绣球花铝转运蛋白HmALMT11的生物信息学及其表达特性分析

doi:10.7668/hbnxb.20194790

华北农学报 ›› 2024, Vol. 39 ›› Issue (4): 94-101. doi: 10.7668/hbnxb.20194790

所属专题： 生物技术；

• 作物遗传育种·种质资源·生物技术 • 上一篇下一篇

绣球花铝转运蛋白HmALMT11的生物信息学及其表达特性分析

王晓玥¹, 陈双双², 齐香玉², 冯景², 陈慧杰², 孙明¹, 邓衍明²

¹ 林木资源高效生产全国重点实验室(北京林业大学),花卉种质创新与分子育种北京市重点实验室,国家花卉工程技术研究中心,城乡生态环境北京实验室,北京林业大学园林学院,北京 100083
² 江苏省高效园艺作物遗传改良重点实验室,江苏省农业科学院休闲农业研究所,江苏南京 210014

收稿日期:2024-01-04 出版日期:2024-08-27
通讯作者:
孙明(1980—),男,山东淄博人,教授,博士,主要从事观赏植物栽培、遗传育种与分子生物学研究。
邓衍明(1976—),男,安徽泗县人,研究员,博士,主要从事观赏植物栽培、遗传育种与分子生物学研究。
作者简介:
王晓玥(1998—),女,内蒙古包头人,在读硕士,主要从事观赏植物遗传育种与分子生物学研究。
基金资助:
国家自然科学基金项目(32371956); 国家自然科学基金项目(2035); 江苏省种业振兴“揭榜挂帅”项目(JBGS[2021]097)

Bioinformatics Analysis and Expression Profiling of the Aluminum Transporter HmALMT11 in Hydrangea macrophylla

WANG Xiaoyue¹, CHEN Shuangshuang², QI Xiangyu², FENG Jing², CHEN Huijie², SUN Ming¹, DENG Yanming²

¹ State Key Laboratory of Efficient Production of Forest Resources,Beijing Forestry University,Key Laboratory of Ornamental Plants Germplasm Innovation & Molecular Breeding,National Engineering Research Center for Floriculture,Laboratory of Urban and Rural Ecological Environment,School of Landscape Architecture,Beijing Forestry University,Beijing 100083,China
² Jiangsu Provincial Key Laboratory for the Genetics and Improvement of Horticultural Crops,Institute of Leisure Agriculture,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China

Received:2024-01-04 Published:2024-08-27

摘要/Abstract

摘要：

铝激活苹果酸转运蛋白(ALMT)是植物中广泛存在的一个基因家族,其编码的蛋白质在调控植物根部酸分泌和对铝离子的响应方面起着重要作用。为了明晰大花绣球无尽夏HmALMT11的序列特征及其在逆境胁迫后的表达特征,进一步探索大花绣球无尽夏的生物学功能,为后续HmALMT11功能鉴定提供理论依据。以大花绣球无尽夏为材料,克隆得到HmALMT11基因,并对其进行生物信息学、亚细胞定位和铝胁迫响应分析。结果表明,HmALMT11包含1个1 590 bp的开放阅读框,编码529个氨基酸,蛋白分子质量为59.1 ku,理论等电点为9.10。为不稳定的碱性蛋白;HmALMT11蛋白含有6个跨膜结构域,定位于细胞质膜;实时荧光定量PCR分析发现,低浓度100 μmol/L和高浓度800 μmol/L的Al₂(SO₄)₃处理都可诱导HmALMT11在无尽夏根、茎、叶中的上调表达,且在根中相对表达量最高,具有明显的组织表达特异性。研究表明,HmALMT11蛋白可能在绣球花适应铝胁迫过程中发挥重要调控作用。

关键词: 绣球花, ALMT, 铝胁迫, 生物信息学分析, 亚细胞定位, 实时荧光定量PCR

Abstract:

Aluminum-activated malate transporter(ALMT)gene family,as a widely distributed gene family in plants,the encoded protein plays an important role in regulating plant root acid secretion and response to aluminum ions.In order to clarify the sequence characteristics and expression features of H. macrophylla Endless Summer(HmALMT11)under abiotic stress, further exploring the biological functions of H. macrophylla Endless Summer, and providing a theoretical basis for the subsequent identification of the function of HmALMT11.HmALMT11 gene was cloned from the leaves of Hydrangea macrophylla Endless Summer,and the bioinformatics,subcellular localization and aluminum stress response analysis of this gene were also carried out.HmALMT11 contained an open reading frame of 1 590 bp and encoded 529 amino acids,with a molecular weight of 59.1 ku and a theoretical isoelectric point of 9.10 was an unstable basic protein containing 6 transmembrane domains.The subcellular localization analysis revealed that the protein encoded by HmALMT11 was specifically targeted to the plasma membrane.The qRT-PCR analysis revealed that treatments with both low(100 μmol/L) and high(800 μmol/L) concentrations of Al₂(SO₄)₃ increased the expression level of HmALMT11 in the roots,stems,and leaves of Hydrangea macrophylla Endless Summer.Notably,the highest expression level was observed in the roots,demonstrating a clear tissue-specific expression pattern.These results suggested that HmALMT11 protein might play an important role during the regulation of aluminum tolerance in hydrangea.

