserved domain，and is located in the nucleus. CsIDM2 and CsIDM3，which belong to the heat shock protein family，have typical ACD conserved domains，which are located in the nucleus，cell membrane and cytoplasm. The promoter region of CsIDMs contains a variety of cis-acting elements that respond to environmental signals，including light response，plant hormone response，stress response and plant growth-related elements. Expression analysis showed that CsIDMs expressed higher in flower buds，axillary buds and stems，but lower in fully open flowers，which suggests a certain tissue specificity. CsIDMs were differentially expressed under drought，high temperature and biotic stresses. During the formation and release of dormancy in overwintering buds，the expression of CsIDM1 showed a high-low-high expression pattern in the stages of paradormancy，endodormancy and ecodormancy. It was confirmed that CsIDM2 interacts with CsIDM3 to form a heterodimer by bimolecular fluorescence complementation. CsIDM2 and CsIDM3 interact with CsMBD5 and CsMBD16，respectively. In summary，CsIDMs play an important role in the stress response and bud dormancy removal of tea plants，and may participate in the regulation of methylation through the formation of protein complexes.

Key words: Camellia sinensis, demethylation, IDM complex, subcellular localization, expression analysis, BiFC assay

李宇腾, 陈瑶, 任恒泽, 李聪聪, 王浩乾, 曹红利, 岳川, 郝心愿, 王新超. 茶树CsIDM的鉴定、表达分析及互作验证[J]. 园艺学报, 2023, 50(8): 1679-1696.

LI Yuteng, CHEN Yao, REN Hengze, LI Congcong, WANG Haoqian, CAO Hongli, YUE Chuan, HAO Xinyuan, WANG Xinchao. Identification，Expression Analysis and Interaction Validation of CsIDM in Tea Plants[J]. Acta Horticulturae Sinica, 2023, 50(8): 1679-1696.

图/表 13

表1 引物信息

Table 1 Primers information

用途 Application	序列名称 Sequence name	引物序列（5′-3′） Primer sequence
基因克隆 Gene clone	CsIDM1	F：GGTACTGGTGTTCATTGATTAGAGCAAAGAAAG R：TTATTTTTATTTCTCATTATCTTGCAACATAGTGG
	CsIDM2	F：ATGGATGACTTGTCTCATGCT R：TTATATCCCTTTCATCACAATTCCTTC
	CsIDM3	F：ATTATTCTGGTTGATTTCTAGCCCCA R：TCATCTGCATAAAATATGAACACCATTC
qRT-PCR	Q-CsIDM1	F：ACTTTCGGATCAACTACCAAACGAT R：AAACATGGACCACTTCCCGTCA
	Q-CsIDM2	F：GATCTATTTCCTCCCCGGCTT R：TTCTAGCCCAGTTAGCCGCTTG
	Q-CsIDM3	F：GATGGGCCTTTCATGATGCC R：ATGCAGATTTGCCGACTCCA
内参基因 Reference gene	Q-CsPTB	F：TGACCAAGCACACTCCACACTATCG R：TGCCCCCTTATCATCATCCACAA
亚细胞定位 Subcellular localization	CsIDM1-GFP	F：CGAGAGTGTCGGAGCTATGTTATTCAACAAAGAAATCG R：GCCCTTGCTCACCATGTCGACTTTCTCATTATCTTGCAACA
	CsIDM2-GFP	F：GGTACCCGGGGATCCTCTAGAATGGATGACTTGTCTCATGC R：GCCCTTGCTCACCATGTCGACTATCCCTTTCATCACAATTC
	CsIDM3-GFP	F：GGTACCCGGGGATCCTCTAGAATGTGTACGTGCCCAAAGCA R：GCCCTTGCTCACCATGTCGACCTCATATTTCATGATGATAG
BIFC载体构建 BIFC vector construction	CsIDM1-BIFC	F：CGAACGATAGTTAATTAAATGTTATTCAACAAAGAAATC R：ACTGCCACCTCCTCCACTAGTTTTCTCATTATCTTGCAACA
	CsIDM2-BIFC	F：CGAACGATAGTTAATTAAATGGATGACTTGTCTCATGC R：CTGCCACCTCCTCCACTAGTTATCCCTTTCATCACAATTC
	CsIDM3-BIFC	F：CGAACGATAGTTAATTAAATGTGTACGTGCCCAAAGCAAAG R：CTGCCACCTCCTCCACTAGTCTCATATTTCATGATGATAG
	CsMBD5-BIFC	F：TACGAACGATAGTTAATTAAATGTCGACCTGCGAATCC R：CTGCCACCTCCTCCACTAGTGCTGCAAGAGAAATGAAATG
	CsMBD16-BIFC	F：TACGAACGATAGTTAATTAAATGGATGACAATGGCGCATC R：CACCACTGCCACCTCCTCCACTAGTCCATTCAAAATTTGG

