玉米ACO基因家族生物信息学及表达模式分析

doi:10.3864/j.issn.0578-1752.2024.07.008

中国农业科学 ›› 2024, Vol. 57 ›› Issue (7): 1308-1318.doi: 10.3864/j.issn.0578-1752.2024.07.008

玉米ACO基因家族生物信息学及表达模式分析

王程泽¹(), 张燕¹(), 付伟², 贾京哲¹, 董金皋¹, 申珅¹(), 郝志敏¹()

¹ 河北农业大学生命科学学院/华北作物改良与调控国家重点实验室/河北省农业微生物生物信息利用技术创新中心/河北省植物生理与分子病理学重点实验室，河北保定 071000
² 邯郸学院，河北邯郸 056005

收稿日期:2023-10-31 接受日期:2023-12-09 出版日期:2024-04-01 发布日期:2024-04-09
通信作者:
郝志敏，E-mail：haozhimin@hebau.edu.cn。
申珅，E-mail：shenshen0428@163.com
联系方式: 王程泽，E-mail：2060848550@qq.com。张燕，E-mail：2419298400@qq.com。王程泽和张燕为同等贡献作者。
基金资助:
河北省自然科学基金(C2021204112); 河北省省级科技计划(23567601H); 国家玉米产业技术体系(CARS-02)

Bioinformatics and Expression Pattern Analysis of Maize ACO Gene Family

WANG ChengZe¹(), ZHANG Yan¹(), FU Wei², JIA JingZhe¹, DONG JinGao¹, SHEN Shen¹(), HAO ZhiMin¹()

¹ College of Life Science, Hebei Agricultural University/State Key Laboratory of North China Crop Improvement and Regulation/ Hebei Bioinformatic Utilization and Technological Innovation Center for Agricultural Microbes/Hebei Key Laboratory of Plant Physiology and Molecular Pathology, Baoding 071000, Hebei
² Handan College, Handan 056005, Hebei

Received:2023-10-31 Accepted:2023-12-09 Published:2024-04-01 Online:2024-04-09

摘要/Abstract

摘要：

【目的】对玉米1-氨基环丙烷-1-羧酸氧化酶ACO基因家族进行全基因组鉴定，分析其在玉米不同器官和不同发育时期以及响应外源激素和病菌侵染中的表达模式，为明确玉米ACO基因家族功能打下基础。【方法】利用生物信息学方法，在玉米B73自交系基因组中鉴定ACO，对其基因结构、蛋白质理化性质、家族成员间的亲缘关系以及保守基序进行分析，利用实时荧光定量PCR（real-time fluorescence quantitative PCR，qRT-PCR）技术分析ZmACO基因家族的表达模式。【结果】除ZmACO11外，ZmACO家族成员均具有Fe²⁺离子结合位点和底物抗坏血酸结合位点。系统发育分析显示，ZmACO2与ScACO在同一分支，亲缘关系较近，Bootstrap值达98。基因表达分析表明，ZmACO2、5、9、15、20、35在各发育时期均活跃表达，且在叶片中呈优势表达，因此选择上述6个基因进行下一步检测。喷施乙烯利后，上述6个基因的表达均有所波动，其中ZmACO2的表达量受影响较大，变化幅度在8倍左右。在乙烯利处理的0—24 h内这6个基因的表达量存在波动，但在处理后24 h，6个基因的表达量均接近0。水杨酸处理后，ZmACO5的表达量受影响较大，变化倍数在2倍左右。其他基因的表达量在处理后24 h均接近0。ZmACO9、35在3—12 h的表达量存在波动，ZmACO2、15、20表达量呈下调趋势。在响应生物胁迫方面，接种玉米大斑病菌（Setosphaeria turcica）后，ZmACO5、9的表达量变化幅度最大，在接种后第10天，这两个基因的表达量分别升至对照组的50和60倍。接种玉米小斑病菌（Cochlibolus heterostrophus）后，ZmACO5的表达量变化幅度较大，变化倍数在40—90倍。接种立枯丝核菌（Rhizoctonia solani）后，ZmACO5、35表达量变化幅度最大，在病菌接种的第3天达到200倍。【结论】ZmACO2、5、20、35在玉米生长发育过程中表达变化最活跃；施加外源乙烯利和水杨酸可以对ZmACO的表达水平造成显著影响。病菌侵染玉米后ZmACO的表达水平产生显著变化，与生物胁迫应答关系密切。

