Identification and Expression Analysis of R2R3-MYB Gene Family in Chinese Chestnut

doi:10.13304/j.nykjdb.2024.0477

Abstract

Abstract:

R2R3-MYB transcription factors are the largest MYB subfamily in plants and are widely involved in regulating plant growth and development and stress response. R2R3-MYB gene family of Chinese chestnut was genome-widely identified， and the chromosome distribution of these genes， subcellular localization prediction， phylogenetic relationships， protein motif， gene structure and sequence alignment of conserved domains were analyzed. Finally， the expression of the CmR2R3-MYB gene family under drought stress was analyzed by transcriptome and RT-qPCR. The results showed that total 141 CmR2R3-MYB genes were unequally distributed on 12 chromosomes and 1 contig fragment. And the phylogenetic tree divided them into 6 groups. There were 7 CmR2R3-MYB genes differentially expressed under drought stress. Among them， CmMYB89 was upregulated， while CmMYB32， CmMYB95， CmMYB91， CmMYB119， CmMYB131 and CmMYB104 were downregulated， which indicated that these 7 genes could be involved in drought resistance of Chinese chestnut. Above results provided theoretical foundations for further exploring the role of the R2R3-MYB gene family in response to drought stress of Chinese chestnut.

Key words: Chinese chestnut, drought, R2R3-MYB gene family

摘要：

R2R3-MYB转录因子是植物中最大的MYB亚家族，广泛参与调节植物生长发育和胁迫响应。对板栗R2R3-MYB基因家族进行全基因组鉴定，分析其在染色体上分布、亚细胞定位、系统进化关系、蛋白基序、基因结构、保守结构域，最后通过转录组和RT-qPCR分析CmR2R3-MYB基因在干旱胁迫下的表达。结果表明，共鉴定出141个CmR2R3-MYB基因，不均等地分布于12条染色体与1条contig片段。系统进化树将其分为6个亚家族。有7个CmR2R3-MYB基因在干旱胁迫下差异表达，其中CmMYB89表达量上调，CmMYB32、CmMYB95、CmMYB91、CmMYB119、CmMYB131及CmMYB104表达量下调，表明这些基因可能与板栗抗旱性密切相关。以上研究结果为进一步探究R2R3-MYB基因家族在板栗响应干旱胁迫方面的作用奠定了理论基础。

关键词: 板栗, 干旱, R2R3-MYB基因家族

CLC Number:

S664.2

Yaming LIU, Yong YANG, Xiaoxiao KANG, Meng WANG, Dongsheng WANG, Haie ZHANG. Identification and Expression Analysis of R2R3-MYB Gene Family in Chinese Chestnut[J]. Journal of Agricultural Science and Technology, 2025, 27(8): 47-59.

刘亚明, 杨涌, 康肖肖, 王萌, 王东升, 张海娥. 板栗R2R3-MYB基因家族的鉴定及表达分析[J]. 中国农业科技导报, 2025, 27(8): 47-59.

Figures/Tables 13

Table 1 Primer sequences used in the study

基因Gene	上游引物Forward primer （5’-3’）	下游引物Reverse primer （5’-3’）
CmMYB119	TGGTCAGCAATCGCAGCT	TGTTTCTGGGTTGGCTTGGT
CmMYB91	GCAGACTCCGCTGGACAA	GCTGCAATTGCTGACCACC
CmMYB89	AGCTGCAGGGCTTCTTCG	CCCTGGCAATCTCCCAGC
CmMYB131	TGGTGACCCTCAATGCCC	CCGGATCACATGCGTGGA
CmMYB104	TGCGGAGAGGACCATGGA	ATTCCAACGGCCCTCACC
CmMYB32	GGCTTTGACACTTGCGCC	AACTCTCCGACCTCCGCT
CmMYB95	GAGCAGCCCATCCGGATC	TCTCGCCACGGGTCTGTA
Actin	ATTCACGAGACCACCTACA	TGCCACAACCTTAATCTTCAT

Fig. 1 Distribution of the genes on the chromosomeNote：1~12 are chromosomes； 13 is contig fragment.