图1 CsIDM的扩增

Fig. 1 Amplification of the CsIDM

表2 茶树与拟南芥IDM基因的基本特征比较

Table 2 Comparison of basic characteristics of IDM in tea plant and Arabidopsis thaliana

基因名称 Gene name	登录号 GenBank	氨基酸数 Amino acids number	分子质量/kD Molecular weight	pI	稳定系数 Instability index	亲水性 GRAVY	亚细胞定位 Subcellular localization
CsIDM1 AtIDM1	OK649843 AT3G14980	1 251 1 189	139.38 131.33	7.69 6.69	46.94 44.49	-0.451 -0.489	细胞核Nucleus 细胞核Nucleus
CsIDM2 AtIDM2	OK649845 AT1G54840	419 349	46.94 39.39	5.10 6.04	45.75 44.83	-0.251 -0.374	细胞核Nucleus 细胞核Nucleus
CsIDM3 AtIDM3	OK649846 AT1G20870	211 463	23.22 51.93	8.95 7.07	45.73 51.10	-0.470 -0.616	叶绿体Chloroplast 细胞核Nucleus

图2 IDM进化树和保守结构域

Fig. 2 Phylogenetic trees and conserved domain of IDMs

图3 CsIDM 在茶树不同组织中的qRT-PCR相对表达量和转录组表达丰度不同字母表示0.05水平上差异显著。

Fig. 3 Relative expression of qRT-PCR and transcriptome expression abundanceof CsIDMs in different tissues of tea plants Different letters indicate significant difference at 0.05 level.

图4 茶树‘龙井43’CsIDM在生物胁迫下的表达分析不同小写字母表示同一处理不同时间的基因表达量在0.05水平上差异显著。

Fig. 4 Expression analysis of CsIDMs under biotic stress in‘Longjing 43’ Different lowercase letters in the same treatment indicate significant differences among different treatment time at 0.05 level.

图5 茶树‘龙井43’CsIDM在干旱胁迫下的表达处理组与对照组在不同处理时间的基因表达差异进行统计分析（t检验），*表示0.05水平上差异显著。

Fig. 5 Expression analysis of CsIDM under drought stress in‘Longjing 43’ In comparisons of treatment group and control group, differences in gene expression at different treatment times were statistically analyzed by t-test and the sign of * in the figure indicate significant difference at 0.05 level.

图6 茶树‘龙井43’CsIDM在高温胁迫下的表达处理组与对照组在不同处理时间的基因表达差异进行统计分析（t检验），*表示0.05水平上差异显著。

Fig. 6 Expression analysis of CsIDM under high temperature stress in‘Longjing 43’ In comparisons of treatment group and control group, differences in gene expression at different treatment times were statistically analyzed by t-test and the sign of * in the figure indicate significant difference at 0.05 level.

图7 CsIDM在6个茶树品种越冬芽休眠阶段的表达 I：休眠形成阶段;Ⅱ：深休眠阶段;Ⅲ：休眠解除阶段。

Fig. 7 Expression of CsIDM in buds of different tea cultivars at different dormancy periods I：Paradormancy stage;Ⅱ：Endodormancy stage;Ⅲ：Encodormancy stage.

图8 茶树CsIDM蛋白在烟草叶片中的亚细胞定位

Fig. 8 Subcellular localization of CsIDM proteins in tobacco leaf

图9 茶树CsIDM蛋白BiFC互作验证

Fig. 9 BiFC assey between CsIDMs

图10 茶树CsIDM蛋白与CsMBD5的BiFC互作验证

Fig. 10 BiFC assey between CsIDMs and CsMBD5

图11 茶树CsIDM蛋白与CsMBD16的BiFC互作验证

Fig. 11 BiFC assey between CsIDMs and CsMBD16

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