AOX基因家族的结构和功能特征分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-1285

摘要/Abstract

摘要：

交替氧化酶（alternative oxidase,AOX）是植物呼吸途径上的一种末端氧化酶,通过对玉米、水稻、拟南芥、二穗短柄草、番茄、马铃薯、高粱、大豆等8个物种中AOX基因进行鉴定分析,以期进一步挖掘AOX基因的功能以及调控机制。在8个物种中共鉴定到35个编码AOX蛋白的基因,通过聚类分析将其划分为5个亚家族。通过分析和比较AOX基因的系统进化关系、染色体位置、外显子-内含子结构以及保守基序,进一步验证了该亚家族划分模式,同时也揭示了AOX基因在进化过程中具有较强的保守性。通过顺式作用元件预测、miRNA靶位点预测以及AOX蛋白的GO注释分析,发现AOX基因可能在植物生长发育以及应激反应中发挥重要作用。在玉米苗期进行低温、高温、干旱以及盐碱胁迫处理后,对玉米中4个AOX基因进行qRT-PCR分析表明,4个AOX基因呈现不同程度的诱导表达,暗示它们在逆境胁迫应答上存在密切关系。该研究结果为深入理解AOX基因的生物学功能以及响应逆境胁迫的调控机制奠定了基础。

关键词: AOX, 基因家族鉴定, 表达分析, 非生物胁迫

Abstract:

AOX（alternative oxidase,AOX）is a terminal oxidase in the respiratory pathway of plants. Through the identification and analysis of AOX genes in 8 species of Zea mays,Oryza sativa,Arabidopsis thaliana,Brachypodium distachyon,Solanum lycopersicum,Solanum tuberosum,Sorghum bicolor and Glycine max,we aim to further explore the function and regulatory mechanism of AOX gene. A total of 35 genes encoding AOX protein in these 8 species were identified,and these 35 genes were divided into 5 subfamilies by cluster analysis. The phylogenetic analysis and comparison of the chromosome position exon-intron structure and the conserved motif composition of AOX further supported the subfamily division model,and revealed that the AOX gene maintained a strong conserved nature during the evolution process. The prediction of cis-acting elements,miRNA target sites and the GO annotation analysis of AOX protein revealed that AOX gene may play an important role in plant growth and development and stress response. After stress treatment at the seedling stage of maize,the qRT-PCR analysis of 4 AOX genes in maize showed that the 4 AOX genes presented different degrees of inducible expression,suggesting that they were closely related in response to abiotic stress. These results lay a foundation for further understanding the biological function of AOX genes and their regulatory mechanism in response to abiotic stress.

Key words: AOX, gene family identification, expression analysis, abiotic stress

王慧, 马艺文, 乔正浩, 常彦彩, 术琨, 丁海萍, 聂永心, 潘光堂. AOX基因家族的结构和功能特征分析[J]. 生物技术通报, 2022, 38(7): 160-170.

WANG Hui, MA Yi-wen, QIAO Zheng-hao, CHANG Yan-cai, ZHU Kun, DING Hai-ping, NIE Yong-xin, PAN Guang-tang. Structural and Functional Characterization of AOX Gene Family[J]. Biotechnology Bulletin, 2022, 38(7): 160-170.

图/表 10

参考文献 32

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引物名称Gene name	上游引物Upstream primer（5'-3'）	下游引物Downstream primer（5'-3'）
18S	CTGAGAAACGGCTACCACA	CCCAAGGTCCAACTACGAG
Zm00001d017727qRT	TGGACCGTCAAATTACTGCG	ATGCGTTCATTCTCCGCCTC
Zm00001d002434qRT	CTACTGGGGCATCGACACG	GCAGCGACTTGACGATCAG
Zm00001d002435qRT	AGGGGCAGGACAAGAAGGCCG	TACGTGTCCCATGGCCTGAA
Zm00001d002436qRT	GCTTATGTCCACGTCCTCCC	TGTACGTCTCCCATGGCCTA

引物名称Gene name	上游引物Upstream primer（5'-3'）	下游引物Downstream primer（5'-3'）
18S	CTGAGAAACGGCTACCACA	CCCAAGGTCCAACTACGAG
Zm00001d017727qRT	TGGACCGTCAAATTACTGCG	ATGCGTTCATTCTCCGCCTC
Zm00001d002434qRT	CTACTGGGGCATCGACACG	GCAGCGACTTGACGATCAG
Zm00001d002435qRT	AGGGGCAGGACAAGAAGGCCG	TACGTGTCCCATGGCCTGAA
Zm00001d002436qRT	GCTTATGTCCACGTCCTCCC	TGTACGTCTCCCATGGCCTA

