Cloning and Expression Analysis of the CrMYB4 Gene in Carex rigescens

doi:10.13560/j.cnki.biotech.bull.1985.2025-0080

Abstract

Abstract:

Objective To explore the role of CrMYB4 in the growth, development and abiotic stress of Carex rigescens may provide theoretical guidance for further research on its function in the stress resistance mechanism of C. rigescens. Method Based on the previous results of salt-responsive omics of C. rigescens, a CrMYB4 protein was screened, and its bioinformatics characteristics, subcellular localization, promoter cis-acting element analysis, and expression patterns under different tissues, abiotic stresses, and exogenous hormone treatments were evaluated. Result The coding region of the CrMYB4 sequence was 747 bp, encoding 248 amino acids. Phylogenetic analysis revealed that it belonged to the typical R2R3-MYB group and was closely related to plants in the Cyperaceae family. Subcellular localization analysis demonstrated that the CrMYB4 protein was localized in the nucleus. CrMYB4 was highly expressed in new and old leaves, while its expressions were relatively low in tissues such as roots, young buds, and leaf sheaths. Analysis of promoter cis-acting elements showed that the promoter region of CrMYB4 contained multiple functional elements associated with hormone response, plant physiology, and transcriptional recognition. Expression analysis under different abiotic stresses showed that the CrMYB4 gene responded rapidly to salt, drought, and low-temperature stresses, with its expression increasing rapidly after 1 h of treatment. It is speculated that CrMYB4 may be involved in the response of C. rigescens toabiotic stress. Under treatments with different plant hormones (ABA, SA, IAA, and GA3), the expression of CrMYB4 was significantly upregulated and reached its maximum value at 6 d. It is speculated that CrMYB4 may be involved in the hormone response of C. rigescens. Conclusion CrMYB4 has potential roles in the growth and development, stress responses to adversity, and hormone responses of C. rigescens. It plays a crucial role in salt stress and the IAA signaling pathway.

Key words: Carex rigescens, MYB transcription factor, MYB4, gene cloning, NaCl stress, lignin, growth and development, abiotic stress

WEI Yu-jia, LI Yan, KANG Yu-han, GONG Xiao-nan, DU Min, TU Lan, SHI Peng, YU Zi-han, SUN Yan, ZHANG Kun. Cloning and Expression Analysis of the CrMYB4 Gene in Carex rigescens[J]. Biotechnology Bulletin, 2025, 41(7): 248-260.

Figures/Tables 11

Table 1 Primers and their sequences used in this study

引物名称 Primer name	上游引物 Forward primer （5′‒3′）	下游引物 Reverse primer （5′‒3′）
CrMYB4-CDS	ATGGGAAGAGCACCTTGCTG	CTATAATTGAGACATGTCCATTACTCCA
CrMYB4-qPCR	TAGATGGTCAGCAATAGCAGCAC	ACTCGGGTTGTGGCGTATTA
pGD-CrMYB4-GFP	CTCTCTACAAGATCTATGGGAAGAGCACCTTGCTG	CAGAATTCGAAGCTTGTAATTGAGACATGTCCATTACTCCA
CreIF-4α	CTCACGGGTTGGAGCGAGAA	CAGCAGAGAGGCATAGTCCCC

Fig. 1 PCR electrophoresis pattern of CrMYB4, as well as its nucleotide and amino acid sequencesA: PCR electrophoresis pattern. B: Nucleotide and amino acid sequences

Table 2 Physicochemical properties analysis of CrMYB4 predicted protein

理化性质 Physicochemical property	预测结果 Predict result
氨基酸数量 Amino acids number	248
分子量 Molecular weight （kD）	28.00
等电点 Isoelectric point	5.16
分子式 Formula	C₁₂₁₆H₁₈₉₈N₃₃₈O₃₉₈S₁₂
不稳定性系数 Instability index	57.00
脂肪族系数 Aliphatic index	62.14
平均亲水性系数 Grand average of hydropathicity	-0.784

Fig. 2 Bioinformatics analysis of the protein encoded by CrMYB4A: Prediction of the signal peptide. B: Prediction of the hydrophobicity. C: Prediction of the tertiary structure. D: Prediction of the transmembrane structural peptide

Fig. 3 Domain analysis (A) and conservative analysis (B) of CrMYB4

Fig. 4 Phylogenetic tree analysis of CrMYB4

Fig. 5 Subcellular localization of CrMYB4A: Schematic diagram of subcellular localization vector. B: Subcellular localization

Fig. 6 Expression analysis of CrMYB4 in different tissuesR: Root. RC: Root crown. RS: Root system. NGP: New generation plant. LS: Leave sheath. NL: New leave. OL: Old leave. The data are the mean values ± standard errors of three biological replicates. * P<0.05, ** P<0.01. The same below

