杜仲查尔酮异构酶基因家族全基因组鉴定及其表达模式分析

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

摘要/Abstract

摘要：

目的查尔酮异构酶（CHI）是类黄酮生物合成途径中的关键限速酶。对杜仲CHI基因家族成员进行全基因组鉴定，系统分析其表达特征，以探究其在杜仲生长发育及逆境响应中的生物学功能。方法基于杜仲基因组数据，采用生物信息学方法系统鉴定CHI基因家族成员，并全面分析其理化性质、染色体定位、启动子顺式作用元件、进化关系、保守基序和基因结构等特征。同时，结合转录组数据和RT-qPCR技术，探究EuCHIs在不同组织及非生物胁迫处理下的表达模式。结果在杜仲基因组中共鉴定出7个EuCHI基因家族成员（EuCHI-1-EuCHI-7），其编码蛋白的氨基酸长度为84-388 aa，分子量为9 473.24-43 248.30 Da，等电点范围为4.81-9.42，不稳定指数范围为23.09-46.43，亲疏水性范围为-0.381-0.206，不均匀地分布在5条染色体上。亚细胞定位预测显示，除EuCHI-2定位于细胞核中；其余成员均定位于叶绿体中，此外，EuCHI-1在细胞膜中也有分布，EuCHI-4在细胞质和线粒体中也有分布。系统进化分析将EuCHIs划分为Group Ⅰ、Group Ⅱ和Group Ⅵ 3个亚家族，各成员含有1-10个外显子。启动子分析表明，EuCHIs可能参与生长发育和激素响应等生物学过程，转录组数据表明大部分EuCHIs基因参与杜仲叶色形成以及花发育等多种生物学过程。RT-qPCR结果显示，EuCHIs具有组织表达特异性，除EuCHI-3外，其余均在根部高量表达；与ABA和GA₃胁迫处理相比，干旱胁迫处理更能显著诱导EuCHI基因的表达。结论鉴定出7个EuCHIs基因，分为3个亚家族，EuCHIs在杜仲不同组织和不同胁迫中表达模式存在差异，表明它们在杜仲生长发育和非生物胁迫中发挥重要作用。

关键词: 杜仲, CHI基因家族, 全基因组鉴定, 生物信息学分析, 荧光定量PCR, 激素处理, 非生物胁迫, 表达特征

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

程婷婷, 刘俊, 王利丽, 练从龙, 魏文君, 郭辉, 吴尧琳, 杨晶凡, 兰金旭, 陈随清. 杜仲查尔酮异构酶基因家族全基因组鉴定及其表达模式分析[J]. 生物技术通报, 2025, 41(9): 242-255.

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.

图/表 11

表1 EuCHIs基因引物序列

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

表2 杜仲CHIs蛋白的理化性质分析

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

表3 杜仲CHIs基因家族二级结构分析

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

图1 EuCHIs基因家族染色体定位分析

Fig. 1 Chromosome localization analysis of the EuCHIs gene family

图2 杜仲CHIs基因启动子元件分析A：EuCHIs基因启动子元件定位图；B：EuCHIs基因中每个元件数量图；C：EuCHIs基因每类元件总数图；D：3类元件（从左至右依次为激素响应元件、胁迫响应元件和生长发育响应元件）中各个元件占比图

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)

图3 杜仲CHIs三级结构预测

Fig. 3 Tertiary structure prediction of E. ulmoides CHIs

图4 杜仲、番茄、水稻、拟南芥CHIs蛋白的系统进化树

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

图5 EuCHIs保守基序和基因结构分析A：EuCHIs保守基序；B：EuCHIs基因结构；C：保守基序氨基酸序列

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

图6 杜仲与拟南芥和毛果杨CHIs基因的种间共线性分析

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

图7 杜仲CHIs基因表达模式A：叶片不同发育时期表达模式分析（T01-T03叶芽；T04-T06初生叶；T07-T09幼叶；T10-T12老叶）；B：低和高胶含量叶片的表达模式（T13-T15低胶含量叶片；T16-T18高胶含量叶片）；C：红叶和绿叶的表达模式；D：不同组织的表达模式分析；E：不同花发育的表达模式分析（F1：雌花形态分化初期；F2：雌花诱导期；F3：雌花成熟期；M1：雄花形态分化初期；M2：雄花诱导期；M3：雄花成熟期）

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)

图8 EuCHIs表达基因的RT-qPCR分析A：EuCHIs基因在不同组织中的表达模式；与根相比，*P<0.05，**P<0.01；与茎相比，##P<0.01；B：EuCHIs基因在ABA处理中的表达模式；与0 h相比，*P<0.05，**P<0.01；C：EuCHIs基因在GA3处理中的表达模式；与0 h相比，*P<0.05，**P<0.01；D：EuCHIs基因在干旱处理中的表达模式；与0 h相比，**P<0.01

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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