Identification of the Soybean R2R3-MYB Gene Family Based on the T2T Genome and Their Expression Analysis under Drought and Salt Stress

doi:10.13560/j.cnki.biotech.bull.1985.2024-0745

Abstract

Abstract:

Objective R2R3-MYB transcription factors play an important role in plant response to stress conditions. It is aimed to fully identify the R2R3-MYB transcription factors in soybean and understand the role of soybean R2R3-MYB genes in stress responses. Method The soybean R2R3-MYB family genes were systematically identified and evolutionarily analyzed using the soybean Telomere-to-Telomere (T2T) genome. Real-time fluorescence quantitative PCR (RT-qPCR) was used to analyze the expression of this family of genes under drought and salt stress conditions. Result The soybean genome contains 324 R2R3-MYB genes, 77 of which were newly identified and distributed on 20 chromosomes. The results of evolutionary analysis showed that soybean R2R3-MYB family genes can be divided into four subgroups, and the colinear gene pairs were closer on the branches of the evolutionary tree. Promoter analysis revealed that most cis-acting elements related to ABA and drought resistance were present. Through expression analysis, 10 genes were speculated to be involved in the process of soybean salt tolerance, of which GmMYB19 and GmMYB76 have been reported, and GmMYB89 and GmMYB214 were identified for the first time and found to be related to soybean drought and salt tolerance. Conclusion A total of 321 R2R3-MYB family genes were identified in soybean, and 10 candidate genes for drought and salt tolerance were identified through analysis of their expression patterns under drought and salt stress conditions.

Key words: Glycine max, R2R3-MYB gene family, bioinformatics analysis, drought and salt stress, expression analysis, T2T

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.

Figures/Tables 9

Table 1 Primer information used

引物名称 Primer name	正向引物 Forward sequence （5'-3'）	反向引物 Reverse sequence （5'-3'）
Gm_MYB114	GGACCATACCTCCTTGGCAG	GGTGATGGTGGTGTGTCCAA
Gm_MYB137	AGCTGCCAGGAAGAACAGAC	ACGCAACGTGTCTCTGAACT
Gm_MYB169	TCCTCTGATGGAAGCTGCAC	TGGCTAAACCACTCACCAGC
Gm_MYB19	ATCGGTGGGCCAAAATAGCA	TTAGCCTGCTTCACCACTCG
Gm_MYB208	CCCAGTGACACACAGTCCTC	TACTGGTGCATGCCAAGGAG
Gm_MYB214	TGGTTCAACAGGAAGTGCGT	GCTTGAGGCCAGGATGTAGG
Gm_MYB249	GGCATGTGATGATGGCAAGG	GTGACACAAGGGTGGCTGTA
Gm_MYB51	GGCAAAGGACATGGATCGGA	AACCAACCTCCGTAACGCTT
Gm_MYB76	GCCAGGCAGAACAGACAATG	GGCATAGGAGTTGCCTGGTT
Gm_MYB89	GATGGTCTACCATTGCGGCT	GTGTGGTGTGCCAATGGTTC
GmActin	CGGTGGTTCTATCTTGGCATC	GTCTTTCGCTTCAATAACCCTA

Fig. 1 Chromosome location of soybean R2R3-MYB geneRed indicates those that have been identified, and black indicates those newly identified

Fig. 2 Phylogenetic tree of soybean and Arabidopsis thalianaR2R3-MYB genesDifferent colors indicate different groups. The red star indicates the soybean R2R3-MYB gene, and the green star indicates the Arabidopsis thalianaR2R3-MYB gene

Fig. 3 Collinearity analysis of soybean R2R3-MYB genes

Fig. 4 Gene structure and motif analysis of the R2R3-MYB gene family

Fig. 5 Analysis of cis-acting elements in the promoters of soybean R2R3-MYB gene familyA: Number of cis-acting elements in different categories of promoters. B: cis-acting elements related to stress. C: cis-acting elements related to growth and development. D: cis-acting elements related to hormones

Fig. 6 Expression analysis of genes in soybean R2R3-MYB gene family under drought and salt stressA: Expression calorimetry under drought stress. B: Expression calorimetry under salt stress. C: Number of common and specific differentially expressed R2R3-MYB genes under drought and salt stress

Fig. 7 Expression patterns of 10 candidate genes under drought stressThe results are presented as means±SD (n=3, *P<0.05, **P<0.01), the same below