Fig. 7 Analysis of cis-acting elements in the promoter region of CrMYB4A: Number of cis-acting elements in the promoter region (Cr: Carex rigescens; At: Arabidopsis thaliana; Cl: Carex littledalei; Rt: Rhynchospora tenuis; Os: Oryza sativa). B: Distribution and function of cis-acting elements in the promoter region

Fig. 8 Expression analysis of CrMYB4 under different stresses

Fig. 9 Expression analysis of CrMYB4 under different exogenous hormone treatments

References 43

[1]	米海莉, 许兴, 马雅琴, 等. 小麦品种耐盐性的研究 [J]. 干旱地区农业研究, 2003, 21(1): 134-138.
	Mi HL, Xu X, Ma YQ, et al. Study on salt-tolerance of wheat varieties [J]. Agric Res Arid Areas, 2003, 21(1): 134-138.
[2]	熊毅, 熊艳丽, 杨晓鹏, 等. 外源褪黑素对盐胁迫下老化燕麦种子萌发及幼苗的影响 [J]. 中国草地学报, 2020, 42(1): 7-14.
	Xiong Y, Xiong YL, Yang XP, et al. Effects of exogenous melatonin on seeds germination and seedling of aged oat under salt stress [J]. Chin J Grassland, 2020, 42(1): 7-14.
[3]	Dubos C, Stracke R, Grotewold E, et al. MYB transcription factors in Arabidopsis [J]. Trends Plant Sci, 2010, 15(10): 573-581.
[4]	Paz-Ares J, Ghosal D, Wienand U, et al. The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators [J]. EMBO J, 1987, 6(12): 3553-3558.
[5]	许文杰, 黄远浩, 韩蓉蓉, 等. 基于全基因组的栀子苷生物合成相关MYB转录因子系统分析 [J]. 药学学报, 2023, 58(8): 2522-2531.
	Xu WJ, Huang YH, Han RR, et al. Systematic analysis of MYB transcription factors related to the geniposide biosynthesis in Gardenia jasminoides Ellis based on whole genome [J]. Acta Pharm Sin, 2023, 58(8): 2522-2531.
[6]	关范圆, 郑语嫣, 米芯雨, 等. R2R3型MYB转录因子调控植物胁迫应答的研究进展 [J]. 植物医学, 2024, 3(3): 1-9.
	Guan FY, Zheng YY, Mi XY, et al. Research advance of the functions of R2R3-MYB transcription factors in plant response to biotic and abiotic stresses [J]. Plant Health Med, 2024, 3(3): 1-9.
[7]	Yu L, Chen HX, Guan QL, et al. AtMYB2 transcription factor can interact with the CMO promoter and regulate its downstream gene expression [J]. Biotechnol Lett, 2012, 34(9): 1749-1755.
[8]	Dai XY, Xu YY, Ma QB, et al. Overexpression of an R1R2R3 MYB gene, OsMYB3R-2, increases tolerance to freezing, drought, and salt stress in transgenic Arabidopsis [J]. Plant Physiol, 2007, 143(4): 1739-1751.
[9]	Mu RL, Cao YR, Liu YF, et al. An R2R3-type transcription factor gene AtMYB59 regulates root growth and cell cycle progression in Arabidopsis [J]. Cell Res, 2009, 19(11): 1291-1304.
[10]	牛义岭, 姜秀明, 许向阳. 植物转录因子MYB基因家族的研究进展 [J]. 分子植物育种, 2016, 14(8): 2050-2059.
	Niu YL, Jiang XM, Xu XY. Reaserch advances on transcription factor MYB gene family in plant [J]. Mol Plant Breed, 2016, 14(8): 2050-2059.
[11]	Jin H, Martin C. Multifunctionality and diversity within the plant MYB-gene family [J]. Plant Mol Biol, 1999, 41(5): 577-585.
[12]	Yang L, Zhao X, Yang F, et al. PtrWRKY19, a novel WRKY transcription factor, contributes to the regulation of pith secondary wall formation in Populus trichocarpa [J]. Sci Rep, 2016, 6: 18643.
[13]	Zhou JL, Lee CH, Zhong RQ, et al. MYB58 and MYB63 are transcriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis [J]. Plant Cell, 2009, 21(1): 248-266.
[14]	Shen H, Poovaiah CR, Ziebell A, et al. Enhanced characteristics of genetically modified switchgrass (Panicum virgatum L.) for high biofuel production [J]. Biotechnol Biofuels, 2013, 6(1): 71.
