香蕉Ca2+-ATPase基因家族的鉴定及功能分析

doi:10.3864/j.issn.0578-1752.2025.07.013

摘要/Abstract

摘要：

【目的】Ca²⁺-ATPase是植物中重要的Ca²⁺外排通道，对于维持细胞稳态、诱导植物对胁迫的响应等方面起着重要的作用。利用生物信息学和分子生物学等手段，对香蕉A基因组中Ca²⁺-ATPase家族成员进行分析，检测基因表达量的变化，并验证基因功能，为探究香蕉耐盐机制提供参考。【方法】通过基因家族名称并结合拟南芥和水稻Ca²⁺-ATPase序列，在香蕉基因组数据库中BLAST香蕉A基因组中该家族成员；利用Pfam、ExPASy、Cell-PLoc、NCBI、MEGA-X、TBtools等在线网站或软件，预测分析香蕉Ca²⁺-ATPase的蛋白理化性质、亚细胞定位、保守结构域、顺式作用元件等；采用qRT-PCR技术该家族基因的表达情况，对关键基因进行亚细胞定位验证和原核表达分析。【结果】在香蕉A基因组鉴定到20个Ca²⁺-ATPase家族成员，其中，有13个MaACAs和7个MaECAs；蛋白理化性质分析显示，Ca²⁺-ATPase含有6—11个不等的跨膜结构，编码857—1 103个氨基酸，亚细胞定位预测MaACAs可能定位在细胞质膜、叶绿体、内质网及液泡膜上，MaECAs均定位于内质网膜；保守基序高度一致，除MaACA13外均含有该家族特有的4个保守结构域；含有光、激素、防御和应激等响应元件。qRT-PCR结果表明，100 mmol·L^-1 NaCl处理巴西蕉细胞，除MaECA3外，Ca²⁺-ATPase基因家族其他成员在处理期间均出现了不同程度的上调表达，其中MaACA5和MaACA10的上调幅度较大，并且这两个基因的表达均受Ca²⁺影响。烟草叶片亚细胞定位结果显示，MaACA5和MaACA10定位在细胞质膜上；重组质粒pET28a-MaACA5、pET28a-MaACA10和转入大肠杆菌BL21中，诱导表达后的重组菌株在800 mmol·L^-1 NaCl、800 mmol·L^-1甘露醇和50 ℃高温条件下的生长情况优于对照菌株。【结论】在香蕉A基因组中鉴定到20个Ca²⁺-ATPase家族成员，基因结构具有较高的保守性，分布在10条染色体上，包含激素、防御和应激相关的响应元件；筛选到与香蕉耐盐相关的基因MaACA5和MaACA10。

关键词: 巴西蕉, 盐胁迫, 钙离子, Ca²⁺-ATPase, 表达分析, 基因克隆, 亚细胞定位

Abstract:

【Objective】Ca²⁺-ATPase is a vital Ca²⁺ efflux channel in plants, which plays an important role in maintaining cell homeostasis and inducing plant response to stress. In this study, bioinformatics and molecular biology were used to analyze the members of Ca²⁺-ATPase family in banana A genome, detect the changes of gene expression and verify gene function, so as to provide reference for exploring the mechanism of banana salt tolerance.【Method】BLAST the members of banana A genome in banana genome database by gene family name combined with Arabidopsis and rice Ca²⁺-ATPase sequences, and use Pfam, ExPASy, Cell-PLoc, NCBI, MEGA-X, TBtools and other online websites or software to predict and analyze the protein physicochemical properties, subcellular localization, conservative domain and cis-acting elements of banana Ca²⁺-ATPase. The expression of the family genes was analyzed by qRT-PCR, and the subcellular localization and prokaryotic expression of the key genes were analyzed.【Result】In this study, 20 members of Ca²⁺-ATPase family were identified in banana A genome, including 13 MaACAs and 7 MaECAs; proteins analysis of physicochemical properties showed that Ca²⁺-ATPase contained 6-11 transmembrane structures, encoding amino acids between 857 and 1 103. Subcellular localization predicted that MaACAs might be located on plasma membrane, chloroplast, endoplasmic reticulum and tonoplast, while MaECAs was located in endoplasmic reticulum. The conserved motifs are highly consistent, including four conserved domains peculiar to the family except MaACA13, responses to light, hormone, defense and stress. qRT-PCR results showed that other members of Ca²⁺-ATPase gene family except MaECA3 were up-regulated in Brazilian banana cells treated with 100 mmol·L^-1 NaCl, in which MaACA5 and MaACA10 were up-regulated to a large extent, and the expression of these two genes was affected by Ca²⁺. The results of subcellular localization of tobacco leaves showed that MaACA5 and MaACA10 were located on the plasma membrane, and the recombinant plasmids pET28a-MaACA5, pET28a-MaACA10 and transformed into E. coli BL21 were superior to the control strains under the conditions of 800 mmol·L^-1NaCl, 800 mmol·L^-1 mannitol and 50 ℃. 【Conclusion】 20 Ca²⁺-ATPase family members were identified in banana A genome, the gene structure was highly conservative, including hormone, defense and stress-related responses, MaACA5 and MaACA10 related to banana salt tolerance were screened and prokaryotic expression to verify the gene function, which provides a reference for further study of banana salt tolerance.