Fig. 2 Phylogenetic tree of CmR2R3-MYB proteins

Fig. 3 Conserved motifs of CmMYBproteins

Table 2 Motifs of different groups

类群Group	基序Motif
Group 1	motif 6，motif 10
Group 2	motif 6， motif 7， motifm 10
Group 3	motif 6， motif 7， motif 10
Group 4	motif 6， motif 10
Group 5	motif 6， motif 8~10
Group 6	motif 5， motif 8

Fig. 4 Sequence alignment of conserved R2 and R3 domainsNote： * indicates conserved amino acid.

Fig. 5 Cis-acting elements of gene family members and distribution on promoters

Fig. 6 Collinearity analysis among CmR2R3-MYB gene family members and duplication typesA； Collinearity in genome； B： Type of gene family duplications； C： Replacement rate

Fig. 7 Collinearity analysis of R2R3-MYB gene family between chestnut and other speciesA： Castanea mollissima with Arabidopsis thaliana， Oryza sativa； B： Castanea mollissima with Castanea dentata and Castanea crenata

Fig. 8 RSCU values of codon of CmR2R3-MYB genes

Fig. 9 Codon bias analysis of CmR2R3-MYB gene familyA： Neutrality plot analysis； B： ENC-plot analysis； C： PR2-plot analysis

Fig. 10 Transcriptome analysis of CmR2R3-MYB under drought stressA： Volcano map of transcriptome analysis after drought stress； B： Heat map of differentially expressed CmR2R3-MYB genes

Fig. 11 Relative expressions of differentially CmR2R3-MYB expressed genes under different treatmentsNote：** and *** indicate significant differences compared with CK at P<0.01 and P<0.001 levels， respectively.

