Identification and Functional Analysis of the MpPP2A-C Gene in Marchantia polymorpha

doi:10.13560/j.cnki.biotech.bull.1985.2025-0820

Abstract

Abstract:

Objective To identify the MpPP2A-C gene of Marchantia polymorpha, analyze its expressions and study its interaction with the MpPP2A-A protein, so as to provide a theoretical basis for clarifying the function of the MpPP2A-C gene. Method The phylogenetic trees of PP2A-C of Marchantia polymorpha and other plants were constructed, and homology alignment was performed using the PP2A-C gene sequence of Arabidopsis thaliana to identify the PP2A-C gene of M. polymorpha, and bioinformatics analysis was conducted on it. The expression pattern of the MpPP2A-C gene in tissue specificity and response to abscisic acid was explored by RT-qPCR. The interaction relationship between the MpPP2A-C subunit and the MpPP2A-A subunit was explored by using yeast two-hybrid experiments. Result Three MpPP2A-C genes (MpPP2A-C1, MpPP2A-C2, and MpPP2A-C3) were identified in the M. polymorpha genome. These genes were highly conserved in bryophytes, and their encoded proteins had conserved structural features, though they differed in physicochemical properties and subcellular localization. The MpPP2A-C gene was expressed in the apical notch, gemma cup and the rest of thallus. Among them, the expressions of MpPP2A-C1 and MpPP2A-C3 were the highest at the apical notch, while there was no significant difference in the expressions of MpPP2A-C2 among various tissues. ABA treatment significantly inhibited the growth of gemma. The expressions of MpPP2A-C1 increased with the rise of ABA concentration, there was no significant difference in the expression of MpPP2A-C2, while the expression of MpPP2A-C3 first increased and then decreased. The yeast two-hybrid experiment inferred that MpPP2A-A interacted with MpPP2A-C1 and MpPP2A-C3. Conclusion The MpPP2A-C is highly conserved, can respond to abscisic acid, and interacts with the A subunit. These results lay the foundation for future research on the function of the PP2A-C gene.

Key words: Marchantia polymorpha, PP2A-C gene, gene expression, abscisic acid, yeast two-hybrid

JIANG Xin-hua, FANG Tian-yu, ZHANG Jing-jing, LI Xiang-yuan, ZHANG Bang-yue, LIAO Xiao-shan, RONG Duo-yan. Identification and Functional Analysis of the MpPP2A-C Gene in Marchantia polymorpha[J]. Biotechnology Bulletin, 2026, 42(4): 216-226.

Figures/Tables 9

Table 1 Basic information of PP2A-C in M. polymorpha

基因名称

Gene name

基因ID

Locus ID

分子量

Molecular weight （Da）

等电点

Point isoelectric

脂肪系数

Aliphatic index

不稳定系数

Instability index

亲水性

GRAVY

亚细胞定位

Subcellular localization

细胞质 Cytoplasm

线粒体 Mitochondrion

细胞核 Nucleus

细胞质 Cytoplasm

线粒体 Mitochondrion

细胞核 Nucleus

细胞质 Cytoplasm

细胞核 Nucleus

Fig. 1 Evolutionary analysis of PP2A-C amino acid sequences in M. polymorpha and other speciesC. Eustigma: Chlamydomonas eustigma; C. vulgaris: Chlorellavulgaris; M. Polymorpha: Marchantia polymorpha; P. patens: Physcomitrella patens; A. capillus-veneris: Adiantum capillus-veneris; C. richardii: Ceratopteris richardii; A. thaliana: Arabidopsis thaliana; O. sativa: Oryza sativa; M. domestica: Malus domestica; S. tuberosum: Solanum tuberosum

Fig. 2 Conserved domain and sequence alignment of PP2A-C proteins between A. thaliana and M. polymorphaA: Schematic diagram of structural domains in proteins. PP2A-like: Serine/Threonine protein phosphatase PP2A-like. Metallo-depent PP-like: Metallo-dependent phosphatase-like. MPP_PP2A_PP4_PP6: PP2A, PP4, and PP6 phosphoprotein phosphatases, metallophosphatase domain. Calcineurin-like_PHP: Calcineurin-like, phosphoesterase domain. B: Multiple sequence alignment of proteins. The conserved motifs of the catalytic core domain (GDXHG and GDXDRG) are indicated by red boxes; the conserved motif of the A-subunit binding domain (LVEEYNVQEV) is underlined in blue. The blue triangles indicate the metal ion binding site Asp, while the green triangles denote the metal ion binding site Glu

