Genome-wide Identification of the Chalcone Isomerase Gene Family in Eucommia ulmoides and Analysis of Their Expression Patterns

doi:10.13560/j.cnki.biotech.bull.1985.2025-0202

Abstract

Abstract:

Objective Chalcone isomerase (CHI) is a key rate-limiting enzyme in the flavonoid biosynthesis pathway. The aim of this study is to conduct a genome-wide identification of the CHI gene family members in Eucommia ulmoides, systematically analyze their expression characteristics, and explore their biological functions in the growth, development, and stress response of E. ulmoides. Method Based on the genome data of E. ulmoides, bioinformatics methods were used to systematically identify the members of the CHI gene family, and comprehensively analyze their physicochemical properties, chromosomal localization, cis-acting elements in promoters, evolutionary relationships, conserved motifs, and gene structures. Meanwhile, transcriptome data and RT-qPCR technology were combined to investigate the expression patterns of EuCHIs in different tissues and under abiotic stress treatments. Result A total of 7 EuCHI gene family members (EuCHI-1 to EuCHI-7) were identified in the genome of E. ulmoides. The amino acid lengths of their encoded proteins ranged from 84 to 388 aa, the molecular weights ranged from 9 473.24 to 43 248.30 Da, the isoelectric points ranged from 4.81 to 9.42, the instability indices ranged from 23.09 to 46.43, and the hydrophilicity and hydrophobicity values ranged from -0.381 to 0.206. They were unevenly distributed on 5 chromosomes. Subcellular localization prediction showed that except for EuCHI-2, which was localized in the nucleus; the other members were all localized in the chloroplast, additionally, EuCHI-1 was also distributed in the cell membrane, EuCHI-4 was also distributed in both the cytoplasm and mitochondria. In the phylogenetic analysis, EuCHIs were classified into three subfamilies: Group Ⅰ, Group Ⅱ, and Group Ⅵ, and each member contained 1 to 10 exons. Promoter analysis indicated that EuCHIs might be involved in biological processes such as growth, development, and hormone response. Transcriptome data showed that most EuCHIs genes were involved in various biological processes of E. ulmoides, such as leaf color formation and floral development. The RT-qPCR results showed that EuCHIs had tissue expression specificity. Except for EuCHI-3, the other genes were highly expressed in the roots. Compared with the treatments of ABA and GA₃ stress, the drought stress treatment could more significantly induce the expression of EuCHI genes. Conclusion Seven EuCHIs genes are identified and divided into three subfamilies. The expression patterns of EuCHIs differ among different tissues and under different stress conditions in E. ulmoides, indicating that they play important roles in the growth, development, and abiotic stress responses of E. ulmoides.

Key words: Eucommia ulmoides, CHI gene family, genome-wide identification, bioinformatics analysis, quantitative real-time PCR (RT-qPCR), hormone treatment, abiotic stress, expression characteristics

CHENG Ting-ting, LIU Jun, WANG Li-li, LIAN Cong-long, WEI Wen-jun, GUO Hui, WU Yao-lin, YANG Jing-fan, LAN Jin-xu, CHEN Sui-qing. Genome-wide Identification of the Chalcone Isomerase Gene Family in Eucommia ulmoides and Analysis of Their Expression Patterns[J]. Biotechnology Bulletin, 2025, 41(9): 242-255.

Figures/Tables 11

Table 1 Primer sequences of EuCHIs genes

基因名称 Gene name	正向引物 Forward primer （5′-3′）	反向引物 Reverse primer （5′-3′）
EuCHI-1	TCAGAAGTGCAAAACGATGC	GTCGGGCCAAGTCAAAAATA
EuCHI-2	TGCTGATCACGAGAAACTGG	CGGTGAGTTTCCTGATGGAT
EuCHI-3	ACTGGAACCTACACCGATGC	GCCCAATTTCAGGAAATGAA
EuCHI-4	AATTCGATGTCCAAGCGTTC	ACGGACTCCATCAACTCCTC
EuCHI-5	CCGTCGTCACTACCTCCAAT	GAAACGGGACAGCAACAAAT
EuCHI-6	TTTCCTCCGGAGATCAACAC	TTTTCAACTGGGGCAGAAAC
EuCHI-7	GAGCCTATTACGGGCATTGA	TCATGGCATTTGTTCAGCTC
UBC	AGTGGGTGGTGCTGTAGTCC	AACTCCCGTTTCGTTTGTTG

