红花质膜H+-ATPase基因家族成员鉴定及响应低氮低磷胁迫的表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0057

摘要/Abstract

摘要：

目的对红花质膜H⁺-ATPase（CtPMA）基因家族进行全基因组鉴定与分析，为研究该基因家族及其在响应低氮低磷胁迫中的功能奠定基础。方法利用生物信息学方法对CtPMA基因家族成员进行鉴定和系统的分析；利用实时荧光定量PCR（RT-qPCR）分析其组织表达特异性及在低氮低磷胁迫下的响应。结果在红花中共鉴定到10个CtPMAs（命名为CtPMA1-CtPMA10），其编码蛋白质的氨基酸数为785-958 aa，相对分子质量为85.23-105.52 kD，等电点为5.26-7.91，亚细胞位点预测显示全部位于细胞膜；都含有Cation_ATPase_N结构域、E1_E2 ATPase结构域和HAD-superfamily hydrolase subfamily ⅢA结构域；顺式作用元件预测显示CtPMA基因家族内广泛存在与生长发育、激素和胁迫相关的调控元件；基因共线性显示全基因组复制和片段复制在CtPMA基因家族进化中起到了重要作用。RT-qPCR结果表明，在根中高表达的CtPMA1和CtPMA7的表达量显著受到低氮低磷胁迫的诱导，说明CtPMA1和CtPMA7可能与红花根部吸收氮磷的功能密切相关。结论红花全基因组中共鉴定到10个CtPMA基因，其进化相对保守，在不同的组织部位具有不同的表达模式。当红花遭受低氮低磷胁迫时在根部高表达的CtPMA1和CtPMA7的转录水平显著被诱导，暗示其通过诱导自身转录水平以提高对氮磷的吸收和利用效率，用来抵御营养等逆境胁迫。

关键词: 红花, 质膜H⁺-ATPase基因家族, 生物信息学分析, 低氮胁迫, 低磷胁迫

Abstract:

Objective Genome-wide identification and analysis of the plasma membrane H⁺-ATPase (PMA) gene family in safflower (Carthamus tinctorius L.) may provide the basis for investigating this gene family and its function in response to low nitrogen and low phosphorus stress. Method Bioinformatics was used to identify and systematically analyze the members of the CtPMA gene family. Real-time fluorescence quantitative PCR (RT-qPCR) was applied to analyze the tissue-specific expression pattens and the responses to low nitrogen and low phosphorus stress. Result A total of 10 CtPMAs (named CtPMA1-CtPMA10) were identified in safflower, encoding proteins ranging from 785 to 958 aa, with relative molecular weight of 85.23 to 105.52 kD, and isoelectric points of 5.26 to 7.91. All were predicted to be localized to the plasma membrane by the subcellular localization. All members contained conserved domains, including the Cation_ATPase_N domain, the E1_E2 ATPase domain, and the HAD-superfamily hydrolase subfamily ⅢA domain. Cis-acting element analysis revealed a lot of regulatory elements related to growth, development, hormones and stress responses. Gene collinearity indicated that whole-genome duplication and segmental duplication played key roles in the evolution of the CtPMA gene family. RT-qPCR results showed that the CtPMA1 and CtPMA7, which were highly expressed in the roots, were significantly induced by low nitrogen and low phosphorus stress. This finding suggests that CtPMA1 and CtPMA7 may play a crucial role in the process of nitrogen and phosphorus uptake in safflower roots. Conclusion A total of 10 CtPMAs members are identified in the safflower genome.These members demonstrate evolutionary conservation and tissue-specific expression patterns. The highly expressed CtPMA1 and CtPMA7 transcripts in the roots were significantly induced under low nitrogen and phosphorus stress. This suggests that safflower may improve the absorption and utilization efficiency of nitrogen and phosphorus by increasing its CtPMAs transcription level in order to resist nutritional stress.

