陆地棉SKS基因家族全基因组特征分析与<i>GhSKS13</i>抗黄萎病功能解析

doi:10.7668/hbnxb.20194046

摘要/Abstract

摘要：

鉴定陆地棉 SKS 基因家族成员,解析其演化规律,为棉花抗性育种提供新的候选基因。基于已公布的陆地棉遗传标准系TM-1基因组数据,以陆地棉品种中棉-14为试验材料。通过生物信息学对陆地棉SKS家族成员进行全基因组鉴定,并对其家族成员的染色体分布、进化关系、基因结构、共线性关系等进行预测分析。通过实时荧光定量聚合酶链式反应分析GhSKS13的表达模式,并利用病毒诱导的基因沉默技术初步探究陆地棉SKS13在棉花抵御大丽轮枝菌过程中的功能。共鉴定到48个陆地棉SKS基因,不均匀分布于陆地棉19条染色体上,聚类分析分为5个亚组,基因序列具有较高保守性,共线性关系分析显示,陆地棉SKS基因家族受到纯化选择。组织模式表达分析显示,GhSKS13在陆地棉根组织中优势表达,并且受大丽轮枝菌诱导显著上调表达。 GhSKS13沉默植株对大丽轮枝菌抗性减弱,同时抑制病程相关基因GhPR1、GhPR2、GhPR3和GhPR5表达。大丽轮枝菌入侵GhSKS13沉默植株,其过氧化氢(H₂O₂)沉积明显低于对照,暗示GhSKS13促进活性氧(ROS)的形成。总之,明确了陆地棉SKS家族成员的系统进化关系、染色体分布特征和基因结构特征;并阐明了GhSKS13参与棉花对大丽轮枝菌抗性反应。

关键词: 陆地棉, 全基因组鉴定, 大丽轮枝菌, SKS基因, 活性氧

Abstract:

Genomic-wide identification of systematic work on the SKS gene family in cotton has not been reported.Identifying SKS (Skewed5 similar) gene family members and analyzing evolutionary relationship in Gossypium hirsutum,and analyzing the mechanism of GhSKS13 gene regulating cotton disease resistance are expected to provide a new direction for the cultivation of resistant plants.Here,the members of upland cotton SKS family were identified by bioinformatics at the genome-wide based on published genomic data of upland cotton genetic standard line TM-1,and G.hirsutum variety CRI-14 was used as the material.And chromosome distribution,evolutionary relationships,gene structure,and collinearity of SKS family members were predicted.Then,the expression pattern of GhSKS13 was analyzed by Real-time Quantitative PCR and virus-induced gene silencing was employed for preliminary investigation of the function of GhSKS13 in cotton resistance to Verticillium dahliae.A total of 48 upland cotton SKS genes were identified,unevenly distributed on 19 chromosomes,clustered into 5 subgroups with highly conserved gene sequences.Collinearity analysis revealed that the upland cotton SKS gene family was subject to purifying selection.Expression pattern analysis showed that GhSKS13 was predominantly expressed in upland cotton root tissues and significantly up-regulated by V.dahliae infection. GhSKS13-silenced reduced plants resistance to V.dahliae and suppressed the expression of the pathogen-related genes including GhPR1, GhPR2,GhPR3 and GhPR5 compared to control plants. GhSKS13-silenced plants invaded by V.dahliae showed significantly lower hydrogen peroxide (H₂O₂) deposition compared to the control,suggesting that GhSKS13 promotes the formation of reactive oxygen species (ROS).In conclusion,this study clarified the phylogenetic relationships,chromosomal distribution characteristics and gene structure characteristics of upland cotton SKS family members,and elucidated the involvement of GhSKS13 in upland cotton plant resistance to V.dahliae.

Key words: Gossypium hirsutum, Genome-wide identification, Verticillium dahliae, SKS gene, Reactive oxygen species

陆承哲, 贾培, 武盼, 唐叶, 石琳芳, 陈爱民, 彭清忠, 吴家和. 陆地棉SKS基因家族全基因组特征分析与GhSKS13抗黄萎病功能解析[J]. 华北农学报, 2023, 38(6): 156-167. doi: 10.7668/hbnxb.20194046.

