Cloning of MYB Transcription Factor Gene CeMYB154 and Analysis of Salt Tolerance Function in Cyperus esculentus

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

Abstract

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

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.

Figures/Tables 8

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

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

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

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

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

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

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

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

References 39

1	张玉娟, 黎冬华, 宫慧慧, 等. 芝麻NAC转录因子基因SiNAC77的克隆及耐盐功能分析 [J]. 生物技术通报, 2023, 39(11): 308-317.
	Zhang YJ, Li DH, Gong HH, et al. Cloning and salt-tolerance analysis of NAC transcription factor SiNAC77 from Sesamum indicum L [J]. Biotechnol Bull, 2023, 39(11): 308-317.
2	杨帆, 朱文学. 油莎豆研究现状及展望 [J]. 粮食与油脂, 2020, 33(7): 4-6.
	Yang F, Zhu WX. Research status and prospect of Cyperus esculentus [J]. Cereals Oils, 2020, 33(7): 4-6.
3	刘玉兰, 王小宁, 舒垚, 等. 不同产地油莎豆性状及组成分析研究 [J]. 中国油脂, 2020, 45(8): 125-129.
	Liu YL, Wang XN, Shu Y, et al. Character and composition of Cyperus esculentus from different origins [J]. China Oils Fats, 2020, 45(8): 125-129.
4	王会伟, 张向歌, 李春鑫, 等. 油莎豆耐盐性评估及盐胁迫下幼苗根系转录组学分析 [J]. 作物学报, 2023, 49(7): 1882-1894.
	Wang HW, Zhang XG, Li CX, et al. Evaluation of salt tolerance in Cyperus esculentus and transcriptomic analysis of seedling roots under salt stress [J]. Acta Agron Sin, 2023, 49(7): 1882-1894.
5	周扬, 王鹏, 李雨欣. 植物耐盐分子机制研究进展 [J]. 广东农业科学, 2023, 50(10): 97-109.
	Zhou Y, Wang P, Li YX. Research progress on molecular mechanism of plant salt tolerance [J]. Guangdong Agric Sci, 2023, 50(10): 97-109.
6	冯凯月, 赵鑫焱, 李子妍, 等. 植物响应盐碱胁迫的分子机制研究进展 [J]. 生物技术通报, 2024, 40(10): 122-138.
	Feng KY, Zhao XY, Li ZY, et al. Research advances in the molecular mechanisms of plant response to saline-alkali stress [J]. Biotechnol Bull, 2024, 40(10): 122-138.
23	李星伟, 王庆江, 吴宇童, 等. 苹果MdMYB113基因响应高盐胁迫的功能鉴定 [J]. 植物生理学报, 2021, 57(3): 579-588.
	Li XW, Wang QJ, Wu YT, et al. Functional identification of MdMYB113 gene in apple in response to high salt stress [J]. Plant Physiol J, 2021, 57(3): 579-588.
24	Cao YP, Han YH, Li DH, et al. MYB transcription factors in Chinese pear (Pyrus bretschneideri rehd.): genome-wide identification, classification, and expression profiling during fruit development [J]. Front Plant Sci, 2016, 7: 577.
25	Zhang ZJ, Zhang L, Liu Y, et al. Identification and expression analysis of R2R3-MYB family genes associated with salt tolerance in Cyclocarya paliurus [J]. Int J Mol Sci, 2022, 23(7): 3429.
26	张红梅, 刘晓庆, 陈华涛, 等. 大豆转录因子GmWRKY58亚细胞定位及在非生物胁迫下的表达分析 [J]. 华北农学报, 2018, 33(2): 49-57.