Table 2 Basic information on the secondary and tertiary structures of PP2A-C in M. polymorpha

蛋白质名称 Protein name	氨基酸数量 Number of amino acids （aa）	蛋白质二级结构 Secondary structure of protein			蛋白质三级结构 Tertiary structure of protein
蛋白质名称 Protein name	氨基酸数量 Number of amino acids （aa）	α-螺旋 Alpha helix（%）	延伸链 Extended strand（%）	不规则卷曲 Random coil（%）	相似性 Identity （%）	全局模型质量评估 GMQE	基于距离约束的定性模型能量分析全局分数 QMEANDisCo Global
MpPP2A-C1	306	37.25	15.03	47.71	82.89	0.91	0.89±0.05
MpPP2A-C2	304	39.47	14.80	45.72	65.35	0.88	0.83±0.05
MpPP2A-C3	303	38.28	16.18	45.54	58.14	0.88	0.83±0.05

Fig. 3 Tertiary structure of MpPP2A-C proteinA: MpPP2A-C1. B: MpPP2A-C2. C: MpPP2A-C3. All structures are colored by the confidence gradient, with blue indicating high confidence (trustworthy) and red indicating low confidence (unreliable)

Fig. 4 Differences of expressions of MpPP2A-C in different tissue parts of M. polymorphaValues are the average±standard error, ****P<0.000 1 (Student's t-test)

Fig. 5 Phenotype and expression analysis of MpPP2A-C gene treated with abscisic acid at different concentrationsA: The comparison of the surface type of the phyllodes of the plant under different concentrations of ABA, and the scale length is 1 mm. B: Statistics of the phyllosoma area under different concentrations of ABA treatment. C: Expressions of MpPP2A-C gene under different ABA concentrations. Values are the average ± standard error, the lowercase letters indicate significant differences between groups (P<0.05). Statistical analysis was performed using one-way analysis of variance (ANOVA) combined with Duncan’s multiple comparison test

Fig. 6 Construction of yeast two-hybrid vectorsA: Amplification of the target fragment of the MpPP2A-A/C1/C2/C3 gene (M: DNA Marker; 1-4: PCR amplified products of the MpPP2A-A/C1/C2/C3 genes). B: Enzymatic digestion of pGADT7 and pGBKT7 plasmids (M: DNA Marker; 1: Undigested pGADT7 plasmid; 2: pGADT7 plasmid after enzymatic digestion; 3: Undigested pGBKT7 plasmid; 4: pGBKT7 plasmid after enzymatic digestion). C: Construction of the pGADT7-MpPP2A-A/C1 vector and pGBKT7-MpPP2A-C2/C3 vectors (M: DNA Marker; 1-4: DH5α-pGADT7-MpPP2A-A bacterial liquid, N1: Negative control, P1: Positive control (the target fragment of the MpPP2A-A); 5-8: DH5α-pGADT7-MpPP2A-C1 bacterial liquid, N2: Negative control, P2: Positive control (the target fragment of the MpPP2A-C1); 9-12: DH5α-pGBKT7-MpPP2A-C2 bacterial liquid. N3: Negative control. P3: Positive control (the target fragment of the MpPP2A-C2); 13-14: DH5α-pGBKT7-MpPP2A-C3 bacterial liquid. N4: Negative control. P4: Positive control (the target fragment of the MpPP2A-C3)

Fig. 7 Growth of colonies in the defective mediumA: Verification of the interaction between AD-MpPP2A-A and BD-MpPP2A-C2/C3. B: Verification of the interaction between AD-MpPP2A-C1 and BD-A. SD-LW: Dideficiency medium (lacking leucine and tryptophan). SD-LWHA: Four-Deficiency medium (lacking leucine, tryptophan, histidine and adenine)

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