Table 2 Analysis of physical and chemical properties of CHI proteins from E. ulmoides

基因ID Gene ID	基因名 Gene name	氨基酸数量Number of amino acids （aa）	分子量 Molecular weight （Da）	等电点 Isoelectric point pI	不稳定指数 Instability index	亚细胞定位 Subcellular localization	亲水性总平均值 Hydrophilicity and hydrophobicity
EUC25438-RA	EuCHI-1	291	31 099.58	9.28	36.93	细胞膜、叶绿体	0.047
EUC24475-RA	EuCHI-2	280	30 689.55	6.29	35.13	细胞核	-0.381
EUC08195-RA	EuCHI-3	259	27 648.28	4.81	30.60	叶绿体	-0.086
EUC08196-RA	EuCHI-4	84	9 473.24	9.42	23.09	叶绿体、细胞质、线粒体	0.206
EUC26357-RA	EuCHI-5	283	30 748.30	9.17	46.43	叶绿体	-0.077
EUC24815-RA	EuCHI-6	203	22 629.79	4.85	33.83	叶绿体	-0.144
EUC14152-RA	EuCHI-7	388	43 248.30	8.22	44.54	叶绿体	-0.146

Table 3 Secondary structure analysis of E. ulmoidesCHIs gene family

基因名 Gene name	α-螺旋α-helix		延伸链Extended chain		无规则卷曲Random coil
基因名 Gene name	数量 Quantity	占比 Proportion （%）	数量 Quantity	占比 Proportion （%）	数量 Quantity	占比 Proportion （%）
EuCHI-1	96	32.99	48	16.49	147	50.52
EuCHI-2	109	38.93	41	14.64	130	46.43
EuCHI-3	108	41.70	45	17.37	106	40.93
EuCHI-4	31	36.90	22	26.19	31	36.90
EuCHI-5	101	35.69	44	15.55	138	48.76
EuCHI-6	87	42.86	41	20.20	75	36.95
EuCHI-7	126	32.47	58	14.95	204	52.58

Fig. 1 Chromosome localization analysis of the EuCHIs gene family

Fig. 2 Analysis of promoter elements of E. ulmoides CHIs genesA: Location map of promoter elements of EuCHIs genes. B: Number map of each elements in EuCHIs genes. C: Total number map of each type of elements in EuCHIs genes. D: Proportion map of each element in three categories of elements (From left to right are hormone response element, stress response element, growth and development response element)

Fig. 3 Tertiary structure prediction of E. ulmoides CHIs

Fig. 4 Phylogenetic tree of CHIs proteins from E. ulmoides, Solanum lycopersicum, Oryza sativa, and Arabidopsis thaliana

Fig. 5 Analysis of conserved motifs and gene structures of EuCHIsA: Conserved motifs of EuCHIs. B: Gene structure of EuCHIs. C: Conserved motif amino acid sequence

Fig. 6 Interspecific collinearity analysis of CHIs gene of E. ulmoides with Arabidopsis thaliana and Populustrichocarpa

Fig. 7 Expression patterns of E. ulmoidesCHIs genesA: Analysis of expression patterns at different leaf development stages (T01-T03: Leaf buds. T04-T06: Primary leaves. T07-T09: Young leaves. T10-T12: Old leaves). B: Expression patterns of leaves with low and high gum contents (T13-T15: Leaves with low gum content. T16-T18: Leaves with high gum content). C: Expression patterns of red and green leaves. D: Analysis of expression patterns in different tissues. E: Analysis of expression patterns during different flower development stages (F1: Early stage of female flower morphological differentiation. F2: Induction stage of female flowers. F3: Maturation stage of female flowers. M1: Early stage of male flower morphological differentiation. M2: Induction stage of male flowers. M3: Maturation stage of male flowers)

Fig. 8 RT-qPCR analysis of the EuCHIs expressed genesA: Expression patterns of the EuCHIs genes in different tissues. * P<0.05 and ** P<0.01 compared with roots; ##P<0.01 compared with stems. B: Expression patterns of the EuCHIs genes during ABA treatment. *P<0.05 and **P<0.01 compared with 0 h. C: Expression patterns of the EuCHIs genes during GA3 treatment. *P<0.05 and **P<0.01 compared with 0 h. D: Expression patterns of the EuCHIs genes under drought treatment. **P<0.01 compared with 0 h

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