	Zhang HM, Liu XQ, Chen HT, et al. Subcellular localization and expression analysis of a soybean transcription factor GmWRKY58 in response to abiotic stresses [J]. Acta Agric Boreali Sin, 2018, 33(2): 49-57.
27	陈彧, 邢文婷, 李雨欣, 等. 铁皮石斛DcNAC1基因克隆、表达及转录自激活活性分析 [J]. 南方农业学报, 2023, 54(6): 1612-1621.
	Chen Y, Xing WT, Li YX, et al. Cloning, expression and trans-activation activity analysis of DcNAC1 gene from Dendrobium catenatum [J]. J South Agric, 2023, 54(6): 1612-1621.
28	Dossa K, Mmadi MA, Zhou R, et al. Ectopic expression of the sesame MYB transcription factor SiMYB305 promotes root growth and modulates ABA-mediated tolerance to drought and salt stresses in Arabidopsis [J]. AoB Plants, 2019, 12(1): plz081.
29	胡雅丹, 伍国强, 刘晨, 等. MYB转录因子在调控植物响应逆境胁迫中的作用 [J]. 生物技术通报, 2024, 40(6): 5-22.
	Hu YD, Wu GQ, Liu C, et al. Roles of MYB transcription factor in regulating the responses of plants to stress [J]. Biotechnol Bull, 2024, 40(6): 5-22.
30	He YX, Dong YS, Yang XD, et al. Functional activation of a novel R2R3-MYB protein gene, GmMYB68, confers salt-alkali resistance in soybean (Glycine max L.) [J]. Genome, 2020, 63(1): 13-26.
7	Zhang S, Wu QR, Liu LL, et al. Osmotic stress alters circadian cytosolic Ca²⁺ oscillations and OSCA1 is required in circadian gated stress adaptation [J]. Plant Signal Behav, 2020, 15(12): 1836883.
8	Jiang ZH, Zhou XP, Tao M, et al. Plant cell-surface GIPC sphingolipids sense salt to trigger Ca²⁺ influx [J]. Nature, 2019, 572(7769): 341-346.
9	Chen K, Gao JH, Sun SJ, et al. BONZAI proteins control global osmotic stress responses in plants [J]. Curr Biol, 2020, 30(24): 4815-4825.e4.
10	Yan JW, Liu Y, Yan JW, et al. The salt-activated CBF1/CBF2/CBF3-GALS1 module fine-tunes galactan-induced salt hypersensitivity in Arabidopsis [J]. J Integr Plant Biol, 2023, 65(8): 1904-1917.
11	Gao F, Zhou J, Deng RY, et al. Overexpression of a Tartary buckwheat R2R3-MYB transcription factor gene, FtMYB9, enhances tolerance to drought and salt stresses in transgenic Arabidopsis [J]. J Plant Physiol, 2017, 214: 81-90.
12	Le Hir R, Castelain M, Chakraborti D, et al. AtbHLH68 transcription factor contributes to the regulation of ABA homeostasis and drought stress tolerance in Arabidopsis thaliana [J]. Physiol Plant, 2017, 160(3): 312-327.
13	Raineri J, Wang SH, Peleg Z, et al. The rice transcription factor OsWRKY47 is a positive regulator of the response to water deficit stress [J]. Plant Mol Biol, 2015, 88(4/5): 401-413.
14	Dubos C, Stracke R, Grotewold E, et al. MYB transcription factors in Arabidopsis [J]. Trends Plant Sci, 2010, 15(10): 573-581.
15	Wang YJ, Zhang Y, Fan CJ, et al. Genome-wide analysis of MYB transcription factors and their responses to salt stress in Casuarina equisetifolia [J]. BMC Plant Biol, 2021, 21(1): 328.
16	Liao Y, Zou HF, Wang HW, et al. Soybean GmMYB76, GmMYB92, and GmMYB177 genes confer stress tolerance in transgenic Arabidopsis plants [J]. Cell Res, 2008, 18(10): 1047-1060.