Fig. 8 Expression patterns of 10 candidate genes under salt stress

References 37

17	Zhang C, Xie L, Yu H, et al. The T2T genome assembly of soybean cultivar ZH13 and its epigenetic landscapes [J]. Mol Plant, 2023, 16(11): 1715-1718.
18	Lu SN, Wang JY, Chitsaz F, et al. CDD/SPARCLE: the conserved domain database in 2020 [J]. Nucleic Acids Res, 2020, 48(D1): D265-D268.
19	Mariethoz J, Alocci D, Gastaldello A, et al. Glycomics@ExPASy: bridging the gap [J]. Mol Cell Proteomics, 2018, 17(11): 2164-2176.
20	Tamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11 [J]. Mol Biol Evol, 2021, 38(7): 3022-3027.
21	Chen CJ, Wu Y, Li JW, et al. TBtools-II: a “one for all, all for one” bioinformatics platform for biological big-data mining [J]. Mol Plant, 2023, 16(11): 1733-1742.
22	Chen SF, Zhou YQ, Chen YR, et al. Fastp: an ultra-fast all-in-one FASTQ preprocessor [J]. Bioinformatics, 2018, 34(17): i884-i890.
23	Pertea M, Kim D, Pertea GM, et al. Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown [J]. Nat Protoc, 2016, 11(9): 1650-1667.
24	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT Method [J]. Methods, 2001, 25(4): 402-408.
25	张丹, 郑平, 邓彪, 等. 百香果(Passiflora edulis)R2R3-MYB基因家族的结构和功能分析 [J]. 果树学报, 2024, 41(1): 12-29.
	Zhang D, Zheng P, Deng B, et al. Structure and function analysis of R2R3-MYB gene family from passion fruit(Passiflora edulis) [J]. J Fruit Sci, 2024, 41(1): 12-29.
26	郭玉婷, 杜长霞. 黄瓜R2R3-MYB亚家族鉴定及生物信息学分析[J]. 浙江农林大学学报, 2024, 41 (2): 286-296.
	Guo YT, Du CX. Identification and bioinformatics analysis of R2R3-MYB subfamily in cucumber [J]. J Zhejiang A F Univ, 2024, 41 (2): 286-296.
1	Dubos C, Stracke R, Grotewold E, et al. MYB transcription factors in Arabidopsis [J]. Trends Plant Sci, 2010, 15(10): 573-581.
2	薛英喜, 魏建华, 姜廷波, 等. 植物次生生长相关MYB转录因子研究进展 [J]. 安徽农业科学, 2012, 40(13): 7650-7655.
	Xue YX, Wei JH, Jiang TB, et al. Research advances in the secondary growth-associated MYB transcription factors in plants [J]. J Anhui Agric Sci, 2012, 40(13): 7650-7655.
3	胡月, 王志富, 曹心如. 植物MYB转录因子的功能研究进展 [J]. 安徽农学通报, 2023, 29(12): 60-62.
	Hu Y, Wang ZF, Cao XR. Progress in the functional study of plant MYB transcription factors [J]. Anhui Agric Sci Bull, 2023, 29(12): 60-62.
4	Wilkins O, Nahal H, Foong J, et al. Expansion and diversification of the Populus R2R3-MYB family of transcription factors [J]. Plant Physiol, 2009, 149(2): 981-993.
5	Abdullah-Zawawi MR, Ahmad-Nizammuddin NF, Govender N, et al. Comparative genome-wide analysis of WRKY, MADS-box and MYB transcription factor families in Arabidopsis and rice [J]. Sci Rep, 2021, 11(1): 19678.
6	Wei QH, Chen R, Wei X, et al. Genome-wide identification of R2R3-MYB family in wheat and functional characteristics of the abiotic stress responsive gene TaMYB344 [J]. BMC Genomics, 2020, 21(1): 792.
7	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 factors in regulating the of plants to stress [J]. Biol Bull, 2024, 40 (6): 5-22.
8	令狐斌. 苦荞黄酮醇代谢转录因子FtMYB7和FtMYB9基因克隆及功能分析 [D]. 太谷: 山西农业大学, 2016.
	Linghu B. Gene cloning and functional analysis of flavonoids alcohol metabolism transcription factors FtMYB7 and FtMYB9 in Fagopyrum tataricum Gaertn [D]. Taigu: Shanxi Agricultural University, 2016.
27	张超凡. MYB基因家族的鉴定及其在开花植物中的进化分析 [D]. 武汉: 华中农业大学, 2022.
	Zhang CF. Identification of MYB gene family and its evolutionary analysis in flowering plants [D]. Wuhan: Huazhong Agricultural University, 2022.
