番茄HMA基因家族的鉴定及SlHMA1镉转运功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2023-0896

摘要/Abstract

摘要：

【目的】Heavy metal ATPase（HMA）基因家族广泛参与植物对金属元素的吸收和转运，系统鉴定番茄HMA基因家族成员及其特征，并研究其在应对镉胁迫过程中的功能，为解析番茄重金属转运机制及番茄低镉积累种质创新提供理论依据。【方法】通过生物信息学鉴定番茄HMA基因家族成员，并分析其系统进化树、蛋白理化性质、基因结构、顺式作用元件、基因表达模式等，通过酵母功能互补试验研究SlHMA1的镉转运活性。【结果】番茄基因组中存在8个SlHMAs，分属2个亚组。在基因结构方面，各SlHMAs间及与拟南芥和水稻的同源基因之间都存在显著差异。SlHMAs成员启动子区域含有较多逆境响应相关的顺式作用元件，RT-qPCR结果也揭示大多数SlHMAs表达对镉胁迫有不同程度的组织特异性响应。酵母功能互补试验表明SlHMA1蛋白具有镉转运活性，进化分析表明HMA1广泛存在于植物界，且其ATP水解酶活性相关的氨基酸保守基序DKTGT也在植物界高度保守。【结论】番茄SlHMAs具有HMA家族基因的典型特征，同时也存在功能多样性。SlHMAs及其氨基酸保守基序DKTGT与金属离子转运及镉胁迫响应密切相关，在低镉作物育种方面具有重要应用潜力。

关键词: 番茄, 金属转运蛋白, 重金属ATP酶, 镉, SlHMA1

Abstract:

【Objective】 The heavy metal ATPase（HMA）gene family is extensively involved in the absorption and transport of metal elements in plants. Therefore, this study is aimed to systematically identify the members of the tomato HMA gene family and their characteristics, as well as to investigate their functions in response to cadmium stress.【Method】 Tomato HMA gene family members were identified through bioinformatics, and their systematic evolution tree, protein physicochemical properties, gene structure, cis-regulatory elements, and gene expression patterns, etc., were analyzed. Additionally, yeast functional complementation experiments were conducted to validate the Cd²⁺ transport activity of SlHMA1.【Result】 Eight SlHMAs were identified in the tomato genome, categorized into 2 subgroups. Remarkable differences in gene structure were observed among SlHMAs genes and their homologs in Arabidopsis and rice. Notably, the promoter regions of SlHMAs members were found to harbor numerous cis-regulatory elements associated with stress responses, and RT-qPCR analysis also indicated that most SlHMAs genes showed tissue-specific responses to cadmium stress. Furthermore, yeast functional complementation experiments provided conclusive evidence for Cd²⁺ transport activity of SlHMA1, while evolutionary analysis indicated the widespread presence of HMA1 in the plant kingdom, with the motif DKTGT related to ATP hydrolysis activity within HMA1 also being highly conserved across the plant kingdom.【Conclusion】 The eight SlHMAs present typical characteristics of the HMA gene family while also having functional diversity. SlHMAs, along with their conserved amino acid motif DKTGT, are closely associated with metal ion transport and cadmium stress response, offering significant potential for application in the breeding of low cadmium-accumulating crop varieties.

Key words: tomato, metal transporter, heavy metal ATPase, cadmium, SlHMA1

赵曜, 文朗, 骆少丹, 栗子杏, 刘超超. 番茄HMA基因家族的鉴定及SlHMA1镉转运功能研究[J]. 生物技术通报, 2024, 40(2): 212-222.

ZHAO Yao, WEN Lang, LUO Shao-dan, LI Zi-xing, LIU Chao-chao. Identification of HMA Gene Family and Cadmium Transport Function of SlHMA1 in Tomato[J]. Biotechnology Bulletin, 2024, 40(2): 212-222.

图/表 10

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基因Gene	正向引物Forward primer（5'-3'）	反向引物 Reverse primer（5'-3'）
SlHMA1	CAGGAGGCCTTATAATGGTAGG	TAGTACATCAGCAACAGCTACC
SlHMA3	GTGTTCTGACTCAACAGACAAC	TTTCCACATGTCATTGAGCTTG
SlHMA4	GATGGCGTCGTTATTAATGGTC	ACAGTTCCACCAATGACCTTAT
SlHMA5.1	ATTCATCATTGGACGACGTTTC	AGCTGGTTCTTAACACCGAATA
SlHMA5.2	GCTTATGGATTTAGCCCCAAAG	TCGTTCTTTTGTATCAATCGGC
SlHMA6	TAGTCAGTGGAGATTTAGGGGA	CAATGATCTGATCTCCGACTGA
SlHMA7	CTGCAGAATACGTGTTGATGAG	GGGATGGAAATCACATTGTACG
SlHMA8	ATTCTCTCTCTCACACGATCAC	CAATAGCTGACTAGTACGACGT
SlActin	CCATTCTCCGTCTTGACTTGG	TCTTTCCTAATATCCACGTCAC

