茶树泛素连接酶基因CsPUB21对非生物胁迫的响应

doi:10.16420/j.issn.0513-353x.2024-0378

摘要/Abstract

摘要：

以‘陕茶1号’茶树品种为材料，克隆得到U-box家族泛素E3连接酶基因CsPUB21并进行功能分析。序列分析发现CsPUB21编码区长度为1 242 bp，共编码413个氨基酸，在N端含有U-box保守结构域、C端含有ARM结构域，为不稳定的亲水性蛋白。启动子区含有多个响应干旱、高盐、低温胁迫的顺式作用元件。亚细胞定位结果表明，CsPUB21定位于细胞膜和细胞质。基因表达模式分析显示，在5个供试茶树品种中，CsPUB21受干旱和盐胁迫诱导表达上调，而在低温胁迫下降低。在拟南芥中过表达CsPUB21，显著增强了植株对干旱和盐胁迫的抗性。qRT-PCR结果显示，CsPUB21过表达拟南芥在干旱和盐胁迫后CsPUB21表达量上调。与野生型相比，CsPUB21过表达拟南芥在干旱、盐胁迫处理条件下叶绿素含量、可溶性糖含量较高，抗氧化酶SOD、POD、CAT活性增强，相对电导率和MDA含量降低。研究结果说明CsPUB21在茶树应对干旱和盐胁迫时发挥正向调节作用。

关键词: 茶树, CsPUB21, 基因克隆, 非生物胁迫, 表达分析, 异源功能验证

Abstract:

The U-box ubiquitin E3 ligase gene CsPUB21 were cloned and functionally analyzed in Camellia sinensis‘Shaancha 1’. The length of the coding sequence of CsPUB21 is 1 242 bp，encoding a total of 413 amino acids with a conserved U-box domain at the N terminus and an ARM domain at the C terminus. The encoded protein，an unstable hydrophilic protein. The promoter region contains multiple cis-acting elements in response to drought，high salt and low temperature. The subcellular localization results indicated that CsPUB21 is localized on the cell membrane and cytoplasm. Gene expression pattern analysis showed that CsPUB21 was up-regulated by drought and salt stress treatments，and down-regulated by low temperature in different cultivars of C. sinensis. Overexpression of CsPUB21 gene in Arabidopsis significantly enhanced plants resistance to drought and salt stress. The qRT-PCR results showed that the expression level of CsPUB21 was up-regulated after drought and salt stress. Compared with the wild type，overexpression of CsPUB21 in Arabidopsis resulted in higher chlorophyll content and soluble sugar content under drought and salt stress，enhanced activities of antioxidant enzymes SOD，POD，and CAT，and decreased relative conductivity and MDA content. The research results indicated that the CsPUB21 plays a positive regulatory role in tea plant response to drought and salt stress.

Key words: Camellia sinensis, CsPUB21, gene cloning, abiotic stress, expression analysis, heterologous function verification

邓淑琴, 高莹瑞, 李雨桐, 王瑛, 龚春梅, 白娟. 茶树泛素连接酶基因CsPUB21对非生物胁迫的响应[J]. 园艺学报, 2025, 52(3): 655-670.

DENG Shuqin, GAO Yingrui, LI Yutong, WANG Ying, GONG Chunmei, BAI Juan. Response of Ubiquitin-ligase Gene CsPUB21 to Different Abiotic Stress in Camellia sinensis[J]. Acta Horticulturae Sinica, 2025, 52(3): 655-670.

图/表 12

表1 本研究中所用的引物序列信息

Table 1 Primers sequence information used in this study

引物名称 Primer name	引物序列（5′-3′） Primer sequence	用途 Usage
1300-CsPUB21-F	GAGAACACGGGGGAC GAGCTCATGATTTCGTCTTGGCGGAG	植物过表达 Plant overexpression
1300-CsPUB21-R	CGCATAGCTAATCTG GGATCCTCAATTTTGAAGAGCTTCAACAAGA	植物过表达 Plant overexpression
qRT-CsPUB21-F	GAAGGGTTTGGCGAAGGAGA	荧光定量PCR Quantitative real time PCR
qRT-CsPUB21-R	AGCCCTCTCGCATATGCTTC
qRT-Csβ-actin-F	GGTAACATTGTGCTCAGTGGTGG
qRT-Csβ-actin-R	AACGACCTTAATCTTCATGCTGC