[15]	Vannini C, Locatelli F, Bracale M, et al. Overexpression of the rice Osmyb4 gene increases chilling and freezing tolerance of Arabidopsis thaliana plants [J]. Plant J, 2004, 37(1): 115-127.
[16]	孟祥宇, 谢红梅, 才可新, 等. 人参PgMYB4基因表达载体构建及其对拟南芥的抗旱性分析 [J]. 中草药, 2016, 47(17): 3094-3097.
	Meng XY, Xie HM, Cai KX, et al. Construction of expression vector of PgMYB4 gene in Panax ginseng and analysis on drought resisting [J]. Chin Tradit Herb Drugs, 2016, 47(17): 3094-3097.
[17]	Zhang FR, Zhang XC, Wan WH, et al. MYB4 in Lilium pumilum affects plant saline-alkaline tolerance [J]. Plant Signal Behav, 2024, 19(1): 2370724.
[18]	田宇. 冬小麦TaMYB4-TaMDHAR模块抗寒功能探究 [D]. 哈尔滨: 东北农业大学, 2022.
	Tian Y. Study on cold resistance function of TaMYB4-TaMDHAR module in winter wheat [D]. Harbin: Northeast Agricultural University, 2022.
[19]	朱英洁. 陆地棉抗紫外线MYB基因挖掘及MYB4功能初探 [D]. 曲阜: 曲阜师范大学, 2022.
	Zhu YJ. Mining of ultraviolet-resistant MYB gene in upland cotton and preliminary study on the function of MYB4 [D]. Qufu: Qufu Normal University, 2022.
[20]	马万里, 韩烈保, 罗菊春. 草坪植物的新资源——苔草属植物 [J]. 草业科学, 2001, 18(2): 43-45, 56.
	Ma WL, Han LB, Luo JC. A new lawn plant resource: genus Carex L [J]. Pratacultural Sci, 2001, 18(2): 43-45, 56.
[21]	杨学军, 武菊英, 滕文军, 等. 北京薹草属植物资源调查与园林应用评价 [J]. 广西植物, 2014, 34(5): 664-669.
	Yang XJ, Wu JY, Teng WJ, et al. Investigation and landscape application evaluation on Carex resources in Beijing [J]. Guihaia, 2014, 34(5): 664-669.
[22]	李明娜. 基于蛋白质组学的白颖苔草盐胁迫下应激调控机理的研究 [D]. 北京: 中国农业大学, 2019.
	Li MN. Study on stress regulation mechanism of Carex albuginosa under salt stress based on protein omics [D]. Beijing: China Agricultural University, 2019.
[23]	刘雪梅, 李文, 黄管大, 等. 相异度算法结合邻接法构建系统进化树的评估 [J]. 华南理工大学学报: 自然科学版, 2019, 47(6): 136-141, 148.
	Liu XM, Li W, Huang GD, et al. Feasibility evaluation of dissimilarity algorithm and adjacency method for constructing evolutionary tree [J]. J South China Univ Technol Nat Sci Ed, 2019, 47(6): 136-141, 148.
[24]	Yoo SD, Cho YH, Sheen J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis [J]. Nat Protoc, 2007, 2(7): 1565-1572.
[25]	李岩, 米鑫丰, 闫丽, 等. 白颖苔草UDP-糖基转移酶UGT72B3基因的克隆与耐盐性分析 [J]. 中国草地学报, 2023, 45(8): 10-22.
	Li Y, Mi XF, Yan L, et al. Cloning and salt tolerance analysis of UDP-glycosyltransferases UGT72B3 gene in Carex rigescens [J]. Chin J Grassland, 2023, 45(8): 10-22.
[26]	崔宝禄, 陈忠平, 孙婷婷, 等. 番茄GT-1基因组织表达模式及非生物胁迫、植物生长调节剂响应分析 [J]. 福建农业学报, 2023, 38(2): 144-150.
	Cui BL, Chen ZP, Sun TT, et al. Expressions and responses to abiotic stresses and plant growth regulator of tomato GT-1 [J]. Fujian J Agric Sci, 2023, 38(2): 144-150.
[27]	石文昕, 郎俊凯, 许玲兴, 等. 叶面预喷施水杨酸缓解西瓜幼苗高温胁迫的生理效应 [J]. 西北植物学报, 2024, 44(11): 1692-1702.
	Shi WX, Lang JK, Xu LX, et al. Physiological effects of foliar pre spray of salicylic acid on alleviating high temperature stress in Citrullus lanatus seedlings [J]. Acta Bot Boreali Occidentalia Sin, 2024, 44(11): 1692-1702.
[28]	赵永晶, 杨洁, 赵晗茜, 等. 荷花CKX基因家族的鉴定及非生物胁迫下的表达模式分析 [J]. 植物生理学报, 2024, 60(12): 1783-1798.
	Zhao YJ, Yang J, Zhao HQ, et al. Genome-wide identification and expression pattern of the CKX gene family under abiotic stress in Nelumbo nucifera [J]. Plant Physiol J, 2024, 60(12): 1783-1798.