LU Chengzhe, JIA Pei, WU Pan, TANG Ye, SHI Linfang, CHEN Aimin, PENG Qingzhong, WU Jiahe. Genome-wide Characterization of SKS Gene Family in Gossypium hirsutum and Functional Analysis of GhSKS13 in Plant Resistance to Verticillium Wilt[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(6): 156-167. doi: 10.7668/hbnxb.20194046.

http://www.hbnxb.net/CN/Y2023/V38/I6/156

图/表 11

参考文献 30

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引物名称 Primer name	序列(5'—3') Sequence(5'—3')	用途 For use
V-GhSKS13-F	CCTCCATGGGGATCCATCCACAGCTTTACATAGGG	VIGS
V-GhSKS13-R	CGTGAGCTCGGTACCTTGTTTAACCCCAACTTTAC	VIGS
GhSKS13-F	AGGCATACGGTGCAAGTGTT	qPCR
GhSKS13-R	GCGTGTTCTCAGGGATGTTG	qPCR
ITS1-F	AAAGTTTTAATGGTTCGCTAAGA	qPCR
ST-VE1-R	CTTGGTCATTTAGAGGAAGTAA	qPCR
GhUBQ7-F	GAAGGCATTCCACCTGACCAAC	qPCR
GhUBQ7-R	CTTGACCTTCTTCTTCTTGTGCTTG	qPCR
GhPR1-F	GGGGCAGTGCTGACCTATCG	qPCR
GhPR1-R	TTAGCACAACCAAGATGGACAGAGT	qPCR
GhPR2-F	CCACCAGCAGCAGAAGTTATCG	qPCR
GhPR2-R	TTCAAGGTTTGCACTCGGAAGA	qPCR
GhPR3-F	GATGACTCCACAATCACCGAAGC	qPCR
GhPR3-R	GCGGTCTTCTACCTGGGCATT	qPCR
GhPR5-F	GGCAGTAGAACCCGAACTGG	qPCR
GhPR5-R	CAGGGAACCAGGCCATACTG	qPCR

引物名称 Primer name	序列(5'—3') Sequence(5'—3')	用途 For use
V-GhSKS13-F	CCTCCATGGGGATCCATCCACAGCTTTACATAGGG	VIGS
V-GhSKS13-R	CGTGAGCTCGGTACCTTGTTTAACCCCAACTTTAC	VIGS
GhSKS13-F	AGGCATACGGTGCAAGTGTT	qPCR
GhSKS13-R	GCGTGTTCTCAGGGATGTTG	qPCR
ITS1-F	AAAGTTTTAATGGTTCGCTAAGA	qPCR
ST-VE1-R	CTTGGTCATTTAGAGGAAGTAA	qPCR
GhUBQ7-F	GAAGGCATTCCACCTGACCAAC	qPCR
GhUBQ7-R	CTTGACCTTCTTCTTCTTGTGCTTG	qPCR
GhPR1-F	GGGGCAGTGCTGACCTATCG	qPCR
GhPR1-R	TTAGCACAACCAAGATGGACAGAGT	qPCR
GhPR2-F	CCACCAGCAGCAGAAGTTATCG	qPCR
GhPR2-R	TTCAAGGTTTGCACTCGGAAGA	qPCR
GhPR3-F	GATGACTCCACAATCACCGAAGC	qPCR
GhPR3-R	GCGGTCTTCTACCTGGGCATT	qPCR
GhPR5-F	GGCAGTAGAACCCGAACTGG	qPCR
GhPR5-R	CAGGGAACCAGGCCATACTG	qPCR

