利用中华猕猴桃杂交后代转录组测序筛选抗溃疡病相关基因

doi:10.16420/j.issn.0513-353x.2023-0593

园艺学报 ›› 2024, Vol. 51 ›› Issue (8): 1743-1757.doi: 10.16420/j.issn.0513-353x.2023-0593

• 遗传育种·种质资源·分子生物学 • 上一篇下一篇

利用中华猕猴桃杂交后代转录组测序筛选抗溃疡病相关基因

匡美美¹^,², 李黎², 马建伟³, 刘原³, 蒋鸿霏³, 雷瑞², 满玉萍², 王一帆⁴, 黄波³, 王彦昌²^,^*(), 刘世彪¹^,^*()

¹ 吉首大学生物资源与环境科学学院，湖南吉首 416000
² 中国科学院武汉植物园，武汉 430074
³ 四川省苍溪猕猴桃研究所，四川苍溪 628400
⁴ 中国农业大学农学院，北京 100193

收稿日期:2024-01-12 修回日期:2024-05-20 出版日期:2024-08-25 发布日期:2024-08-21
通讯作者:
^*E-mail：kiwifruit@wbgcas.cn，
liushibiao_1@163.com
基金资助:
国家重点研发计划(2019YFD1000200); 中国科学院国际合作项目(151542KYSB20210004); 新疆维吾尔自治区区域协同创新专项（科技援疆）项目(2020E0224)

Screening of Kiwifruit Canker Resistance-Related Genes Based on the Transcriptome Sequencing Analysis of Hybrids from Actinidia chinensis

KUANG Meimei¹^,², LI Li², MA Jianwei³, LIU Yuan³, JIANG Hongfei³, LEI Rui², MAN Yuping², WANG Yifan⁴, HUANG Bo³, WANG Yanchang²^,^*(), LIU Shibiao¹^,^*()

¹ College of Biology and Environmental Sciences，Jishou University，Jishou，Hunan 416000，China
² Wuhan Botanical Garden，Chinese Academy of Sciences，Wuhan 430074，China
³ Sichuan Cangxi Kiwifruit Research Institute，Cangxi，Sichuan 628400，China
⁴ College of Agronomy and Biotechnology，China Agricultural University，Beijing 100193，China

Received:2024-01-12 Revised:2024-05-20 Published:2024-08-25 Online:2024-08-21

摘要/Abstract

摘要：

猕猴桃细菌性溃疡病是猕猴桃产业中的毁灭性病害。不同遗传背景的猕猴桃材料抗性差异极大。为研究猕猴桃响应溃疡病菌（Pseudomonas syringae pv. actinidiae，Psa）的早期反应，挖掘抗病相关基因，对接种溃疡病菌6和24 h后的中华猕猴桃‘金农’× 中华猕猴桃雄株杂交后代中的抗病个体‘21-2-4’及感病个体‘19-3-11’进行转录组测序比较分析。结果表明抗病个体对Psa防御主要集中在24 h，而感病个体主要集中在6 h。KEGG通路分析表明差异表达基因主要富集于植物—病原菌相互作用、苯丙烷生物合成、淀粉和蔗糖代谢途径等初生、次生产物的代谢。对比抗、感病个体中的基因表达模式，对植物—病原菌相互作用、植物激素信号转导及苯丙烷生物合成等抗病相关途径中可能的候选基因进行初步筛选。以中华猕猴桃抗性品种‘金农’和感病品种‘红昇’为材料，通过qRT-PCR验证了初步筛选出来的基因，确定PR1（Acc06864/Acc06865/Acc06866）、FLS2（Acc06484）可作为抗病候选基因深入研究。

关键词: 中华猕猴桃, 细菌性溃疡病, 杂交后代, 转录组, 抗病相关基因

Abstract:

Kiwifruit bacterial canker is a devastating disease in the kiwifruit industry，causing huge losses to the industry. Great differences in resistance are found in materials with different genetic background. To study the early response of kiwifruit to canker and to explore the genes related to resistance，transcriptome sequencing and comparative analysis was performed on the resistant‘21-2-4’and the susceptible‘19-3-11’in the offspring of Actinidia chinensis‘Jinnong’× male after 6 h and 24 h inoculation with Pseudomonas syringae pv. actinidiae（Psa）. The results showed that the defense of‘21-2-4’and‘19-3-11’against Psa mainly focused on 24 h and 6 h respectively. KEGG analysis showed that DEGs were mainly enriched in the primary and secondary metabolic pathways such as plant-pathogen interaction，phenylpropanoid biosynthesis，starch and sucrose metabolism. Gene expression patterns in resistant and susceptible materials were compared，and possible candidate genes in disease- esistant pathways such as plant-pathogen interaction，plant hormone signal transduction，and phenylpropane biosynthesis were screened. By using the resistant‘Jinnong’and susceptible‘Hongsheng’，the genes screened were verified by qRT-PCR，and then PR1（Acc06864/Acc06865/Acc06866）and FLS2（Acc06484）were selected as candidate genes for further study.