17	Yoo JH, Park CY, Kim JC, et al. Direct interaction of a divergent CaM isoform and the transcription factor, MYB2, enhances salt tolerance in Arabidopsis [J]. J Biol Chem, 2005, 280(5): 3697-3706.
18	Zhu N, Cheng SF, Liu XY, et al. The R2R3-type MYB gene OsMYB91 has a function in coordinating plant growth and salt stress tolerance in rice [J]. Plant Sci, 2015, 236: 146-156.
19	Zhang X, Chen LC, Shi QH, et al. SlMYB102, an R2R3-type MYB gene, confers salt tolerance in transgenic tomato [J]. Plant Sci, 2020, 291: 110356.
20	Zhao YY, Yang ZE, Ding YP, et al. Over-expression of an R2R3 MYB gene, GhMYB73, increases tolerance to salt stress in transgenic Arabidopsis [J]. Plant Sci, 2019, 286: 28-36.
21	Dong W, Liu XJ, Li DL, et al. Transcriptional profiling reveals that a MYB transcription factor MsMYB4 contributes to the salinity stress response of alfalfa [J]. PLoS One, 2018, 13(9): e0204033.
22	Du BY, Liu H, Dong KT, et al. Over-expression of an R2R3 MYB gene, MdMYB108L, enhances tolerance to salt stress in transgenic plants [J]. Int J Mol Sci, 2022, 23(16): 9428.
31	Zhang WX, Wang N, Yang JT, et al. The salt-induced transcription factor GmMYB84 confers salinity tolerance in soybean [J]. Plant Sci, 2020, 291: 110326.
32	Du YT, Zhao MJ, Wang CT, et al. Identification and characterization of GmMYB118 responses to drought and salt stress [J]. BMC Plant Biol, 2018, 18(1): 320.
33	Pi EX, Xu J, Li HH, et al. Enhanced salt tolerance of rhizobia-inoculated soybean correlates with decreased phosphorylation of the transcription factor GmMYB183 and altered flavonoid biosynthesis [J]. Mol Cell Proteomics, 2019, 18(11): 2225-2243.
34	Pi EX, Zhu CM, Fan W, et al. Quantitative phosphoproteomic and metabolomic analyses reveal GmMYB173 optimizes flavonoid metabolism in soybean under salt stress [J]. Mol Cell Proteomics, 2018, 17(6): 1209-1224.
35	Wang C, Wang LJ, Lei J, et al. IbMYB308, a sweet potato R2R3-MYB gene, improves salt stress tolerance in transgenic tobacco [J]. Genes, 2022, 13(8): 1476.
36	Hossain MA, Bhattacharjee S, Armin SM, et al. Hydrogen peroxide priming modulates abiotic oxidative stress tolerance: insights from ROS detoxification and scavenging [J]. Front Plant Sci, 2015, 6: 420.
37	Liu P, Wu XL, Gong BB, et al. Review of the mechanisms by which transcription factors and exogenous substances regulate ROS metabolism under abiotic stress [J]. Antioxidants, 2022, 11(11): 2106.
38	Li WH, Zhong JL, Zhang LH, et al. Overexpression of a Fragaria vesca MYB transcription factor gene (FvMYB82) increases salt and cold tolerance in Arabidopsis thaliana [J]. Int J Mol Sci, 2022, 23(18): 10538.
39	程钰鑫. R2R3 MYB转录因子MYB71、MYB79和MYB121对拟南芥ABA及干旱胁迫响应的调控 [D]. 长春: 东北师范大学, 2022.
	Cheng YX. Regulation of R2R3 MYB transcription factors MYB71, MYB79 and MYB121 on ABA and drought stress response in Arabidopsis thaliana [D]. Changchun: Northeast Normal University, 2022.