序列ID Sequence ID	染色体 Chromosome	氨基酸数量/个 Number of amino acid	相对分子质量/ku Molecular weight	理论等电点 Theoretical pI	不稳定指数 Instability index	脂肪指数 Aliphatic index	总平均亲水性 Grand average of hydropathicity
Gh_D13G240200.1	D13	551	61.62	8.92	32.58	79.93	-0.29
Gh_D13G240400.1	D13	551	61.62	8.92	32.58	79.93	-0.29
Gh_D13G240000.1	D13	551	61.62	8.92	32.58	79.93	-0.29
Gh_D13G240100.1	D13	551	61.72	8.93	32.96	81.51	-0.26
Gh_A13G237100.1	A13	551	61.74	9.06	32.66	80.44	-0.30
Gh_A13G237000.1	A13	560	62.69	9.17	29.58	78.98	-0.32
Gh_A13G237300.1	A13	551	61.69	8.93	33.15	81.69	-0.28
Gh_D01G163800.1	D01	521	58.35	9.08	35.06	77.62	-0.31
Gh_D13G240300.1	D13	551	61.43	8.88	32.11	80.98	-0.22
Gh_A13G237500.1	A13	551	61.45	8.79	34.85	80.11	-0.27
Gh_A13G237200.1	A13	551	61.46	8.79	35.00	80.64	-0.26
Gh_A08G092100.1	A08	396	44.92	8.99	46.13	82.20	-0.29
Gh_A13G237600.1	A13	550	61.69	8.79	32.90	85.95	-0.24
Gh_A11G200600.1	A11	539	60.37	8.72	34.32	89.11	-0.14
Gh_D11G204500.1	D11	539	60.48	8.72	34.86	90.35	-0.11
Gh_D12G073800.1	D12	537	60.33	9.64	32.45	96.18	-0.10
Gh_A12G074300.1	A12	537	60.43	9.63	31.84	96.72	-0.11
Gh_A11G050000.1	A11	538	59.83	9.00	35.82	86.58	-0.16
Gh_A11G200700.1	A11	536	59.99	9.49	34.30	85.78	-0.21
Gh_D11G204600.1	D11	539	60.17	9.49	34.46	86.40	-0.19
Gh_A10G081300.1	A10	548	61.11	8.95	38.03	82.50	-0.21
Gh_D10G099800.1	D10	538	60.17	9.13	35.94	82.94	-0.21
Gh_A05G155700.1	A05	524	59.63	9.71	44.84	79.60	-0.26
Gh_D05G172400.1	D05	524	59.70	9.88	45.70	77.37	-0.31
Gh_A06G175400.1	A06	542	61.21	9.62	44.29	79.65	-0.26
Gh_D06G177900.1	D06	542	61.10	9.52	42.73	79.65	-0.25
Gh_D12G191300.1	D12	550	61.01	8.57	34.82	85.25	-0.17
Gh_A12G195200.1	A12	550	60.92	8.56	35.40	85.80	-0.14
Gh_D01G108800.1	D01	541	60.21	9.43	31.47	83.44	-0.25
Gh_A01G112100.1	A01	541	60.18	9.52	30.84	83.25	-0.25
Gh_A05G240100.1	A05	541	59.84	9.06	32.37	83.44	-0.25
Gh_D05G255900.1	D05	541	59.76	9.07	32.63	83.81	-0.25
Gh_A12G037000.1	A12	543	61.07	8.82	34.56	81.45	-0.24
Gh_D12G037400.1	D12	543	60.92	9.08	33.59	84.33	-0.23
Gh_D01G190400.1	D01	545	61.18	9.31	35.00	81.71	-0.29
Gh_A01G192200.1	A01	545	61.43	9.18	35.47	80.44	-0.29
Gh_A05G169300.1	A05	545	61.52	5.56	41.55	92.66	-0.09
Gh_D05G186200.1	D05	545	61.38	5.31	41.98	93.38	-0.07
Gh_A05G282100.1	A05	562	63.42	5.70	31.16	86.99	-0.18
Gh_D05G294500.1	D05	1 102	123.46	5.17	33.60	84.50	-0.20
Gh_A05G282000.1	A05	445	49.93	5.17	36.80	76.45	-0.31
Gh_A07G099100.1	A07	649	72.29	6.87	28.80	84.95	-0.16
Gh_D07G096200.1	D07	590	65.97	5.79	29.10	84.83	-0.15
Gh_A07G045500.1	A07	585	65.70	6.54	35.32	87.11	-0.17
Gh_D07G046500.1	D07	585	65.80	6.60	34.42	85.62	-0.18
Gh_D12G238000.1	D12	589	65.76	8.77	37.32	84.23	-0.24
Gh_A09G214200.1	A09	593	66.69	8.88	39.75	82.01	-0.27
Gh_D09G206100.1	D09	593	66.79	8.76	41.09	81.99	-0.28

