陆地棉ACX基因家族的鉴定与功能分析

doi:10.11963/cs20220012

摘要/Abstract

摘要：

【目的】通过对陆地棉酰基辅酶A氧化酶（acyl coenzyme A oxidase, ACX）基因家族进行鉴定和表达分析,为后续研究ACX基因的功能奠定基础。【方法】采用生物信息学方法对陆地棉基因组中ACX基因家族成员进行鉴定,并系统分析其理化性质、基因结构、进化关系、基因复制、启动子区顺式作用元件和表达模式等。利用病毒诱导的基因沉默技术初步探究GhACX16基因的功能。【结果】陆地棉基因组中鉴定到20个ACX基因,分布在13条染色体上,聚类分析将其分为4个亚族。非同义突变率/同义突变率（K_a/K_s）分析结果表明陆地棉ACX家族基因经历了较强烈的纯化选择。ACX基因的启动子区含有热应激、干旱、植物激素响应等相关的顺式作用元件。非生物胁迫下的表达模式分析结果显示,陆地棉ACX基因明显响应高温、低温、盐和模拟干旱胁迫;分析高温胁迫下耐高温与高温敏感棉花材料不同发育时期的花药中ACX基因的表达模式,发现GhACX5和GhACX16基因的表达量变化明显。与阴性对照相比,高温胁迫下GhACX16基因沉默的棉苗表现出明显的耐高温特征,且叶片中脯氨酸含量、叶绿素含量和过氧化氢酶活性显著升高,而丙二醛含量显著降低。【结论】陆地棉ACX基因的鉴定及表达模式分析表明,ACX基因广泛参与非生物胁迫响应;经基因沉默试验和生理生化分析初步推测其中的GhACX16基因可能在高温胁迫响应中发挥重要的功能。

关键词: 陆地棉; ACX基因; 生物信息学; 表达模式分析; GhACX16; 病毒诱导的基因沉默

Abstract:

[Objective] The acyl-coenzyme A oxidase (ACX) gene family was identified in Gossypium hirsutum and their expression patterns were analyzed, which would lay the foundation for further studies of the function of ACX genes. [Method] The members of ACX gene family in G. hirsutum genome were identified by bioinformatics method, and their physical and chemical properties, gene structure, evolutionary relationships, gene replication, cis-acting elements in promoter region and expression patterns were analyzed systematically, and the virus-induced gene silencing (VIGS) technique was used to preliminary explore the function of GhACX16 gene. [Results] A total of 20 ACX genes were identified in G. hirsutum genome and distributed on 13 chromosomes, which were divided into 4 subfamilies by cluster analysis. The results of non-synonymous mutation rate/synonymous mutation rate (K_a/K_s) analysis showed that the ACX genes in G. hirsutum had undergone strong purifying selection. The promoter region of ACX genes contain cis-acting elements related to heat stress, drought, plant hormone responses, etc. The expression patterns under abiotic stresses showed that the ACX genes responded obviously to high-temperature, low-temperature, salt, and simulated drought stress. The expression patterns of ACX genes in the anthers at different developmental stages of high temperature-tolerant/sensitive cotton lines under high temperature conditions were analyzed, and it was found that the expression levels of GhACX5 and GhACX16 changed obviously. Compared with the negative control, the GhACX16 silenced cotton seedlings showed obvious characteristics of high temperature tolerance, and the proline content, chlorophyll content, and catalase activity were significantly higher, while the malondialdehyde content was significantly lower in the leaves. [Conclusion] The identification and expression patterns of ACX genes in G. hirsutum showed that ACX genes were widely involved in abiotic stresses response, and GhACX16 gene was preliminary speculated to play an important role in high temperature stress response according to the VIGS verification and physiological and biochemical analysis.

Key words: upland cotton; ACX gene; bioinformatics; expression pattern analysis; GhACX16; virus-induced gene silencing

田一波,潘奥,陈劲,周仲华,袁小玲,刘志. 陆地棉ACX基因家族的鉴定与功能分析[J]. 棉花学报, 2022, 34(3): 215-226.

Tian Yibo,Pan Ao,Chen Jin,Zhou Zhonghua,Yuan Xiaoling,Liu Zhi. Identification and functional analysis of the ACX gene family in Gossypium hirsutum L.[J]. Cotton Science, 2022, 34(3): 215-226.