基因Gene	正向引物Forward primer（5'-3'）	反向引物 Reverse primer（5'-3'）
SlHMA1	CAGGAGGCCTTATAATGGTAGG	TAGTACATCAGCAACAGCTACC
SlHMA3	GTGTTCTGACTCAACAGACAAC	TTTCCACATGTCATTGAGCTTG
SlHMA4	GATGGCGTCGTTATTAATGGTC	ACAGTTCCACCAATGACCTTAT
SlHMA5.1	ATTCATCATTGGACGACGTTTC	AGCTGGTTCTTAACACCGAATA
SlHMA5.2	GCTTATGGATTTAGCCCCAAAG	TCGTTCTTTTGTATCAATCGGC
SlHMA6	TAGTCAGTGGAGATTTAGGGGA	CAATGATCTGATCTCCGACTGA
SlHMA7	CTGCAGAATACGTGTTGATGAG	GGGATGGAAATCACATTGTACG
SlHMA8	ATTCTCTCTCTCACACGATCAC	CAATAGCTGACTAGTACGACGT
SlActin	CCATTCTCCGTCTTGACTTGG	TCTTTCCTAATATCCACGTCAC

基因ID Gene ID	基因名称 Gene name	氨基酸数目 No. of amino acid	分子量 Molecular weight/kD	等电点 pI	稳定性 Instability index	脂肪系数 Aliphatic index	亲水性 GRAVY	亚细胞定位 Subcellular localization	跨膜结构域 TM
Solyc02g092920.2	SlHMA1	821	89.08	8.44	34.91	100.4	0.13	叶绿体 Chloroplast	0
Solyc07g009130.2	SlHMA3	1 302	140.44	6.94	45.63	80.46	-0.231	细胞质膜 Plasma membrane	8
Solyc11g062100.1	SlHMA4	698	76.04	5.68	36.13	104.44	0.227	细胞质膜 Plasma membrane	7
Solyc08g080870.2	SlHMA5.1	954	103.58	5.58	39.69	102.69	0.139	细胞质膜 Plasma membrane	6
Solyc08g080890.2	SlHMA5.2	890	96.58	5.80	32.38	103.96	0.200	细胞质膜 Plasma membrane	8
Solyc01g105160.4	SlHMA6	964	101.94	8.30	33.79	97.68	0.092	叶绿体 Chloroplast	0
Solyc02g068490.3	SlHMA7	1 015	108.59	5.16	30.72	106.97	0.324	细胞质膜 Plasma membrane	10
Solyc08g061610.2	SlHMA8	894	95.20	5.94	38.01	103.66	0.118	叶绿体 Chloroplast	0

基因ID Gene ID	基因名称 Gene name	氨基酸数目 No. of amino acid	分子量 Molecular weight/kD	等电点 pI	稳定性 Instability index	脂肪系数 Aliphatic index	亲水性 GRAVY	亚细胞定位 Subcellular localization	跨膜结构域 TM
Solyc02g092920.2	SlHMA1	821	89.08	8.44	34.91	100.4	0.13	叶绿体 Chloroplast	0
Solyc07g009130.2	SlHMA3	1 302	140.44	6.94	45.63	80.46	-0.231	细胞质膜 Plasma membrane	8
Solyc11g062100.1	SlHMA4	698	76.04	5.68	36.13	104.44	0.227	细胞质膜 Plasma membrane	7
Solyc08g080870.2	SlHMA5.1	954	103.58	5.58	39.69	102.69	0.139	细胞质膜 Plasma membrane	6
Solyc08g080890.2	SlHMA5.2	890	96.58	5.80	32.38	103.96	0.200	细胞质膜 Plasma membrane	8
Solyc01g105160.4	SlHMA6	964	101.94	8.30	33.79	97.68	0.092	叶绿体 Chloroplast	0
Solyc02g068490.3	SlHMA7	1 015	108.59	5.16	30.72	106.97	0.324	细胞质膜 Plasma membrane	10
Solyc08g061610.2	SlHMA8	894	95.20	5.94	38.01	103.66	0.118	叶绿体 Chloroplast	0