白颖苔草CrMYB4基因的克隆和表达分析

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

生物技术通报 ›› 2025, Vol. 41 ›› Issue (7): 248-260.doi: 10.13560/j.cnki.biotech.bull.1985.2025-0080

白颖苔草CrMYB4基因的克隆和表达分析

魏雨佳¹(), 李岩², 康语涵¹, 弓晓楠¹, 杜敏¹, 涂岚¹, 石鹏¹, 于子涵¹, 孙彦³(), 张昆¹()

^1.黄河三角洲草地资源与生态国家林业和草原局重点实验室青岛农业大学草业学院，青岛 266109
^2.中国科学院西北生态环境资源研究院干旱区生态安全与可持续发展重点实验室，兰州 730000
^3.农业农村部牧草与草坪草种子质量监督检验测试中心（北京）中国农业大学草业科学与技术学院，北京 100193

收稿日期:2025-01-18 出版日期:2025-07-26 发布日期:2025-07-22
通讯作者: 孙彦，女，博士，教授，研究方向：草坪草分子育种；E-mail: ctsoffice@163.com
张昆，男，博士，副教授，研究方向：草坪草分子育种；E-mail: zk61603@163.com
作者简介:魏雨佳，女，硕士，研究方向：草坪草分子育种；E-mail: weiyujia0814@163.com
第一联系人：魏雨佳、李岩为共同第一作者
基金资助:
山东省自然科学基金项目(ZR2021QC037);山东省自然科学基金项目(ZR2024MC174);青岛农业大学高层次人才启动基金(665/1121011);国家自然科学基金项目(32101435);国家自然科学基金项目(32271763)

Cloning and Expression Analysis of the CrMYB4 Gene in Carex rigescens

WEI Yu-jia¹(), LI Yan², KANG Yu-han¹, GONG Xiao-nan¹, DU Min¹, TU Lan¹, SHI Peng¹, YU Zi-han¹, SUN Yan³(), ZHANG Kun¹()

^1.Key Laboratory of National Forestry and Grassland Administration on Grassland Resources and Ecology in the Yellow River Delta, College of Grassland Science, Qingdao Agricultural University, Qingdao 266109
^2.Key Laboratory of Ecological Safety and Sustainable Development in Arid Lands, Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences, Lanzhou 730000
^3.Forage and Turfgrass Seed Quality Inspection and Testing Centre (Beijing), Ministry of Agriculture and Rural Affairs, College of Grassland Science and Technology, China Agricultural University, Beijing 100193

Received:2025-01-18 Published:2025-07-26 Online:2025-07-22

摘要/Abstract

摘要：

目的探究CrMYB4在白颖苔草生长发育及非生物胁迫中的作用，为进一步研究其在白颖苔草抗逆机制中的功能提供理论指导。方法基于前期白颖苔草盐响应组学结果，筛选到的1个CrMYB4蛋白，并对其生物信息学特征、亚细胞定位、启动子顺式作用元件分析及不同组织、非生物胁迫和外源激素处理下的表达情况进行评价。结果 CrMYB4序列编码区为747 bp，编码248个氨基酸。进化分析发现其属于典型的R2R3-MYB类群，与莎草科植物亲缘关系较近。亚细胞定位分析显示，CrMYB4蛋白定位于细胞核中。CrMYB4基因在新叶和老叶中高表达，而在根、幼芽、叶鞘等组织中的表达量较低。启动子顺式元件分析显示，CrMYB4启动子区域含有多个参与激素响应、植物生理和转录识别相关的保守功能元件。CrMYB4基因在盐、干旱和低温胁迫下均有着快速响应，在处理1 h后迅速上调，推测CrMYB4可能参与白颖苔草的非生物胁迫响应。在不同植物激素（ABA、SA、IAA和GA3）处理下，CrMYB4均显著上调，在6 d时达到最大值，且在IAA处理后响应最为明显，推测CrMYB4可能参与白颖苔草的激素响应。结论 CrMYB4在白颖苔草生长发育、逆境胁迫和激素响应等方面均具有潜在作用，在盐胁迫和IAA信号通路中起关键作用。

关键词: 白颖苔草, MYB转录因子, MYB4, 基因克隆, 盐胁迫, 木质素, 生长发育, 非生物胁迫

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

魏雨佳, 李岩, 康语涵, 弓晓楠, 杜敏, 涂岚, 石鹏, 于子涵, 孙彦, 张昆. 白颖苔草CrMYB4基因的克隆和表达分析[J]. 生物技术通报, 2025, 41(7): 248-260.

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.

图/表 11

表1 研究中使用的引物及其序列

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

图1 CrMYB4的PCR电泳图及核苷酸与氨基酸序列A：PCR电泳图；B：核苷酸与氨基酸序列

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

表2 CrMYB4编码蛋白的理化性质分析

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

图2 CrMYB4编码蛋白的生物信息学分析A：信号肽预测；B：疏水性预测；C：三级结构预测；D：跨膜结构肽预测

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

图3 CrMYB4的结构域分析（A）与保守性分析（B）

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

图4 CrMYB4的系统进化树分析

Fig. 4 Phylogenetic tree analysis of CrMYB4

图5 CrMYB4亚细胞定位A：亚细胞定位载体示意图；B：亚细胞定位

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

图6 CrMYB4的不同组织表达分析R：根茎；RC：根颈；RS：根；NGP：幼芽；LS：叶鞘；NL：新叶；OL：老叶。数据为3次生物学重复的平均值±标准误差，* P<0.05，** P<0.01，下同

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

图7 CrMYB4启动子区域顺式作用元件分析A：启动子区域顺式作用元件数量（Cr：白颖苔草；At：拟南芥；Cl：康藏嵩草；Rt：刺子莞；Os：水稻）；B：启动子区域顺式作用元件分布及功能

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

图8 CrMYB4不同胁迫下表达分析

Fig. 8 Expression analysis of CrMYB4 under different stresses

图9 CrMYB4在不同外源激素处理下表达分析

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

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白颖苔草CrMYB4基因的克隆和表达分析

Cloning and Expression Analysis of the CrMYB4 Gene in Carex rigescens

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