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链接本文: https://journal.cricaas.com.cn/Jweb_mhxb/CN/10.11963/cs20220012

https://journal.cricaas.com.cn/Jweb_mhxb/CN/Y2022/V34/I3/215

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引物名称 Primer name	序列 Sequence	用途 Purpose
GhACX16-F	ATGCTAACATGCGCCAAGG	CDS扩增
GhACX16-R	GAACCCAATATGGTGCGTGTACT	Amplification of CDS
VIGS-F	AAGGTTACCGAATTCTCTAGACCCCGCTAAGTATTTCGCCA	VIGS载体构建
VIGS-R	GAGCTCGGTACCGGATCCCCAATCCACCATTTGATGGC	Vector construction for VIGS
GhACX16-QF	GCAAATTCGCAACGGTTTTCGC	qRT-PCR
GhACX16-QR	AACTGAACGGAATCGCAGTGCT
Actin9-F	CCTCCGTCTAGACCTTGCTG	qRT-PCR
Actin9-R	TCATTCGGTCAGCAATACCA

基因名称 Gene name	基因ID Gene ID	CDS 长度 CDS length/bp	染色体定位 Chromosome location	氨基酸数量 Amino acid number	等电点 Isoelectric point	蛋白质分子质量 Molecular weight/ku	亚细胞定位 Subcellular localization
GhACX1	Gh_A01G0206	2 013	A01: 1 796 359-1 800 867	670	8.22	75.50	Pero
GhACX2	Gh_A01G0207	2 019	A01: 1 804 843-1 816 831	672	8.78	75.90	Pero
GhACX3	Gh_A03G1765	1 920	A03: 103 416 142-103 419 599	639	9.02	72.00	Pero
GhACX4	Gh_A09G2126	1 995	A09: 78 122 816-78 129 033	664	6.77	74.50	Pero
GhACX5	Gh_A10G0856	2 070	A10: 14 797 765-14 800 475	689	8.47	77.20	Pero
GhACX6	Gh_A11G0258	1 995	A11: 2 261 306-2 266 347	664	7.06	74.70	Pero
GhACX7	Gh_A11G2090	2 067	A11: 40 858 218-40 862 134	688	8.70	77.30	Pero
GhACX8	Gh_A12G1588	2 067	A12: 87 695 524-87 703 039	688	8.52	77.30	Pero
GhACX9	Gh_A12G2360	1 995	A12: 101 992 043-101 997 823	664	7.29	74.90	Pero
GhACX10	Gh_A13G0968	1 302	A13: 42 022 635-42 026 136	433	8.59	47.70	Pero
GhACX11	Gh_D01G0200	2 013	A13: 1 529 855-1 534 363	670	8.35	75.60	Pero
GhACX12	Gh_D01G0201	2 019	D01: 1 536 500-1 543 312	672	8.79	75.80	Pero
GhACX13	Gh_D02G1922	2 025	D02: 62 830 071-62 833 541	674	8.68	75.80	Pero
GhACX14	Gh_D09G1772	1 317	D09: 44 714 315-44 717 800	438	8.62	47.90	Pero
GhACX15	Gh_D09G2062	1 995	D09: 47 571 769-47 578 007	664	6.57	74.50	Pero
GhACX16	Gh_D10G1120	1 944	D10: 14 327 876-14 330 493	647	8.34	72.50	Pero
GhACX17	Gh_D11G0268	1 995	D11: 2 179 426-2 184 528	664	7.05	74.60	Pero
GhACX18	Gh_D11G2339	2 067	D11: 35 740 424-35 744 350	688	8.75	77.20	Pero
GhACX19	Gh_D12G1589	2 031	D12: 45 479 502-45 485 010	676	8.24	75.90	Pero
GhACX20	Gh_D12G2375	1 995	D12: 56 200 689-56 206 438	664	6.89	74.90	Pero

同源基因对	非同义突变率	同义突变率	非同义突变率/同义突变率
Homologous gene pair	K_a	K_s	K_a/K_s
GhACX1/GhACX11	0.009	0.025	0.358
GhACX3/GhACX8	0.103	0.468	0.220
GhACX3/GhACX12	0.130	0.582	0.224
GhACX3/GhACX13	0.010	0.033	0.303
GhACX3/GhACX19	0.100	0.487	0.206
GhACX4/GhACX6	0.082	0.408	0.200
GhACX4/GhACX9	0.075	0.396	0.189
GhACX4/GhACX15	0.004	0.049	0.092
GhACX4/GhACX17	0.083	0.392	0.211
GhACX5/GhACX16	0.009	0.048	0.178
GhACX6/GhACX9	0.055	0.325	0.168
GhACX6/GhACX17	0.007	0.057	0.125
GhACX7/GhACX18	0.009	0.042	0.205
GhACX8/GhACX13	0.098	0.472	0.207
GhACX8/GhACX19	0.010	0.024	0.411
GhACX9/GhACX15	0.077	0.403	0.190
GhACX9/GhACX17	0.056	0.330	0.169
GhACX11/GhACX13	0.116	0.548	0.211
GhACX13/GhACX19	0.095	0.493	0.193
GhACX15/GhACX17	0.085	0.392	0.216