28	梁细妹, 秦双双, 韦范, 等. 转录因子在植物干旱应激中的功能研究进展 [J]. 生物资源, 2024, 46(3): 220-230.
	Liang XM, Qin SS, Wei F, et al. Research progress on the function of transcription factors in plant drought stress [J]. Biotic Resour, 2024, 46(3): 220-230.
29	潘志立, 郭雯, 王婷, 等. 叶片吸收水分的研究进展 [J]. 植物生理学报, 2021, 57(1): 19-32.
	Pan ZL, Guo W, Wang T, et al. Research progress on foliar water uptake [J]. Plant Physiol J, 2021, 57(1): 19-32.
30	刘晓龙, 徐晨, 邵勤, 等. 脱落酸提高水稻抗逆性的研究进展 [J]. 东北农业科学, 2022, 47(6): 29-33.
	Liu XL, Xu C, Shao Q, et al. Research progress of abscisic acid in improving rice stress resistance [J]. J Northeast Agric Sci, 2022, 47(6): 29-33.
31	Daszkowska-Golec A, Szarejko I. Open or close the gate - stomata action under the control of phytohormones in drought stress conditions [J]. Front Plant Sci, 2013, 4: 138.
32	Mohamed HI, Latif HH. Improvement of drought tolerance of soybean plants by using methyl jasmonate [J]. Physiol Mol Biol Plants, 2017, 23(3): 545-556.
33	Sheteiwy MS, Shao HB, Qi WC, et al. Seed priming and foliar application with jasmonic acid enhance salinity stress tolerance of soybean (Glycine max L.) seedlings [J]. J Sci Food Agric, 2021, 101(5): 2027-2041.
34	Wang FB, Ren XQ, Zhang F, et al. A R2R3-type MYB transcription factor gene from soybean, GmMYB12, is involved in flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis [J]. Plant Biotechnol Rep, 2019, 13(3): 219-233.
9	李红强, 罗倩, 杨帅, 等. 过表达NtMYB4a基因增强烟草抗旱能力 [J]. 亚热带植物科学, 2022, 51(1): 13-18.
	Li HQ, Luo Q, Yang S, et al. Over-expression of NtMYB4a gene enhances drought resistance of tobacco [J]. Subtrop Plant Sci, 2022, 51(1): 13-18.
10	伍翀. 黄芩MYB转录因子功能初步研究 [D]. 武汉: 武汉工业学院, 2012.
	Wu C. Preliminary study on the function of MYB transcription factor in Scutellaria baicalensis Georgi [D]. Wuhan: Wuhan Institute of Technology, 2012.
11	Fang Q, Wang XQ, Wang HY, et al. The poplar R2R3 MYB transcription factor PtrMYB94 coordinates with abscisic acid signaling to improve drought tolerance in plants [J]. Tree Physiol, 2020, 40(1): 46-59.
12	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.
13	Abe H, Urao T, Ito T, et al. Arabidopsis AtMYC₂ (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling [J]. Plant Cell, 2003, 15(1): 63-78.
14	Zhang LC, Liu GX, Zhao GY, et al. Characterization of a wheat R2R3-MYB transcription factor gene, TaMYB19, involved in enhanced abiotic stresses in Arabidopsis [J]. Plant Cell Physiol, 2014, 55(10): 1802-1812.
15	Zhang LC, Zhao GY, Xia C, et al. A wheat R2R3-MYB gene, TaMYB30-B, improves drought stress tolerance in transgenic Arabidopsis [J]. J Exp Bot, 2012, 63(16): 5873-5885.
16	刘赵, 沈海军, 周凌晨, 等. 可溶性大豆多糖的提取及在食品中的应用研究进展 [J]. 中国调味品, 2023, 48(6): 199-208.
	Liu Z, Shen HJ, Zhou LC, et al. Research progress on the extraction of soluble soybean polysaccharides and their application in food [J]. China Condiment, 2023, 48(6): 199-208.
35	Wang N, Zhang WX, Qin MY, et al. Drought tolerance conferred in soybean (Glycine max. L) by GmMYB84, a novel R2R3-MYB transcription factor [J]. Plant Cell Physiol, 2017, 58(10): 1764-1776.
36	Shi K, Liu J, Liang H, et al. An alfalfa MYB-like transcriptional factor MsMYBH positively regulates alfalfa seedling drought resistance and undergoes MsWAV3-mediated degradation [J]. J Integr Plant Biol, 2024, 66(4): 683-699.
37	Xiao LY, Shi YY, Wang R, et al. The transcription factor OsMYBc and an E3 ligase regulate expression of a K⁺ transporter during salt stress [J]. Plant Physiol, 2022, 190(1): 843-859.