Key words: Hydrangea macrophylla, ALMT, Aluminum stress, Bioinformatics analysis, Subcellular localization, qRT-PCR

王晓玥, 陈双双, 齐香玉, 冯景, 陈慧杰, 孙明, 邓衍明. 绣球花铝转运蛋白HmALMT11的生物信息学及其表达特性分析[J]. 华北农学报, 2024, 39(4): 94-101. doi: 10.7668/hbnxb.20194790.

WANG Xiaoyue, CHEN Shuangshuang, QI Xiangyu, FENG Jing, CHEN Huijie, SUN Ming, DENG Yanming. Bioinformatics Analysis and Expression Profiling of the Aluminum Transporter HmALMT11 in Hydrangea macrophylla[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(4): 94-101. doi: 10.7668/hbnxb.20194790.

http://www.hbnxb.net/CN/Y2024/V39/I4/94

图/表 8

表1 试验所用的引物

Tab.1 The primers were used in this experiment

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')
HmALMT11-F1	ATGGCCGCAGATGCAGAGAA
HmALMT11-R1	ATCTGTTCCATGGGAAGGCAAAT
HmALMT11-F2	AGCTTGCATGCCTGCAGGTCGACATGGC
	CGCAGATGCAGAGAAG
HmALMT11-R2	CTCGCCCTTGCTCACCATGGATCCATCTG
	TTCCATGGGAAGGCA
HmALMT11-RTF	GATTCAGACTCCTCGCTCAGTACG
HmALMT11-RTR	GCGGCGGTTCTTAATGCTGTTAG
HmUPL7-RTF	GCCACGTACTCACACTTGCT
HmUPL7-RTR	TCTGTGCCTGCAATCCCTTT

图1 HmALMT11编码区片段

Fig.1 Coding region fragment of HmALMT11

表2 HmALMT11蛋白理化性质分析

Tab.2 Analysis of physicochemical properties of HmALMT11 protein

类型Type	数值Value
氨基酸数/个Number of amino acids	529
分子质量/ku Molecular weight	59.1
理论pI值Theoretical pI	9.10
带负电荷的残基总数(Asp+Glu)/个Total number of negatively charged residues(Asp+Glu)	57
带正电荷的残基总数(Arg+Lys)/个Total number of positively charged residues(Arg+Lys)	69
分子式Formula	C₂₆₆₅H₄₂₆₀N₇₁₄O₇₆₄S₁₈
原子总数/个Total number of atoms	8 421
脂肪族氨基酸指数Aliphatic index	98.83
亲水性平均系数(GRAVY)Grand average of hydropathicity(GRAVY)	-0.014
不稳定性指数(不稳定蛋白)Instability index (protein as unstable)	36.03

图2 HmALMT11蛋白结构分析 A.HmALMT11保守结构域预测;B.HmALMT11跨膜结构域预测。

Fig.2 Structure analysis of HmALMT11 A.Conserved domains prediction of HmALMT11;B.Transmembrane domains prediction of HmALMT11.

图3 HmALMT11氨基酸序列同源比对与进化分析 A.HmALMT11氨基酸序列同源性比对;B.HmALMT11系统发育树;C.HmALMT11氨基酸序列保守结构域分析。

Fig.3 Homologous amino acid sequences alignment and phylogenetic tree of HmALMT11 A.Homologous amino acid sequences alignment of HmALMT11;B.Phylogenetic tree of HmALMT11;C.Analysis of conserved domain of HmALMT11 amino acid sequence.

表3 HmALMT11蛋白亚细胞定位预测

Tab.3 Subcellular localization prediction of HmALMT11 protein

亚细胞Subcellular location	分值Score
亚细胞Subcellular location	LocDB	PotLocDB	Neural Nets	Pentamers	Integral
细胞核Nuclear	0	0.0	0	0.00	0.00
质膜Plasma membrane	10	1.4	1	0.37	8.17
胞外Extracellular	0	0.0	1	1.13	0.15
细胞质Cytoplasmic	0	0.0	0	0.46	0.00
线粒体Mitochondrial	0	0.0	0	1.85	0.00
内质网Endoplasmic reticulum	0	0.8	0	1.11	1.14
过氧化物酶体Peroxisomal	0	0.0	1	0.00	0.03
高尔基体Golgi	0	0.0	0	0.78	0.00
叶绿体Chloroplast	0	0.0	0	0.04	0.00
液泡Vacuolar	0	0.8	0	0.00	0.50

图4 HmALMT11亚细胞定位分析 PIP2.膜定位标记对照蛋白,标尺单位10 μm。

Fig.4 Subcellular localization of HmALMT11 PIP2.Control protein for membrane localization,scale unit 10 μm.

图5 Al₂(SO₄)₃处理条件下无尽夏根、茎、叶中HmALMT11基因转录水平分析不同小写字母表示P<0.05水平上差异显著。

Fig.5 Transcriptional levels of HmALMT11 in root,stem and leaf of Endless Summer under different Al₂(SO₄)₃ treatments Different lowercase letters mean significant difference at 0.05 level.

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绣球花铝转运蛋白HmALMT11的生物信息学及其表达特性分析

Bioinformatics Analysis and Expression Profiling of the Aluminum Transporter HmALMT11 in Hydrangea macrophylla

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绣球花铝转运蛋白HmALMT11的生物信息学及其表达特性分析

Bioinformatics Analysis and Expression Profiling of the Aluminum Transporter HmALMT11 in Hydrangea macrophylla

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