油莎豆MYB转录因子基因CeMYB154克隆及耐盐功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-1182

摘要/Abstract

摘要：

目的油莎豆MYB转录因子基因CeMYB154参与盐胁迫响应过程，克隆及表征油莎豆CeMYB154并分析其耐盐功能，为油莎豆耐盐育种提供分子基础和基因资源。方法基于油莎豆参考基因组信息，克隆CeMYB154的全长CDS序列，利用生物信息学软件对其氨基酸序列特征及其启动子上顺式作用元件进行分析，利用分子生物学技术对其进行亚细胞定位和转录激活验证。构建CeMYB154过表达载体，利用农杆菌介导法转化拟南芥，并对过表达株系的耐盐表型和生理指标进行分析。结果成功克隆了长度为780 bp的CeMYB154 CDS序列，其编码蛋白序列中2个典型的MYB结构域，属于R2R3型MYB转录因子。蛋白序列比对分析显示，CeMYB154蛋白在多个物种间序列高度保守。启动子区域含有多种激素（如ABA）响应以及MYB响应相关顺式作用元件。此外，亚细胞定位和转录激活分析显示，CeMYB154是一个具有转录激活活性的核转录因子。成功创制了过表达CeMYB154转基因拟南芥，获得了纯合T₃转基因阳性株系。在盐胁迫下，与野生型拟南芥相比，转基因植株幼苗生长状态（叶片大小、颜色以及根长）较好；并且，转基因植株中MDA和H₂O₂含量均显著降低，而CAT、POD和SOD抗氧化酶活性则显著增强，表明过表达CeMYB154基因提高了拟南芥的耐盐性。结论油莎豆CeMYB154具有正向调控盐胁迫响应的功能，过表达CeMYB154基因可以增强抗氧化酶活性而提高转基因植株的耐盐性。

关键词: 油莎豆, 盐胁迫, MYB转录因子, 转录激活, 功能分析, 过表达, 亚细胞定位, 抗氧化酶

Abstract:

Objective The MYB transcription factor gene CeMYB154 is involved in the salt stress response process in oil chestnut(Cyperus esculentus). Cloning and characterization of CeMYB154 gene in oil chestnut and analysis of its salt tolerance function may provide molecular basis and genetic resources for salt tolerant breeding of oil chestnut. Method Based on the reference genome information of oil chestnut, the full-length CDS sequence of CeMYB154 was cloned. Bioinformatics software was used to analyze its amino acid sequence characteristics and cis acting elements on its promoter, and molecular biology techniques were used to perform subcellular localization and transcriptional activation verification. CeMYB154 overexpression vector was constructed, Arabidopsis thaliana was transformed using Agrobacterium mediated method, and the salt-tolerant phenotype and physiological indicators of the overexpression strain were analyzed. Result The CeMYB154 CDS sequence with a length of 780 bp was successfully cloned, which encodes two typical MYB domains in the protein sequence and belongs to the R2R3 MYB transcription factor. Protein sequence alignment analysis showed that CeMYB154 protein is highly conserved across multiple species. The promoter region contains multiple hormone (such as ABA) response and MYB response related cis-acting elements. In addition, subcellular localization and transcriptional activation analysis showed that CeMYB154 is a nuclear transcription factor with transcriptional activation activity. Transgenic A. thaliana overexpressing CeMYB154 was successfully created and a homozygous T3 transgenic positive strain was obtained. Under salt stress, transgenic plant seedlings showed better growth status (leaf size, color, and root length) compared to wild-type A. thaliana. Moreover, the levels of MDA and H₂O₂ in the transgenic plants significantly reduced, while the activities of CAT, POD, and SOD antioxidant enzymes significantly enhanced, indicating that overexpression of the CeMYB154 gene improved salt tolerance in A. thaliana. Conclusion Preliminary evidence has shown that the CeMYB154 gene in C. esculentus has the function of positively regulating salt stress response. The overexpression of the CeMYB154 gene may enhance antioxidant enzyme activity and improve salt tolerance in transgenic plants.

Key words: Cyperus esculentus, salt stress, MYB transcription factor, transcriptional activation, functional analysis, over-expression, subcellular localization, antioxidase

程珊, 王会伟, 陈晨, 朱雅婧, 李春鑫, 别海, 王树峰, 陈献功, 张向歌. 油莎豆MYB转录因子基因CeMYB154克隆及耐盐功能分析[J]. 生物技术通报, 2025, 41(6): 218-228.

CHENG Shan, WANG Hui-wei, CHEN Chen, ZHU Ya-jing, LI Chun-xin, BIE Hai, WANG Shu-feng, CHEN Xian-gong, ZHANG Xiang-ge. Cloning of MYB Transcription Factor Gene CeMYB154 and Analysis of Salt Tolerance Function in Cyperus esculentus[J]. Biotechnology Bulletin, 2025, 41(6): 218-228.