序列ID Sequence ID	染色体 Chromosome	氨基酸数量/个 Number of amino acid	相对分子质量/ku Molecular weight	理论等电点 Theoretical pI	不稳定指数 Instability index	脂肪指数 Aliphatic index	总平均亲水性 Grand average of hydropathicity
Gh_D13G240200.1	D13	551	61.62	8.92	32.58	79.93	-0.29
Gh_D13G240400.1	D13	551	61.62	8.92	32.58	79.93	-0.29
Gh_D13G240000.1	D13	551	61.62	8.92	32.58	79.93	-0.29
Gh_D13G240100.1	D13	551	61.72	8.93	32.96	81.51	-0.26
Gh_A13G237100.1	A13	551	61.74	9.06	32.66	80.44	-0.30
Gh_A13G237000.1	A13	560	62.69	9.17	29.58	78.98	-0.32
Gh_A13G237300.1	A13	551	61.69	8.93	33.15	81.69	-0.28
Gh_D01G163800.1	D01	521	58.35	9.08	35.06	77.62	-0.31
Gh_D13G240300.1	D13	551	61.43	8.88	32.11	80.98	-0.22
Gh_A13G237500.1	A13	551	61.45	8.79	34.85	80.11	-0.27
Gh_A13G237200.1	A13	551	61.46	8.79	35.00	80.64	-0.26
Gh_A08G092100.1	A08	396	44.92	8.99	46.13	82.20	-0.29
Gh_A13G237600.1	A13	550	61.69	8.79	32.90	85.95	-0.24
Gh_A11G200600.1	A11	539	60.37	8.72	34.32	89.11	-0.14
Gh_D11G204500.1	D11	539	60.48	8.72	34.86	90.35	-0.11
Gh_D12G073800.1	D12	537	60.33	9.64	32.45	96.18	-0.10
Gh_A12G074300.1	A12	537	60.43	9.63	31.84	96.72	-0.11
Gh_A11G050000.1	A11	538	59.83	9.00	35.82	86.58	-0.16
Gh_A11G200700.1	A11	536	59.99	9.49	34.30	85.78	-0.21
Gh_D11G204600.1	D11	539	60.17	9.49	34.46	86.40	-0.19
Gh_A10G081300.1	A10	548	61.11	8.95	38.03	82.50	-0.21
Gh_D10G099800.1	D10	538	60.17	9.13	35.94	82.94	-0.21
Gh_A05G155700.1	A05	524	59.63	9.71	44.84	79.60	-0.26
Gh_D05G172400.1	D05	524	59.70	9.88	45.70	77.37	-0.31
Gh_A06G175400.1	A06	542	61.21	9.62	44.29	79.65	-0.26
Gh_D06G177900.1	D06	542	61.10	9.52	42.73	79.65	-0.25
Gh_D12G191300.1	D12	550	61.01	8.57	34.82	85.25	-0.17
Gh_A12G195200.1	A12	550	60.92	8.56	35.40	85.80	-0.14
Gh_D01G108800.1	D01	541	60.21	9.43	31.47	83.44	-0.25
Gh_A01G112100.1	A01	541	60.18	9.52	30.84	83.25	-0.25
Gh_A05G240100.1	A05	541	59.84	9.06	32.37	83.44	-0.25
Gh_D05G255900.1	D05	541	59.76	9.07	32.63	83.81	-0.25
Gh_A12G037000.1	A12	543	61.07	8.82	34.56	81.45	-0.24
Gh_D12G037400.1	D12	543	60.92	9.08	33.59	84.33	-0.23
Gh_D01G190400.1	D01	545	61.18	9.31	35.00	81.71	-0.29
Gh_A01G192200.1	A01	545	61.43	9.18	35.47	80.44	-0.29
Gh_A05G169300.1	A05	545	61.52	5.56	41.55	92.66	-0.09
Gh_D05G186200.1	D05	545	61.38	5.31	41.98	93.38	-0.07
Gh_A05G282100.1	A05	562	63.42	5.70	31.16	86.99	-0.18
Gh_D05G294500.1	D05	1 102	123.46	5.17	33.60	84.50	-0.20
Gh_A05G282000.1	A05	445	49.93	5.17	36.80	76.45	-0.31
Gh_A07G099100.1	A07	649	72.29	6.87	28.80	84.95	-0.16
Gh_D07G096200.1	D07	590	65.97	5.79	29.10	84.83	-0.15
Gh_A07G045500.1	A07	585	65.70	6.54	35.32	87.11	-0.17
Gh_D07G046500.1	D07	585	65.80	6.60	34.42	85.62	-0.18
Gh_D12G238000.1	D12	589	65.76	8.77	37.32	84.23	-0.24
Gh_A09G214200.1	A09	593	66.69	8.88	39.75	82.01	-0.27
Gh_D09G206100.1	D09	593	66.79	8.76	41.09	81.99	-0.28