References 27

[1]	HONG J C. General aspects of plant transcription factor families [J/OL]. Plant Transcription Factors, 2016, 12:35-56 [2024-05-20]. ‍.
[2]	JIN J P, ZHANG H, KONG L, et al.. PlantTFDB 3.0:a portal for the functional and evolutionary study of plant transcription factors [J]. Nucl. Acids Res., 2014,42(D1):D1182-D1187.
[3]	JOSHI R, WANI S H, SINGH B, et al.. Transcription factors and plants response to drought stress:current understanding and future directions [J/OL]. Front. Plant Sci., 2016,7:1029 [2024-05-20]. .
[4]	DUBOS C, STRACKE R, GROTEWOLD E,et al.. MYB transcription factors in Arabidopsis [J]. Trends Plant Sci., 2010,15(10):573-581.
[5]	DING P, TANG P, LI X,et al..Genome-wide identification,phylogeny and expression analysis of the R 2R3-MYB gene family in quinoa (Chenopodium quinoa) under abiotic stress [J/OL].Funct. Plant Biol., 2024,51:FP23261 [2024-05-20]. .
[6]	LI Y T, ZHANG J, WANG S J, et al.. Genome-wide identification of the Pyrus R 2R3-MYB gene family and PhMYB62 regulation analysis in Pyrus hopeiensis flowers at low temperature [J/OL].Int. J. Biol. Macromol., 2024,257:128611 [2024-05-20]. .
[7]	YANG K Y, DONG Q L, WU J H, et al.. Genome-wide analysis of the R 2R3-MYB transcription factor gene family expressed in Juglans regia under abiotic and biotic stresses [J/OL]. Ind. Crops Prod.,2023,198:116709 [2024-05-20]. .
[8]	WANG Y, ZHANG Y, FAN C, et al.. Genome-wide analysis of MYB transcription factors and their responses to salt stress in Casuarina equisetifolia [J/OL].BMC Plant Biol.,2021,21(1):328 [2024-05-20]. .
[9]	GAO J, ZHAO Y, ZHAO Z, et al.. RRS1 shapes robust root system to enhance drought resistance in rice [J]. New. Phytol., 2023,238(3):1146-1162.
[10]	XU W, TANG W, WANG C, et al.. SiMYB56 confers drought stress tolerance in transgenic rice by regulating lignin biosynthesis and ABA signaling pathway [J/OL]. Front. Plant Sci., 2020,11:785 [2024-05-20]. .
[11]	FANG Q, WANG X Q, WANG H Y, 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]	赵爽,葛朝红,闵卓,等.干旱胁迫对不同板栗品种叶片保水力的影响[J].黑龙江农业科学,2021(2):28-32.
	ZHAO S, GE C H, MIN Z, et al.. Effects of drought stress on leaf water holding capacity of different chestnut varieties [J].Heilongjiang Agric. Sci., 2021(2):28-32.
[13]	CAO F, GUO C, WANG X, et al.. Genome-wide identification,evolution,and expression analysis of the NAC gene family in chestnut (Castanea mollissima) [J/OL]. Front. Genet., 2024,15:1337578 [2024-05-20]. .
[14]	ZHANG P, LIU J, JIA N, et al.. Genome-wide identification and characterization of the bZIP gene family and their function in starch accumulation in Chinese chestnut (Castanea mollissima Blume) [J/OL]. Plant Sci., 2023,14:1166717 [2024-05-20]. .
[15]	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-∆∆Ct method [J]. Methods, 2001, 25: 402-408.
[16]	CHEN C J, WU Y, LI J W, 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.
[17]	NGUYEN L T, SCHMIDT H A, VON HAESELER A, et al.. IQ-TREE:a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies [J]. Mol. Biol. Evol., 2015,32(1):268-274.
[18]	WANG Y P, TANG H B, DEBARRY J D, et al.. MCScanX:a toolkit for detection and evolutionary analysis of gene synteny and collinearity [J/OL]. Nucl. Acids Res., 2012,40(7):e49 [2024-05-20]. .
[19]	MIAO B B, DONG W, GU Y X, et al.. OmicsSuite:a customized and pipelined suite for analysis and visualization of multi-omics big data [J/OL]. Hortic. Res., 2023,10(11):uhad195 [2024-05-20]. .
[20]	QIAO X, LI Q, YIN H, et al.. Gene duplication and evolution in recurring polyploidization-diploidization cycles in plants [J/OL].Genome Biol., 2019,20(1):38 [2024-05-20]. .
[21]	GAO Y, LU Y, SONG Y, et al.. Analysis of codon usage bias of WRKY transcription factors in Helianthus annuus [J/OL]. BMC Genom Data, 2022, 23(1):46 [2024-05-20]..
[22]	ZHANG Y, SHEN Z, MENG X, et al.. Codon usage patterns across seven Rosales species [J/OL]. BMC Plant Biol., 2022,22(1):65 [2024-05-20]. .
[23]	LI Q, LUO Y, SHA A, et al.. Analysis of synonymous codon usage patterns in mitochondrial genomes of nine Amanita species [J/OL]. Front. Microbiol., 2023,14:1134228 [2024-05-20]. .
[24]	ZHU N, DUAN B L, ZHENG H L, et al.. An R 2R3 GhMYBMYBgene3 functions in drought stress by negatively regulating stomata movement and ROS accumulation [J/OL].Plant Physiol. Biochem., 2023,197:107648 [2024-05-20]. .
[25]	QU J, LIU L, GUO Z, et al.. The ubiquitous position effect, synergistic effect of recent generated tandem duplicated genes in grapevine, and their co-response and overactivity to biotic stress [J/OL]. Fruit Res., 2023, 3(1):16 [2024-05-20]. .
[26]	YU L, FEI C, WANG D, et al.. Genome-wide identification,evolution and expression profiles analysis of bHLH gene family in Castanea mollissima [J]. Front. Genet., 2023,14:1193953 [2024-05-20]. .
[27]	SONG Y, ZHANG Y, WANG X, et al.. Telomere-to-telomere reference genome for Panax ginseng highlights the evolution of saponin biosynthesis [J/OL]. Hortic. Res., 2024,11(6):uhae107 [2024-05-20]. .