地钱MpPP2A-C基因的鉴定及功能分析

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

摘要/Abstract

摘要：

目的对地钱MpPP2A-C基因进行鉴定、表达量分析及与MpPP2A-A蛋白的相互作用研究，为阐明MpPP2A-C基因的功能提供理论依据。方法构建地钱和其它植物PP2A-C的系统发育树，并利用拟南芥PP2A-C基因序列进行同源比对，鉴定出地钱PP2A-C基因，对其进行生物信息学分析；通过RT-qPCR探究MpPP2A-C基因在地钱组织特异性及响应脱落酸的表达模式；采用酵母双杂交实验探究MpPP2A-C亚基与MpPP2A-A亚基的相互作用关系。结果地钱基因组中鉴定出3个MpPP2A-C基因（MpPP2A-C1、MpPP2A-C2、MpPP2A-C3），在苔藓植物中高度保守，且其编码蛋白具有保守的结构特征，但在理化性质和亚细胞定位上存在差异。MpPP2A-C基因在地钱顶端缺口、胞芽杯和叶状体中均有表达，其中MpPP2A-C1和MpPP2A-C3在顶端缺口处表达量最高，MpPP2A-C2在各组织中表达无显著差异。ABA处理显著抑制地钱叶状体生长，MpPP2A-C1表达量随ABA浓度升高而上升，MpPP2A-C2表达量无显著差异，MpPP2A-C3表达量则先升后降。酵母双杂交实验证实MpPP2A-A与MpPP2A-C1、MpPP2A-C3均存在相互作用。结论地钱MpPP2A-C具有高度保守性，能响应脱落酸，且与A亚基存在相互作用。这些结果将为未来研究PP2A-C基因功能奠定了基础。

关键词: 地钱, PP2A-C基因, 基因表达, 脱落酸, 酵母双杂交

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 wasexpressed 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

江昕桦, 方天宇, 张晶晶, 李相媛, 张邦跃, 廖晓珊, 荣朵艳. 地钱MpPP2A-C基因的鉴定及功能分析[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0820.

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

图/表 9

表1 地钱PP2A-C的基本信息

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

图1 地钱和其它物种PP2A-C氨基酸序列进化分析C. eustigma：衣藻；C. vulgaris：小球藻；M. polymorpha：地钱；P. patens：小立碗藓；A. capillus-veneris：铁线蕨；C. richardii：水蕨；A. thaliana：拟南芥；O. sativa：水稻；M. domestica：苹果；S. tuberosum：马铃薯

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

图2 拟南芥与地钱的PP2A-C蛋白保守结构域及序列比对A：MpPP2A-C1蛋白结构域示意图；PP2A-like：丝氨酸/苏氨酸蛋白磷酸酶PP2A样；Metallo-depent PP-like：金属依赖性蛋白磷酸酶样结构域；MPP_PP2A_PP4_PP6：金属依赖性蛋白磷酸酶2A/4/6结构域；Calcineurin-like_PHP：钙调神经磷酸酶样磷酸酯酶结构域。B：蛋白序列比对；红色方框标记催化核心结构域保守基序（GDXHG和GDXDRG）；黑色下划线标记A亚基结合结构域保守基序（LVEEYNVQEV）；蓝色三角形为金属离子结合位点Asp，绿色三角形为金属离子结合位点Glu

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

表2 地钱PP2A-C蛋白质二、三级结构基本信息

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%	16.175	45.54%	58.14%	0.88	0.83±0.05

图3 MpPP2A-C蛋白质的三级结构A：MpPP2A-C1；B：MpPP2A-C2；C：MpPP2A-C3。均以可信度梯度进行配色，蓝色表示高置信度（可信），红色表示低置信度（不可信）

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)

图4 MpPP2A-C基因在地钱不同组织部位中表达量的差异数值指平均值±标准误，****P<0.000 1 （Student’s t-test）

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

图5 MpPP2A-C基因在不同浓度脱落酸处理下的表型及表达量分析A：不同浓度ABA处理下的地钱叶状体表型对比，标尺长度为1 mm；B：不同浓度ABA处理下的地钱叶状体面积统计；C：MpPP2A-C基因在不同浓度ABA处理下的表达水平。数值指平均值±标准误，小写字母表示组间显著性差异（P<0.05）；统计分析采用单因素方差分析结合Duncan多重比较法

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 refer to 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

图6 酵母双杂交载体的构建A：MpPP2A-A/C1/C2/C3基因目的片段的扩增（M. DNA Marker；1-4. MpPP2A-A/C1/C2/C3基因的PCR扩增产物）；B：pGADT7和pGBKT7质粒的酶切（M. DNA Marker；1. 未酶切pGADT7质粒；2. 酶切后pGADT7质粒；3. 未酶切pGBKT7质粒；4. 酶切后pGBKT7质粒）；C：pGADT7-MpPP2A-A/C1载体和pGBKT7-MpPP2A-C2/C3载体的构建（M. DNA Marker；1-4. DH5α-pGADT7-MpPP2A-A菌液，N1. 阴性对照，P1. 阳性对照（MpPP2A-A基因目的片段）；5-8. DH5α-pGADT7-MpPP2A-C1菌液，N2. 阴性对照，P2. 阳性对照（MpPP2A-C1基因目的片段）；9-12. DH5α-pGBKT7-MpPP2A-C2菌液，N3. 阴性对照，P3. 阳性对照（MpPP2A-C2基因目的片段）；13-14. DH5α-pGBKT7-MpPP2A-C3菌液，N4. 阴性对照，P4. 阳性对照（MpPP2A-C2基因目的片段）

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 amplification 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)

图7 缺陷培养基菌落生长情况A：AD-MpPP2A-A与BD-MpPP2A-C2/C3的互作验证；B：AD-MpPP2A-C1和BD-MpPP2A-A的互作验证；SD-LW：二缺培养基（缺失亮氨酸和色氨酸）；SD-LWHA：四缺培养基（缺失亮氨酸、色氨酸、组氨酸和腺嘌呤）

Fig. 7 Results 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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