Key words: Carthamus tinctorius L., plasma membrane H⁺-ATPase gene family, bioinformatics analysis, low nitrogen stress, low phosphorus stress

腊贵晓, 赵玉龙, 代丹丹, 余永亮, 郭红霞, 史贵霞, 贾慧, 杨铁钢. 红花质膜H⁺-ATPase基因家族成员鉴定及响应低氮低磷胁迫的表达分析[J]. 生物技术通报, 2025, 41(8): 220-233.

LA Gui-xiao, ZHAO Yu-long, DAI Dan-dan, YU Yong-liang, GUO Hong-xia, SHI Gui-xia, JIA Hui, YANG Tie-gang. Identification of Plasma Membrane H⁺-ATPase Gene Family in Safflower and Expression Analysis in Response to Low Nitrogen and Low Phosphorus Stress[J]. Biotechnology Bulletin, 2025, 41(8): 220-233.

图/表 12

表1 RT-qPCR所用的引物

Table 1 Primers for RT-qPCR

基因 Gene	正向引物 Forward primer （5'-3'）	反向引物 Reverse primer （5'-3'）	长度Length （bp）
CtPMA1	TTGACCAATCTGCCCTCA	CCTTCTGGAAATGCCCAA	189
CtPMA2	CCACCAGATTGGCAAGACT	GGCATCAGCAGGAACGAT	231
CtPMA3	AGATGCCGCCGTGTTAGT	GTCAAAGCCGACTGGTCAA	117
CtPMA4	AGGGATGGGAGATGGTCTG	GCTTCTTGGTGACTGGTAAGG	169
CtPMA5	TGACGACTGATGATGGTAACC	CAATCTGGAGGCTTTCCC	181
CtPMA6	GCAGCCATTCTCGTTCCA	AGGGTGCTTTGTCACAGGA	144
CtPMA7	GGAATCCTCTCTCGTGGGT	GCAAGCATCTTCTTCGCTC	242
CtPMA8	GGTGCTTCTTCTCATAAACTCG	ACGATGTCTCCTGGAACCA	162
CtPMA9	TGAAGGCATTGATAACCTGC	ACACCAGCGGCAAATACC	246
CtPMA10	GGCGAGTCTATTCCAGTAACC	GATTGGTGTTCTCCACAAGG	148
18S	ACACGGGGAGGTAGTGACAA	CCTCCAATGGATCCTCGTTA	135

图1 CtPMAs基因染色体定位

Fig. 1 Localization of CtPMAs across chromosomes

图2 CtPMA基因家族蛋白序列比对分析蓝色代表同源性≥50%，玫红色代表同源性≥75%，深蓝色代表同源性=100%；红色方框代表Cation ATPase N结构域，绿色方框代表E1_E2 ATPase结构域，亮蓝色方框代表HAD-superfamily hydrolase， subfamily ⅢA结构域

Fig. 2 Alignment analysis of protein sequencesin CtPMA gene familyBlue indicates ≥50% homology, rose red indicates ≥75% homology, and dark blue indicates homology = 100%; red boxes indicate cation ATPase N structural domains, green boxes indicate E1_E2 ATPase structural domains, and bright blue boxes indicate HAD-superfamily hydrolase, subfamily IIIA structural domains