关键词: 玉米ACO基因家族, 实时荧光定量PCR, 基因表达, 乙烯利, 水杨酸, 玉米大斑病菌, 玉米小斑病菌, 立枯丝核菌

Abstract:

【Objective】The objective of this study is to perform the genome-wide identification of the maize ACO (1-aminocyclopropane- 1-carboxylate oxidase) gene family, analyze its expression patterns in different organs and developmental stages of maize, as well as in response to exogenous hormones and pathogen infection, and to lay the foundation for clarifying the function of the maize ACO gene family.【Method】Using bioinformatics methods, the ACO was identified in the genome of maize B73 inbred line, and its gene structure, protein physicochemical properties, phylogenetic relationships among family members, and conserved motifs were analyzed. The expression patterns of the ZmACO gene family were analyzed using real-time fluorescence quantitative PCR (qRT-PCR) technology.【Result】Except for ZmACO11, all members of the ZmACO family have Fe²⁺ binding sites and substrate ascorbic acid binding sites. The phylogenetic tree showed that ZmACO2 and ScACO are in the same branch and have a close genetic relationship, with a Bootstrap value of 98. The gene expression analysis indicated that ZmACO2, 5, 9, 15, 20 and 35 were actively expressed at various developmental stages and exhibited dominant expression in leaves, so the six genes mentioned above were selected for the next step of testing. Spraying ethephon resulted in fluctuations in the expression of all six genes mentioned above, the expression level of ZmACO2 was significantly affected, with a variation multiple of about 8 times. The expression levels of these six genes fluctuated within 0-24 h of ethephon treatment. But after 24 h of treatment, all gene expression levels were close to 0. After salicylic acid treatment, the expression level of ZmACO5 was significantly affected, with a variation multiple of about 2 times. The expression levels of other genes were close to 0 at 24 h after treatment. The expression levels of ZmACO9, 35 fluctuated between 3 to 12 h, and the expression levels of ZmACO2, 15, 20 showed a downward trend. In response to biological stress, the expression levels of ZmACO5, 9 showed the greatest changes after inoculation with the Setosphaeria turcica, and on the 10th day after inoculation, the expression levels of these two genes increased by 50 and 60 times, respectively, compared to the control group. After inoculation with the Cochlibolus heterostrophus, the expression level of ZmACO5 changed significantly, with a variation multiple of 40-90 times. After inoculation with Rhizoctonia solani, the expression levels of ZmACO5, 35 showed the greatest changes, reaching 200 times on the 3rd day of inoculation.【Conclusion】The expression changes of ZmACO2, 5, 20 and 35 are most active during the growth and development of maize; The application of exogenous ethephon and salicylic acid can significantly affect the expression level of ZmACO genes. The expression level of ZmACO genes significantly changes after bacterial infection in maize, which is closely related to the response to biological stress.

Key words: maize ACO gene family, qRT-PCR, gene expression, ethephon, salicylic acid, Setosphaeria turcica, Cochlibolus heterostrophus, Rhizoctonia solani

王程泽, 张燕, 付伟, 贾京哲, 董金皋, 申珅, 郝志敏. 玉米ACO基因家族生物信息学及表达模式分析[J]. 中国农业科学, 2024, 57(7): 1308-1318.

WANG ChengZe, ZHANG Yan, FU Wei, JIA JingZhe, DONG JinGao, SHEN Shen, HAO ZhiMin. Bioinformatics and Expression Pattern Analysis of Maize ACO Gene Family[J]. Scientia Agricultura Sinica, 2024, 57(7): 1308-1318.