[1]	XU Hui-zhen, SHANTWANA Ghimire, RAJU Kharel, YUE Yun, SI Huai-jun, TANG Xun. Analysis of the Potato SUMO E3 Ligase Gene Family and Cloning and Expression Pattern of StSIZ1 [J]. Biotechnology Bulletin, 2025, 41(6): 119-129.
[2]	WU Ya, YAO Run, YANG Han-ting, LIU Wei, YANG Shuai, SONG Chi, CHEN Shi-lin. Genome-wide Identification and Expression Analysis of SDR Gene Family in Mentha suaveolens ‘Variegata’ [J]. Biotechnology Bulletin, 2025, 41(5): 175-185.
[3]	LI Zhi-qiang, LUO Zheng-qian, XU Lin-li, ZHOU Guo-hui, QU Si-yu, LIU En-liang, XU Dong-ting. Identification of the Soybean R2R3-MYB Gene Family Based on the T2T Genome and Their Expression Analysis under Drought and Salt Stress [J]. Biotechnology Bulletin, 2025, 41(5): 141-152.
[4]	YANG Chun, WANG Xiao-qian, WANG Hong-jun, CHAO Yue-hui. Cloning， Subcellular Localization and Expression Analysis of MtZHD4 Gene from Medicago truncatula [J]. Biotechnology Bulletin, 2025, 41(5): 244-254.
[5]	SONG Hui-yang, SU Bao-jie, LI Jing-hao, MEI Chao, SONG Qian-na, CUI Fu-zhu, FENG Rui-yun. Cloning and Functional Analysis of the StAS2-15 Gene in Potato under Salt Stress [J]. Biotechnology Bulletin, 2025, 41(5): 119-128.
[6]	BAN Qiu-yan, ZHAO Xin-yue, CHI Wen-jing, LI Jun-sheng, WANG Qiong, XIA Yao, LIANG Li-yun, HE Wei, LI Ye-yun, ZHAO Guang-shan. Cloning of Phytochrome Interaction Factor CsPIF3a and Its Response to Light and Temperature Stress in Camellia sinensis [J]. Biotechnology Bulletin, 2025, 41(4): 256-265.
[7]	LU Yong-jie, XIA Hai-qian, LI Yong-ling, ZHANG Wen-jian, YU Jing, ZHAO Hui-na, WANG Bing, XU Ben-bo, LEI Bo. Cloning and Expression Analysis of AP2/ERF Transcription Factor NtESR2 in Nicotiana tabacum [J]. Biotechnology Bulletin, 2025, 41(4): 266-277.
[8]	LIU Tao, WANG Zhi-qi, WU Wen-bo, SHI Wen-ting, WANG Chao-nan, DU Chong, YANG Zhong-min. Identification and Expression Analysis of the GRAM Gene Family in Potato [J]. Biotechnology Bulletin, 2025, 41(4): 145-155.
[9]	LI Cai-xia, LI Yi, MU Hong-xiu, LIN Jun-xuan, BAI Long-qiang, SUN Mei-hua, MIAO Yan-xiu. Identification and Bioinformatics Analysis of the bHLH Transcription Factor Family in Cucurbita moschata Duch. [J]. Biotechnology Bulletin, 2025, 41(1): 186-197.
[10]	YUAN Liu-jiao, HUANG Wen-lin, CHEN Chong-zhi, LIANG Min, HUANG Zi-qi, CHEN Xue-xue, CHEN Ri-Meng, WANG Li-yun. Effects of Salt Stress on Physiological Characteristics, Ultrastructure and Medicinal Components of Pogostemon cablin Leaves [J]. Biotechnology Bulletin, 2025, 41(1): 230-239.
[11]	QIAO Yan, YANG Fang, REN Pan-rong, QI Wei-liang, AN Pei-pei, LI Qian, LI Dan, XIAO Jun-fei. Cloning and Function Analysis of the ScDHNS Gene of Crotonase/Enoyl-CoA Superfamily from a Wild Potato Species [J]. Biotechnology Bulletin, 2024, 40(9): 92-103.
[12]	XING Li-nan, ZHANG Yan-fang, GE Ming-ran, ZHAO Ling-min, CHEN Yan, HUO Xiu-wen. Analysis of DoWRKY40 Gene Expression Characteristics and Screening of Interacting Proteins in Yam [J]. Biotechnology Bulletin, 2024, 40(8): 118-128.
[13]	WU Shuai, XIN Yan-ni, MAI Chun-hai, MU Xiao-ya, WANG Min, YUE Ai-qin, ZHAO Jin-zhong, WU Shen-jie, DU Wei-jun, WANG Li-xiang. Genome-wide Identification and Stress Response Analysis of Soybean GS Gene Family [J]. Biotechnology Bulletin, 2024, 40(8): 63-73.
[14]	LIN Tong, YUAN Cheng, DONG Chen-wen-hua, ZENG Meng-qiong, YANG Yan, MAO Zi-chao, LIN Chun. Screening and Functional Analysis of Gene CqSTK Associated with Gametophyte Development of Quinoa [J]. Biotechnology Bulletin, 2024, 40(8): 83-94.
[15]	HU Ya-dan, WU Guo-qiang, LIU Chen, WEI Ming. Roles of MYB Transcription Factor in Regulating the Responses of Plants to Stress [J]. Biotechnology Bulletin, 2024, 40(6): 5-22.