Key words: brazil banana, salt stress, calcium, Ca²⁺-ATPase, expression analysis, gene cloning, subcellular localisation

滕梦鑫, 徐亚, 何静, 汪奇, 乔飞, 李敬阳, 李新国. 香蕉Ca²⁺-ATPase基因家族的鉴定及功能分析[J]. 中国农业科学, 2025, 58(7): 1418-1433.

TENG MengXin, XU Ya, HE Jing, WANG Qi, QIAO Fei, LI JingYang, LI XinGuo. Identification and Functional Analysis of Ca²⁺-ATPase Gene Family in Banana[J]. Scientia Agricultura Sinica, 2025, 58(7): 1418-1433.

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蛋白 Protein	氨基酸数量 Amino acids size (aa)	分子量 Mw (Da)	等电点 pI	不稳定系数 The instability index (II)	脂肪系数Aliphatic index	亲水性平均系数 Grand average of hydropathicity	跨膜结构Transmembrane domain	亚细胞定位 Subcellular localization
MaACA1	1095	119281.68	8.36	38.1	101.13	0.05	8	A
MaACA2	1090	119195.56	8.27	36.89	100.53	0.04	11	A
MaACA3	1034	112541.57	5.87	35.13	103.09	0.22	6	B、C、D
MaACA4	1082	118244.8	8.47	40.33	101.71	0.066	7	A
MaACA5	1017	111767.31	5.7	31.68	99.45	0.121	7	A
MaACA6	1020	111212.01	5.88	35.51	103	0.2	6	B、C、D
MaACA7	1020	111518.14	5.91	32.3	102.91	0.158	6	B、C、D
MaACA8	1034	114138.1	6.74	31.15	107.53	0.169	10	B、C、D
MaACA9	1019	111002.4	5.26	32.57	104.26	0.201	6	B、C、D
MaACA10	1024	112321.63	5.85	34.05	95.55	0.091	8	A
MaACA11	1035	114343.58	8.3	30.9	106.03	0.144	10	B、C、D
MaACA12	1103	120673.88	8.87	35.9	102.1	0.072	9	A
MaACA13	857	92860.11	6.01	37.29	109.78	0.266	6	A
MaECA1	1064	116317.6	5.28	35.82	98.15	0.089	7	C
MaECA2	1059	115754.37	5.36	37.3	99.53	0.095	9	C
MaECA3	1051	116158.69	5.4	33.61	100.35	0.131	8	C
MaECA4	1058	115980.52	5.19	36.63	99.5	0.078	7	C
MaECA5	1000	109753.56	5.87	36.37	99.62	0.222	8	C
MaECA6	1070	118771.74	5.85	35.3	97.03	0.107	8	C
MaECA7	1059	115636.05	5.26	36.29	98.32	0.098	9	C

蛋白Protein	α-螺旋Alpha-helix (%)	β-折叠Beta strand (%)	无规则卷曲Loop (%)
MaACA1	44.20	12.24	43.56
MaACA2	46.42	11.83	41.74
MaACA3	49.13	12.38	38.49
MaACA4	47.41	12.20	40.39
MaACA5	46.02	15.44	38.54
MaACA6	48.73	14.51	36.76
MaACA7	50.49	13.14	36.37
MaACA8	48.55	14.02	37.43
MaACA9	50.54	13.35	36.11
MaACA10	46.97	15.43	37.60
MaACA11	48.79	14.30	36.91
MaACA12	47.60	10.79	41.61
MaACA13	49.01	13.54	37.46
MaECA1	51.13	10.90	37.97
MaECA2	51.65	10.86	37.49
MaECA3	52.71	10.56	36.73
MaECA4	50.85	11.15	38.00
MaECA5	47.30	13.30	39.40
MaECA6	53.64	10.84	35.51
MaECA7	47.69	11.43	40.89

香蕉Ca²⁺-ATPase基因家族的鉴定及功能分析

Identification and Functional Analysis of Ca²⁺-ATPase Gene Family in Banana

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