图1 茶树CsPUB21基因的扩增

Fig. 1 Amplification of CsPUB21 gene in Camellia sinensis

表2 CsPUB21基因启动子区顺式作用元件预测

Table 2 Prediction of cis-acting elements in the promoter region of CsPUB21

顺式作用元件 cis-Acting element	序列 Sequence	数量 Amount	功能 Function
MYB	CAACCA CAACTG	6 1	干旱、高盐、低温响应元件Drought，high salt，low temperature response element
MBS	CAACTG	1	干旱响应元件Drought response element
STRE	AGGGG	2	胁迫响应元件Stress response element
WUN-motif	AAATTACT	2	损伤诱导元件Damage inductive element
WRE3	CCACCT	1	损伤诱导元件Damage inductive element
W box	TTGACC	1	损伤与病原响应元件Damage and pathogen response element
ARE	AAACCA	1	厌氧诱导调控元件Anaerobic inducible regulatory element
Box 4	ATTAAT	5	光响应元件Photoresponsive element
ATC-motif	AGTAATCT	1	光响应元件Photoresponsive element
AAGAA-motif	GAAAGAA	1	脱落酸响应元件Abscisic acid response element
ABRE	TACGGTC	1	脱落酸响应元件Abscisic acid response element
MYC	CATGTG	4	抗脱落酸响应元件Abscisic acid resistant response element
F-box	CTATTCTCATT	1	赤霉素响应元件Gibberellin response element
CGTCA-motif	CGTCA	4	茉莉酸响应元件Jasmonic acid response element
TGACG-motif	TGACG	4	茉莉酸响应元件Jasmonic acid response element
ERE	ATTTTAAA	2	乙烯响应元件Ethylene responsive element
as-1	TGACG	4	根特异表达顺式作用调控元件Root specific expression of cis-acting regulatory elements

图2 CsPUB21的系统发育进化树（A）和motif（B）

Fig. 2 Phylogenetic tree（A）and motif（B）of CsPUB21

图3 CsPUB21及其已知近源物种的氨基酸序列多重比对

Fig. 3 Multiple alignments among the amino acid sequences of the CsPUB21 and its known homologs

图4 CsPUB21在茶树原生质体（A）和烟草叶表皮（B）中的亚细胞定位

Fig. 4 Subcellular localization of CsPUB21 in tea leaf mesophyll protoplasts（A）and leaf epidermis of Nicotiana benthamiana（B）

图5 不同茶树品种在干旱、盐（NaCl）、低温（4 ℃）胁迫处理下CsPUB21的表达模式 *、**、***分别表示在P < 0.05、P < 0.01、P < 0.001下的显著差异；ns：非显著差异。下同

Fig. 5 Analysis of CsPUB21 gene expression patterns in different tea cultivars under drought，salt and low temperature stress treatments *，**，*** indicate significant difference as determined via two-way ANOVA at P < 0.05，P < 0.01，P < 0.001，respectively；ns：No significant difference. The same below

图6 拟南芥过表达CsPUB21株系（OE#1和OE#2）及其野生型（Col-0）的鉴定

Fig. 6 Identification of overexpressed CsPUB21 line（OE#1 and OE#2）and wild type（Col-0）in Arabidopsis thaliana

图7 非生物胁迫下拟南芥野生型（Col-0）和过表达CsPUB21株系（OE#1和OE#2）的表型

Fig. 7 Phenotypes of Arabidopsis thaliana widetype（Col-0）and CsPUB21 overexpressing line（OE#1和OE#2）under abiotic stress

图8 非生物胁迫下拟南芥过表达CsPUB21植株中基因的表达量

Fig. 8 Relative expression of CsPUB21 gene in Arabidopsis thaliana with overexpression lines under different abiotic stress

图9 非生物胁迫下拟南芥野生型（Col-0）和过表达CsPUB21株系（OE）中SOD、POD、CAT酶活性

Fig. 9 Activity of SOD,POD and CAT enzyme in Arabidopsis thaliana wild type（Col-0）and CsPUB21 overexpressing lines（OE）under abiotic stress

图10 非生物胁迫下拟南芥野生型（Col-0）和过表达CsPUB21株系（OE）的生理指标

Fig. 10 Physiological indices analysis of Arabidopsis thaliana wild type（Col-0）and CsPUB21 overexpressing lines（OE）under abiotic stress

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