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引物Primer	序列Sequence (5′-3′)
ZmACO1-F/R	TGAAAACCTTGGGCTGGACA/TCACAAGCTCTGGATGGCTG
ZmACO2-F/R	CAAGATGCGTGCGAGAACTG/CGCTTGTAGTGCTCCTTGGT
ZmACO3-F/R	CATCTCCGGCGGCCTAAAA/AGTTCTTTGTCGAATACTCGGT
ZmACO4-F/R	CTACAACCCGGCTAACGAGG/GAATCTGGGCTCCTTGGCTT
ZmACO5-F/R	TACTTCATCCAGCACCACCC/TTGAACCACGGGTGCTTGCC
ZmACO6-F/R	GAGAAGATGCTGGGCGTGAT/ATAGTGGCTGACCTTGGTGC
ZmACO7-F/R	GATGATGGAGTACCGCAGGG/TGACCTTGGTGCCGTAGAAG
ZmACO8-F/R	AGCTGCTTCGAAGAGGTTATCAA/TGACGCCTACGGAAGGTTTAG
ZmACO9-F/R	TCGGGGACCTCTTGCAGATA/AGATGCCGATGGAAACCCTG
ZmACO10-F/R	AAGGTGGCAAAGTACCCCAG/GCCATCCACCTACCGTCTTT
ZmACO11-F/R	CGTCTTCCGCTCCTACATCG/CAGTGAGGTACAGAGCGAGC
ZmACO12-F/R	AGCTCTGTACCTCACTGGAGA/AAAACGCCTCTTGGTAAGCG
ZmACO15-F/R	TGCCTTCTCCATCCTCAAGTC/CGATGACGGGAACCACCATT
ZmACO20-F/R	GGGAGAGCACCTTCTTCGTC/TTCTCCGACAGCTTCTGCAC
ZmACO31-F/R	AAGAAGGTGTGCTCCGACTG/GATGTCCTCCCAGTCCAGGT
ZmACO35-F/R	CTGGGAGAGCACCTTCTTCG/GATCTCCGATGCGAACTGCT
UBQ9-F/R	TACAGTTCTACAAGGTGGACGAC/GCAGTAGTGGCGGTCGAAGT

反应组分Reaction component	反应体系Reaction system	终浓度Final concentration
2×Fast Super EvaGreen Master Mix	10 μL	1×
正向引物Forward primer	1 μL	0.1-0.5 μmol·L^-1 each
反向引物Reverse primer	1 μL	0.1-0.5 μmol·L^-1 each
cDNA (500 ng·μL^-1) template	1 μL
H₂O	补足至20 μL Make up to 20 μL

名称 Name	基因号 Gene ID	氨基酸长度 Size (aa)	开放阅读框 ORF (bp)	基因定位 Gene mapping	分子量 Molecular weight (Da)	等电点 Isoelectric point
ZmACO1	Zm00001d036955	293	1024	scaffold_6:114257252-114258119(+)	33323.18	6.01
ZmACO2	Zm00001d020686	326	1484	scaffold_7:128315057-128316692(-)	36437.57	5.18
ZmACO3	Zm00001d024852	315	1259	scaffold_10:90656251-90657509(-)	35020.67	5.42
ZmACO4	Zm00001d024851	315	948	scaffold_10:90634003-90634950(-)	35020.67	5.42
ZmACO5	Zm00001d011208	317	1532	scaffold_8:142565914-142567685(+)	34646.05	5.06
ZmACO6	Zm00001d004718	310	1234	scaffold_2:133578506-133580227(+)	34557.35	5.13
ZmACO7	Zm00001d000163	313	1451	scaffold_4:209050198-209052191(-)	35045.80	5.01
ZmACO8	Zm00001d005927	226	681	scaffold_2:193010237-193011059(+)	25466.25	6.22
ZmACO9	Zm00001d039575	376	1526	scaffold_3:8204808-8206696(+)	40606.00	5.77
ZmACO10	Zm00001d015860	234	705	scaffold_5:127015024-127015847(+)	26546.42	6.23
ZmACO11	Zm00001d044978	123	372	scaffold_9:9100281-9102917(-)	13700.64	9.47
ZmACO12	Zm00001d046848	260	793	scaffold_9:108298093-108299643(-)	29077.46	9.52
ZmACO15	Zm00001d024853	356	1599	scaffold_10:90488171-90489769(+)	39417.55	6.03
ZmACO20	Zm00001d052136	323	1330	scaffold_4:180604677-180607029(-)	35551.08	4.97
ZmACO31	Zm00001d024843	314	945	scaffold_10:90456450-90457394(-)	34836.40	5.09
ZmACO35	Zm00001d018211	317	1342	scaffold_5:216633702-216636170(+)	35295.88	4.89

玉米ACO基因家族生物信息学及表达模式分析

Bioinformatics and Expression Pattern Analysis of Maize ACO Gene Family

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