[29]	崔正阳, 单颖, 岳晓岚, 等. 毛白杨BIN2.1启动子的组织表达及外源激素响应分析[J/OL]. 分子植物育种, 2024. .
	Cui ZY, Shan Y, Yue XL, et al. Tissue expression and exogenous hormone response of BIN2.1 promoter of Populus tomentosa [J/OL]. Mol Plant Breed, 2024. .
[30]	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.
[31]	Chen BS, Niu FF, Liu WZ, et al. Identification, cloning and characterization of R2R3-MYB gene family in canola (Brassica napus L.) identify a novel member modulating ROS accumulation and hypersensitive-like cell death [J]. DNA Res, 2016, 23(2): 101-114.
[32]	吴琼, 胡明珠, 余晓倩, 等. 赤霞珠葡萄VvMYB4b转录因子的亚细胞定位及表达分析[J/OL].分子植物育种, 2024. .
	Wu Q, Hu MZ, Yu XQ, et al. Subcellular localization and expression analysis of VvMYB4b transcription factor in Cabernet Sauvignon [J/OL] . Mol Plant Breed, 2024. .
[33]	Yu Y, Liu HZ, Zhang N, et al. The BpMYB4 transcription factor from Betula platyphylla contributes toward abiotic stress resistance and secondary cell wall biosynthesis [J]. Front Plant Sci, 2021, 11: 606062.
[34]	Ma LL, Zhang N, Liu P, et al. Single-cell RNA sequencing reveals a key regulator ZmEREB14 affecting shoot apex development and yield formation in maize [J]. Plant Biotechnol J, 2025, 23(3): 766-779.
[35]	李泽凤, 向南星, 魏春梅, 等. 绿萼凤仙花MYB4的克隆及表达分析 [J]. 山东农业科学, 2023, 55(1): 8-14.
	Li ZF, Xiang NX, Wei CM, et al. Cloning and expression analysis of MYB4 genes in Impatiens chlorosepala [J]. Shandong Agric Sci, 2023, 55(1): 8-14.
[36]	Zhang CX, Bian MD, Yu H, et al. Identification of alkaline stress-responsive genes of CBL family in sweet sorghum (Sorghum bicolor L.) [J]. Plant Physiol Biochem, 2011, 49(11): 1306-1312.
[37]	郭飞翔, 李春霞, 周爽, 等. 绿豆R2R3-MYB转录因子家族鉴定及其类黄酮合成调控基因的筛选 [J]. 作物学报, 2025, 51(1): 117-133.
	Guo FX, Li CX, Zhou S, et al. Identification of the R2R3-MYB transcription factor family and screening of genes regulating flavonoid synthesis in mung bean [J]. Acta Agron Sin, 2025, 51(1): 117-133.
[38]	方森. 苹果转录因子MhMYB4抗旱功能鉴定及其作用机制解析 [D]. 郑州: 河南农业大学, 2023.
	Fang S. Identification of drought resistance function of apple transcription factor MhMYB4 and its mechanism analysis [D]. Zhengzhou: Henan Agricultural University, 2023.
[39]	Wang WW, Ma JY, Liu HX, et al. Genome-wide analysis of the switchgrass YABBY family and functional characterization of PvYABBY14 in response to ABA and GA stress in Arabidopsis [J]. BMC Plant Biol, 2024, 24(1): 114.
[40]	Feng L, Teng F, Li N, et al. A reference-grade genome of the xerophyte Ammopiptanthus mongolicus sheds light on its evolution history in legumes and drought-tolerance mechanisms [J]. Plant Commun, 2024, 5(7): 100891.
[41]	Nashaat Al-Attala M, Wang XJ, Abou-Attia MA, et al. A novel TaMYB4 transcription factor involved in the defence response against Puccinia striiformis f. sp. tritici and abiotic stresses [J]. Plant Mol Biol, 2014, 84(4/5): 589-603.
[42]	Ma GL, Li MZ, Wu YL, et al. Camellia sinensis CsMYB4a participates in regulation of stamen growth by interaction with auxin signaling transduction repressor CsAUX/IAA4 [J]. Crop J, 2024, 12(1): 188-201.
[43]	Hao SX, Lu YF, Peng Z, et al. McMYB4 improves temperature adaptation by regulating phenylpropanoid metabolism and hormone signaling in apple [J]. Hortic Res, 2021, 8(1): 182.