基因复制类型 Types of gene duplications	全基因组复制 WGD	串联复制 Tandem duplications	其他复制 Other duplication
基因对/簇	Gh_A01G112100/Gh_D01G108800	Gh_A05G282000、Gh_A05G282100	Gh_A01G112100/Gh_A05G240100
Pairs/Clusters	Gh_A01G192200/Gh_D01G190400		Gh_A01G192200/Gh_A12G037000
of genes	Gh_A01G166300/Gh_D01G163800		Gh_A01G112100/Gh_D01G190400
	Gh_A05G155700/Gh_D05G172400		Gh_A01G112100/Gh_D05G255900
	Gh_A05G169300/Gh_D05G186200	Gh_A13G237000、Gh_A13G237100、	Gh_A01G192200/Gh_D12G037400
	Gh_A05G240100/Gh_D05G255900	Gh_A13G237200、Gh_A13G237300、	Gh_A05G155700/Gh_A06G175400
	Gh_A05G282000/Gh_D05G294500	Gh_A13G237500、Gh_A13G237600	Gh_A05G240100/Gh_D01G108800
	Gh_A06G175400/Gh_D06G177900		Gh_A06G175400/Gh_D05G172400
	Gh_A07G045500/Gh_D07G046500		Gh_A08G015300/Gh_A13G237000
	Gh_A07G099100/Gh_D07G096200		Gh_A09G214200/Gh_A12G243300
	Gh_A09G214200/Gh_D09G206100		Gh_A10G081300/Gh_A11G200700
	Gh_A10G081300/Gh_D10G099800		Gh_A10G081300/Gh_A11G050000
	Gh_A11G050000/Gh_A11G200700		Gh_A10G081300/Gh_D11G204600
	Gh_A11G200600/Gh_D11G204500	Gh_D13G240000、Gh_D13G240100、	Gh_A11G200600/Gh_A12G074300
	Gh_A11G050000/Gh_D11G204600	Gh_D13G240200、Gh_D13G240300、	Gh_A11G200700/Gh_D10G099800
	Gh_A12G195200/Gh_D12G191300	Gh_D13G240400
	Gh_A12G243300/Gh_D12G238000
	Gh_A12G074300/Gh_D12G073800
	Gh_A12G037000/Gh_D12G037400
	Gh_A13G237000/Gh_D13G240000

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陆地棉SKS基因家族全基因组特征分析与GhSKS13抗黄萎病功能解析

Genome-wide Characterization of SKS Gene Family in Gossypium hirsutum and Functional Analysis of GhSKS13 in Plant Resistance to Verticillium Wilt

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陆地棉SKS基因家族全基因组特征分析与GhSKS13抗黄萎病功能解析

Genome-wide Characterization of SKS Gene Family in Gossypium hirsutum and Functional Analysis of GhSKS13 in Plant Resistance to Verticillium Wilt

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