表2 CtPMA基因家族成员理化性质

Table 2 Physicochemical properties of CtPMA gene family

基因名称 Gene name	基因号 Gene ID	蛋白质长度 Protein length （aa）	相对分子质量 Molecular mass （kD）	等电点 Theoretical point	亲水性系数 Hydrophilicity coefficient	不稳定系数 Instability index	亚细胞定位 Subcellular localization
CtPMA1	CtAH01T0012700.1	886	97.78	5.75	0.034	37.94	细胞膜 Plasma membrane
CtPMA2	CtAH03T0170800.1	937	102.76	6.63	0.054	39.45	细胞膜 Plasma membrane
CtPMA3	CtAH05T0036500.1	858	93.95	5.26	0.205	39.82	细胞膜 Plasma membrane
CtPMA4	CtAH05T0146800.1	956	105.47	6.49	0.069	33.32	细胞膜 Plasma membrane
CtPMA5	CtAH05T0252100.1	785	85.23	5.34	0.150	36.06	细胞膜 Plasma membrane
CtPMA6	CtAH06T0267900.1	906	99.49	6.20	0.034	33.52	细胞膜 Plasma membrane
CtPMA7	CtAH09T0032200.1	911	100.36	5.98	0.122	35.71	细胞膜 Plasma membrane
CtPMA8	CtAH09T0161100.1	957	105.49	6.03	0.116	38.33	细胞膜 Plasma membrane
CtPMA9	CtAH11T0113500.1	889	97.10	5.87	0.068	36.90	细胞膜 Plasma membrane
CtPMA10	CtAH12T0151100.1	958	105.52	7.91	0.087	36.00	细胞膜 Plasma membrane

图3 红花、拟南芥和水稻中PMA基因家族系统进化树

Fig. 3 Phylogenetic tree of PMA gene family in safflower (Carthamus tinctorius L.), Arabidopsis, and rice (Oryza sativa L.)

图4 CtPMAs基因结构和保守基序分析A：CtPMA基因家族的系统进化树；B：CtPMAs基因结构，其中外显子、内含子和5'/3'非翻译区分别用黄色方框、黑色线条和绿色方框标记；C和D：CtPMA基因家族的保守基序分布

Fig. 4 Gene structure and conserved motif of CtPMAsA: Phylogenetic tree of CtPMA gene family in safflower. B: Gene structure of CtPMA gene family in safflower, the yellow box refer to exons, the black lines to introns, and green box to 5'/3' untranslated regions. C and D: Distribution of conserved motifs of CtPMA gene family

图5 CtPMAs基因启动子顺式作用元件分析A：CtPMAs起始密码子上游2 000 bp启动子序列中顺式作用元件鉴定；B：各种顺式作用元件的分析

Fig. 5 Analysis of cis-acting elements in the promoter of CtPMAsA: Identification of cis-acting elements in the promoter sequence 2 000 bp upstream of the start codon of CtPMAs. B: Analysis of various cis-acting elements

图6 CtPMAs共线性分析A：红花PMA基因家族共线性分析；B：PMA基因家族在不同物种间的比较共线性图

Fig. 6 Collinearity analysis of CtPMAsA: Collinearity analysis of PMAs gene family in safflower. B: Comparative collinearity analysis of PMA gene familyin different species

表4 CtPMA基因物种内共线性关系

Table 4 Interspecies collinearity relationship among CtPMA genes

基因对

Gene pair

复制类型

Type of duplication

Ka/Ks

选择压力类型

Type of selective pressure

CtPMA3/CtPMA5

片段重复Segmental duplication

0.130 145 43

1.213 732 375

0.107 227 452

纯化选择Purifying selection

CtPMA4/CtPMA10

片段重复Segmental duplication

0.029 828 973

0.740 740 112

0.040 269 148

纯化选择Purifying selection

CtPMA6/CtPMA7

片段重复Segmental duplication

0.062 455 14

1.531 402 62

0.040 782 965

纯化选择Purifying selection

图7 CtPMAs在红花不同组织部位的表达模式L：叶；R：根：S：茎；F：花

Fig. 7 Expression pattern of CtPMAs in different tissues of safflowerL: Leaf; R: root; S: stem; F: flower

图8 CtPMA基因在低氮和磷胁迫下的响应LP：低磷胁迫，LN：低氮胁迫，LP+LN：同时低氮、磷胁迫。数值为平均值±标准差（n=6），利用Duncan’s做方差分析，不同字母表示各组之间差异显著（P<0.05）

Fig. 8 Responses of CtPMA genes under low phosphorus and low nitrogen stressLP, low phosphorus stress; LN, low nitrogen stress; LP+LN, low nitrogen and phosphorus stress. The data indicate mean ± standard deviation (n=6); different letters indicate significant differences between groups (P<0.05)

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