图/表 8

表1 本研究所用的引物序列

Table 1 Primer sequences used in this study

引物名称 Primer name	引物序列 Primer sequence （5′‒3′）	用途 Purpose
MYB154-F	ATGGAGGAAGTGGCATGGAG	基因克隆 Gene clone
MYB154-R	CTAGTATAAAGCAAATTCTGCTGCTTGC	基因克隆 Gene clone
2300-F	gtacccggggatcctctagagtcgacATGGAGGAAGTGGCATGGAG	转基因植株检测 Transgenic plant detection
2300-R	ttgctcaccatggtactagtgtcgacGTATAAAGCAAATTCTGCTGCTTGC	转基因植株检测 Transgenic plant detection
GFP-F	ctatttacaattacggatccATGGAGGAAGTGGCATGGAG	亚细胞定位 Subcellular localization
GFP-R	agatcctcctccggagaccTAAAGCAAATTCTGCTGC	亚细胞定位 Subcellular localization
BD-F	atggccatggaggccgaattcATGGAGGAAGTGGCATGGAG	转录激活活性检测 Transcriptional activation activity assays
BD-R	tcgacggatccccgggaattcGTATAAAGCAAATTCTGCTGCTTGC	转录激活活性检测 Transcriptional activation activity assays
QP-F	GATGGAATTCTGTTGCT	荧光定量PCR引物 Fluorescent quantitative PCR primer
QP-R	CTTTATCTCGTTGTCGG	荧光定量PCR引物 Fluorescent quantitative PCR primer
CetActin-F	ACTCTGGTGATGGTGTGAGC	油莎豆内参引物 Internal primer of Cyperus esculentus
CetActin-R	CCCTCTCTCCGTCAGGATCT	油莎豆内参引物 Internal primer of Cyperus esculentus
AtActin-F	GGTAACATTGTGCTCAGTGGTGG	拟南芥内参引物 Internal primer of Arabidopsis thaliana
AtActin-R	AACGACCTTAATCTTCATGCTGC	拟南芥内参引物 Internal primer of Arabidopsis thaliana

图1 CeMYB154的克隆与生物信息学分析A：CeMYB154基因克隆；B：不同浓度NaCl处理下油莎豆根系中CeMYB154的相对表达水平；C：CeMYB154蛋白结构域分析；D：CeMYB154亚细胞定位预测；E：多物种序列比对；Ce：油莎豆；Ah：花生；Gm：大豆；Zm：玉米；Bna：油菜；At：拟南芥；OS：水稻；St：马铃薯；Ib：甘薯；**表示以WT为对照在P<0.01水平上差异显著，下同

Fig. 1 Cloning and bioinformatics analysis of CeMYB154A: CeMYB154 gene cloning. B: The expressions of CeMYB154 in Cyperus esculentus roots under different concentrations of NaCl treatment. C: CeMYB154 protein domain analysis. D: CeMYB154 subcellular localization prediction. E: Multi-species sequence alignment. Ce: Cyperus esculentus. Ah:: Arachis hypogaea. Gm: Glycine max. Zm: Zea mays. Bna: Brassica napus. At: Arabidopsis thaliana. Os: Oryza sativa. St: Solanum tuberosum. Ib: Ipomoea batatas. ** indicates a significant difference at the P<0.01 level with WT as control, the same below

表2 CeMYB154启动子顺式作用元件分析

Table 2 Analysis of cis-acting elements in CeMYB154 promoter

顺式作用元件 Cis-acting element	序列 Sequence （5′‒3′）	功能 Function	数量 Quantity
CAAT-box	CAAT	启动子和增强子区域共同的顺式作用元件	23
TATA-box	TATATA； ATATAT	转录起始-30附近的核心启动子元件	156
MYB-like	TAACCA	MYB识别和结合元件	1
MRE	AACCTAA	MYB响应元件	1
TCCC-motif	TCTCCCT	光响应元件	1
GT1-motif	GGTTAAT	光响应元件	5
G-box	CACGTC	光响应元件	1
GATA-motif	GATAGGG	光响应元件	1
AAGAA-motif	GAAAGAA	脱落酸响应元件	2
ABRE	ACGTG	脱落酸响应元件	1
TGA-box	TGACGTAA	生长素响应元件	1
TGACG-motif	TGACG	茉莉酸甲酯响应元件	2
ARE	AAACCA	厌氧诱导调节元件	3

图2 亚细胞定位EGFP代表绿色荧光；Cy5代表红色荧光；TD代表明场；ALL代表3种光源叠加

Fig. 2 Subcellular localizationEGFP indicates green fluorescence; Cy5 indicates red fluorescence; TD indicates bright field; ALL indicates the superposition of three light sources

图3 CeMYB154转录激活活性鉴定1、102、103分别代表酵母菌液稀释倍数

Fig. 3 Identification of CeMYB154 transcriptional activation activity1, 102, 103 indicate the dilution multiple of yeast liquid, respectively

图4 转基因拟南芥植株的鉴定A：卡那霉素筛选T1转基因苗；B：T1转基因植株的PCR鉴定（M 为DL2000 marker，1‒10为转基因植株，WT为野生型植株）；C：T2转基因拟南芥植株中CeMYB154的表达水平检测，OE1、OE2和OE3分别代表1个独立的T2代转基因植株，下同

Fig. 4 Identification of transgenic A. thaliana plantsA: Hygromycin screening T1 transgenic vaccines. B: PCR identification of T1 transgenic plants (M is DL2000 marker, 1‒10 are transgenic plants, and WT is wild-type plants). C: Detection of CeMYB154 expression in T2 transgenic A. thaliana plants, OE1, OE2 and OE3 indicate an independent T2 generation transgenic plant, respectively, the same below

图5 CeMYB154转基因拟南芥植株耐盐表型A：盐胁迫对拟南芥植株幼苗生长的影响；B：盐胁迫对转基因拟南芥植株根系生长的影响；C：盐胁迫下WT和转基因拟南芥植株的根长

Fig. 5 Phenotype of salt-tolerant in CeMYB154 transgenic A. thaliana plantsA: Effect of salt stress on the seedling growth of A. thaliana plants. B: Effect of salt stress on the root growth of transgenic A. thaliana plants. C: Root length of WT and transgenic A.thaliana plants under salt stress

图6 盐胁迫下过表达CeMYB154拟南芥的相关生理指标测定

Fig. 6 Determination of physiological indexes related to the overexpression of CeMYB154 in A. thaliana under salt stress

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