基因ID Gene ID	染色体编号 Chrom No.	氨基酸长 Length/ aa	分子质量 Molecular weight/ kD	等电点 pI	重力值 GRAVY	亚细胞定位 Location
AtAOX1a_AT3G22370.1	3	354	39.98	8.56	-0.33	Cytoplasmic
AtAOX1b_AT3G22360.1	3	325	37.43	8.55	-0.35	Cytoplasmic
AtAOX1c_AT3G27620.1	3	329	37.82	6.91	-0.33	Cytoplasmic
AtAOX1d_AT1G32350.1	1	318	36.20	8.65	-0.29	Cytoplasmic
AtAOX2_AT5G64210.1	5	353	40.09	9.14	-0.17	Cytoplasmic
BdAOX_KQJ84389	5	333	37.34	8.37	-0.21	Periplasmic
BdAOX_KQJ84390	5	324	36.14	7.88	-0.19	Periplasmic
BdAOX_KQJ84391	5	330	36.77	7.29	-0.22	Periplasmic
BdAOX_KQK00893	3	394	43.98	9.37	-0.24	Cytoplasmic
BdAOX_PNT69270	3	391	43.67	9.49	-0.29	Cytoplasmic
GmAOX_KRH42153	8	326	37.12	8.68	-0.32	Cytoplasmic
GmAOX_KRH42154	8	333	38.14	9.36	-0.27	Cytoplasmic
GmAOX_KRH58272	5	317	36.01	9.01	-0.18	Cytoplasmic
GmAOX_KRH62683	4	321	36.48	8.57	-0.24	Cytoplasmic
OsAOX_Os02t0318100-00	2	353	39.46	9.46	-0.17	Periplasmic
OsAOX_Os02t0700400-01	2	345	37.89	8.31	-0.15	Cytoplasmic
OsAOX_Os04t0600200-01	4	332	37.14	7.82	-0.12	Cytoplasmic
OsAOX_Os04t0600300-01	4	335	37.25	7.23	-0.21	Periplasmic
SbAOX_EES05672	4	346	38.47	7.82	-0.15	Cytoplasmic
SbAOX_EES12781	6	331	37.16	8.63	-0.23	Periplasmic
SbAOX_EES12782	6	314	35.58	8.78	-0.23	Cytoplasmic
SbAOX_EES12783	6	332	36.84	7.96	-0.20	Cytoplasmic
SlAOX_Solyc01g105220.3.1	1	348	39.91	8.56	-0.26	Cytoplasmic
SlAOX_Solyc08g005560.3.1	8	753	85.44	8.07	-0.14	Cytoplasmic
SlAOX_Solyc08g075540.3.1	8	642	72.72	8.30	-0.30	Periplasmic
StAOX_PGSC0003DMT400019707	8	321	36.72	6.76	-0.38	Cytoplasmic
StAOX_PGSC0003DMT400019708	8	356	39.82	8.29	-0.28	Periplasmic
StAOX_PGSC0003DMT400019709	8	355	39.64	7.75	-0.22	Periplasmic
StAOX_PGSC0003DMT400019710	8	174	19.68	5.44	0.02	Cytoplasmic
StAOX_PGSC0003DMT400032705	1	353	40.36	8.45	-0.19	Cytoplasmic
StAOX_PGSC0003DMT400047562	8	279	31.91	9.08	-0.12	Cytoplasmic
ZmAOX_Zm00001d002434_P001	2	332	36.77	8.02	-0.16	Cytoplasmic
ZmAOX_Zm00001d002435_P001	2	329	37.04	8.98	-0.23	Periplasmic
ZmAOX_Zm00001d002436_P001	2	329	36.90	8.84	-0.20	Periplasmic
ZmAOX_Zm00001d017727_P001	5	347	38.72	8.37	-0.16	Cytoplasmic