[1]	ZHAO Jing, GUO Qian, LI Rui-qi, LEI Ying-yang, YUE Ai-qin, ZHAO Jin-zhong, YIN Cong-cong, DU Wei-jun, NIU Jing-ping. Sequence Analysis and Induced Expression Analysis of GmGST Gene Cluster Genes in Soybean [J]. Biotechnology Bulletin, 2025, 41(5): 129-140.
[2]	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.
[3]	SUN Tian-guo, YI Lan, QIN Xu-yang, QIAO Meng-xue, GU Xin-ying, HAN Yi, SHA Wei, ZHANG Mei-juan, MA Tian-yi. Genome-wide Identification of the DABB Gene Family in Brassica rapa ssp. pekinensis and Expression Analysis under Saline and Alkali Stress [J]. Biotechnology Bulletin, 2025, 41(4): 156-165.
[4]	WANG Tian-tian, CHANG Xue-rui, HUANG Wan-yang, HUANG Jia-xin, MIAO Ru-yi, LIANG Yan-ping, WANG Jing. Identification and Analysis of GASA Gene Family in Pepper （Capsicum annuum L.） [J]. Biotechnology Bulletin, 2025, 41(4): 166-175.
[5]	HUANG Jin-heng, HUANG Xi, ZHANG Jia-yan, ZHOU Xin-yu, LIAO Pei-ran, YANG Quan. Identification and Expression Analysis of the C3H Gene Family in Grona styracifolia across Different Varieties [J]. Biotechnology Bulletin, 2025, 41(4): 243-255.
[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]	QIN Yue, YANG Yan, ZHANG Lei, LU Li-li, LI Xian-ping, JIANG Wei. Identification and Comparative Analysis of the StGAox Genes in Diploid and Tetraploid Potatoes [J]. Biotechnology Bulletin, 2025, 41(3): 146-160.
[9]	WANG Chen, LIU Guo-mei, CHEN Chang, ZHANG Jin-long, YAO Lin, SUN Xuan, DU Chun-fang. Genome-wide Identification and Expression Analysis of CCDs Family in Brassia rapa L. [J]. Biotechnology Bulletin, 2025, 41(3): 161-170.
[10]	MA Tian-yi, XU Jia-jia, LU Wen-jing, WU Yan, SHA Wei, ZHANG Mei-juan, PENG Yi-fang. Expression Analysis and Resistance Identification of BrcGASA3 in Chinese Cabbage ‘Jinxiaotong’ Cultivar under Saline-alkali Stress [J]. Biotechnology Bulletin, 2025, 41(2): 127-138.
[11]	XU Yuan-meng, MAO Jiao, WANG Meng-yao, WANG Shu, REN Jiang-ling, LIU Yu-han, LIU Si-chen, QIAO Zhi-jun, WANG Rui-yun, CAO Xiao-ning. Cloning and Expression Characteristics Analysis of Millet Genes PmDEP1 and PmEP3 [J]. Biotechnology Bulletin, 2025, 41(2): 150-162.
[12]	JIA Zi-jian, WANG Bao-qiang, CHEN Li-fei, WANG Yi-zhen, WEI Xiao-hong, ZHAO Ying. Expression Patterns of CHX Gene Family in Quinoa in Response to NO under Saline-alkali Stress [J]. Biotechnology Bulletin, 2025, 41(2): 163-174.
[13]	QIAN Zheng-yi, WU Shao-fang, CAO Shu-yi, SONG Ya-xin, PAN Xin-feng, LI Zhao-wei, FAN Kai. Identification of the NAC Transcription Factors in Nymphaea colorata and Their Expression Analysis [J]. Biotechnology Bulletin, 2025, 41(2): 234-247.
[14]	HUANG Ying, YU Wen-jing, LIU Xue-feng, DIAO Gui-ping. Bioinformatics and Expression Pattern Analysis of Glutathione S-transferase in Populus davidiana × P. bolleana [J]. Biotechnology Bulletin, 2025, 41(2): 248-256.
[15]	XIANG Chun-fan, LI Le-song, WANG Juan, LIANG Yan-li, YANG Sheng-chao, LI Meng-fei, ZHAO Yan. Functional Identification and Expression Analysis of Cinnamonyl Alcohol Dehydrogenase AsCAD in Angelica sinensis [J]. Biotechnology Bulletin, 2025, 41(2): 295-308.