基因ID Gene ID	染色体编号 Chrom No.	氨基酸长 Length/ aa	分子质量 Molecular weight/ kD	等电点 pI	重力值 GRAVY	亚细胞定位 Location
AtAOX1a_AT3G22370.1	3	354	39.98	8.56	-0.33	Cytoplasmic
AtAOX1b_AT3G22360.1	3	325	37.43	8.55	-0.35	Cytoplasmic
AtAOX1c_AT3G27620.1	3	329	37.82	6.91	-0.33	Cytoplasmic
AtAOX1d_AT1G32350.1	1	318	36.20	8.65	-0.29	Cytoplasmic
AtAOX2_AT5G64210.1	5	353	40.09	9.14	-0.17	Cytoplasmic
BdAOX_KQJ84389	5	333	37.34	8.37	-0.21	Periplasmic
BdAOX_KQJ84390	5	324	36.14	7.88	-0.19	Periplasmic
BdAOX_KQJ84391	5	330	36.77	7.29	-0.22	Periplasmic
BdAOX_KQK00893	3	394	43.98	9.37	-0.24	Cytoplasmic
BdAOX_PNT69270	3	391	43.67	9.49	-0.29	Cytoplasmic
GmAOX_KRH42153	8	326	37.12	8.68	-0.32	Cytoplasmic
GmAOX_KRH42154	8	333	38.14	9.36	-0.27	Cytoplasmic
GmAOX_KRH58272	5	317	36.01	9.01	-0.18	Cytoplasmic
GmAOX_KRH62683	4	321	36.48	8.57	-0.24	Cytoplasmic
OsAOX_Os02t0318100-00	2	353	39.46	9.46	-0.17	Periplasmic
OsAOX_Os02t0700400-01	2	345	37.89	8.31	-0.15	Cytoplasmic
OsAOX_Os04t0600200-01	4	332	37.14	7.82	-0.12	Cytoplasmic
OsAOX_Os04t0600300-01	4	335	37.25	7.23	-0.21	Periplasmic
SbAOX_EES05672	4	346	38.47	7.82	-0.15	Cytoplasmic
SbAOX_EES12781	6	331	37.16	8.63	-0.23	Periplasmic
SbAOX_EES12782	6	314	35.58	8.78	-0.23	Cytoplasmic
SbAOX_EES12783	6	332	36.84	7.96	-0.20	Cytoplasmic
SlAOX_Solyc01g105220.3.1	1	348	39.91	8.56	-0.26	Cytoplasmic
SlAOX_Solyc08g005560.3.1	8	753	85.44	8.07	-0.14	Cytoplasmic
SlAOX_Solyc08g075540.3.1	8	642	72.72	8.30	-0.30	Periplasmic
StAOX_PGSC0003DMT400019707	8	321	36.72	6.76	-0.38	Cytoplasmic
StAOX_PGSC0003DMT400019708	8	356	39.82	8.29	-0.28	Periplasmic
StAOX_PGSC0003DMT400019709	8	355	39.64	7.75	-0.22	Periplasmic
StAOX_PGSC0003DMT400019710	8	174	19.68	5.44	0.02	Cytoplasmic
StAOX_PGSC0003DMT400032705	1	353	40.36	8.45	-0.19	Cytoplasmic
StAOX_PGSC0003DMT400047562	8	279	31.91	9.08	-0.12	Cytoplasmic
ZmAOX_Zm00001d002434_P001	2	332	36.77	8.02	-0.16	Cytoplasmic
ZmAOX_Zm00001d002435_P001	2	329	37.04	8.98	-0.23	Periplasmic
ZmAOX_Zm00001d002436_P001	2	329	36.90	8.84	-0.20	Periplasmic
ZmAOX_Zm00001d017727_P001	5	347	38.72	8.37	-0.16	Cytoplasmic

玉米AOX ZmAOXs	染色体编号 Chrom No.	其他物种AOX Other AOXs	染色体编号 Chrom No.	N	S	Ka	Ks	Ka/Ks	Mya
ZmAOX_Zm00001d002434_P001	2	SbAOX_EES12783	6	842.4	126.6	0.05	0.47	0.10	36.49
ZmAOX_Zm00001d002435_P001	2	BdAOX_KQJ84390	5	824.5	141.5	0.09	1.86	0.05	143.31
ZmAOX_Zm00001d002435_P001	2	OsAOX_Os04t0600300-01	4	858.2	128.8	0.10	0.79	0.13	60.89
ZmAOX_Zm00001d002435_P001	2	SbAOX_EES12782	6	826	113	0.02	0.24	0.08	18.32
ZmAOX_Zm00001d002436_P001	2	AtAOX1a_AT3G22370.1	3	729.6	248.4	0.20	65.03	0.00	5 002.48
ZmAOX_Zm00001d002436_P001	2	BdAOX_KQJ84389	5	839.3	147.7	0.09	1.09	0.09	84.08
ZmAOX_Zm00001d002436_P001	2	GmAOX_KRH62683	4	738.7	218.3	0.21	72.34	0.00	5 564.66
ZmAOX_Zm00001d002436_P001	2	OsAOX_Os04t0600200-01	4	838.9	139.1	0.08	1.12	0.07	86.45
ZmAOX_Zm00001d002436_P001	2	SbAOX_EES12781	6	850.2	136.8	0.01	0.29	0.03	22.22
ZmAOX_Zm00001d002436_P001	2	SlAOX_Solyc01g105220.3.1	1	747.2	230.8	0.26	23.83	0.01	1 832.88
ZmAOX_Zm00001d017727_P001	5	BdAOX_KQK00893	3	856.1	157.9	0.08	1.44	0.06	110.84
ZmAOX_Zm00001d017727_P001	5	BdAOX_PNT69270	3	848.1	156.9	0.11	1.85	0.06	142.36
ZmAOX_Zm00001d017727_P001	5	GmAOX_KRH62683	4	727.2	226.8	0.21	24.26	0.01	1 866.37
ZmAOX_Zm00001d017727_P001	5	OsAOX_Os02t0700400-01	2	864.9	161.1	0.10	1.45	0.07	111.63
ZmAOX_Zm00001d017727_P001	5	SbAOX_EES05672	4	865.8	169.2	0.02	0.22	0.08	16.71
ZmAOX_Zm00001d017727_P001	5	StAOX_PGSC0003DMT400019708	8	760.2	262.8	0.28	25.43	0.01	1 955.81
ZmAOX_Zm00001d017727_P001	5	StAOX_PGSC0003DMT400019709	8	752.6	261.4	0.32	15.51	0.02	1 193.40