Key words: Actinidia chinensis, bacterial canker, progeny, transcriptome, resistance-related gene

匡美美, 李黎, 马建伟, 刘原, 蒋鸿霏, 雷瑞, 满玉萍, 王一帆, 黄波, 王彦昌, 刘世彪. 利用中华猕猴桃杂交后代转录组测序筛选抗溃疡病相关基因[J]. 园艺学报, 2024, 51(8): 1743-1757.

KUANG Meimei, LI Li, MA Jianwei, LIU Yuan, JIANG Hongfei, LEI Rui, MAN Yuping, WANG Yifan, HUANG Bo, WANG Yanchang, LIU Shibiao. Screening of Kiwifruit Canker Resistance-Related Genes Based on the Transcriptome Sequencing Analysis of Hybrids from Actinidia chinensis[J]. Acta Horticulturae Sinica, 2024, 51(8): 1743-1757.

图/表 9

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基因编号 Gene ID	引物序列 Primer sequence	产物大小/bp Product length	退火温度/℃ T_m
Actin	F：5′-TGAGAGATTCCGTTGCCCAGAAGT-3′/ R：5′-TTCCTTACTCATGCGGTCTGCGAT-3′	193	57.4/57.4
Acc23292	F：5′-GGGAGCTGTCATTCGAGGAA-3′/ R：5′-GATAATCGCCCAGCCTTTCG-3′	140	53.8/55.0
Acc14142	F：5′-TCAATCCACCATGCCAGAGT-3′/ R：5′-CAGCTGCCTTCGTGTTCTTT-3′	181	51.8/51.8
Acc28207	F：5′-ATGGACCATCTACTTTGCTT-3′/ R：5′-GTGCTGAGTGCAATCTGCGG-3′	128	51.4/55.3
Acc04756	F：5′-ATGTCGACGAGGAAGGATGT-3′/ R：5′-CGGATCGATCTTGCTAATGC-3′	126	56.5/58.3
Acc26787	F：5′-CGTATGCTCTCCGAGTCACC-3′/ R：5′-ACTTGGCCGGAGAATTGGTT-3′	221	57.8/60.9
Acc04136	F：5′-GACGAGTCGTGGGTTGTAGG-3′/ R：5′-TTTGGTCACTCCAGCTTCCG-3′	159	57.6/60.9
Acc06864	F：5′-CTAGCCACGGTTCACTCCTC-3′/ R：5′-ATGTTCTCGCCGTACTTCCC-3′	203	55.9/53.8
Acc06865	F：5′-AACACCGTTCGTGACCAAGT-3′/ R：5′-TGGTTAGCGTAGTTCTCGGC-3′	83	51.8/53.8
Acc06866	F：5′-AACCAGAGGTCTGGCGATTG-3′/ R：5′-AACCTTACCTGAGGCACACG-3′	171	53.8/53.8
Acc06484	F：5′-ATCACTCAGCCTGCCTCAAC-3′/ R：5′-CTTGGTCACGCAATACGCAG-3′	251	53.8/53.8

基因编号 Gene ID	引物序列 Primer sequence	产物大小/bp Product length	退火温度/℃ T_m
Actin	F：5′-TGAGAGATTCCGTTGCCCAGAAGT-3′/ R：5′-TTCCTTACTCATGCGGTCTGCGAT-3′	193	57.4/57.4
Acc23292	F：5′-GGGAGCTGTCATTCGAGGAA-3′/ R：5′-GATAATCGCCCAGCCTTTCG-3′	140	53.8/55.0
Acc14142	F：5′-TCAATCCACCATGCCAGAGT-3′/ R：5′-CAGCTGCCTTCGTGTTCTTT-3′	181	51.8/51.8
Acc28207	F：5′-ATGGACCATCTACTTTGCTT-3′/ R：5′-GTGCTGAGTGCAATCTGCGG-3′	128	51.4/55.3
Acc04756	F：5′-ATGTCGACGAGGAAGGATGT-3′/ R：5′-CGGATCGATCTTGCTAATGC-3′	126	56.5/58.3
Acc26787	F：5′-CGTATGCTCTCCGAGTCACC-3′/ R：5′-ACTTGGCCGGAGAATTGGTT-3′	221	57.8/60.9
Acc04136	F：5′-GACGAGTCGTGGGTTGTAGG-3′/ R：5′-TTTGGTCACTCCAGCTTCCG-3′	159	57.6/60.9
Acc06864	F：5′-CTAGCCACGGTTCACTCCTC-3′/ R：5′-ATGTTCTCGCCGTACTTCCC-3′	203	55.9/53.8
Acc06865	F：5′-AACACCGTTCGTGACCAAGT-3′/ R：5′-TGGTTAGCGTAGTTCTCGGC-3′	83	51.8/53.8
Acc06866	F：5′-AACCAGAGGTCTGGCGATTG-3′/ R：5′-AACCTTACCTGAGGCACACG-3′	171	53.8/53.8
Acc06484	F：5′-ATCACTCAGCCTGCCTCAAC-3′/ R：5′-CTTGGTCACGCAATACGCAG-3′	251	53.8/53.8

基因编号 Gene ID	表达量 FPKM（‘21-2-4’）			KO	途径 Pathway	NR注释 NR_annotation
基因编号 Gene ID	0 h	6 h	24 h	KO	途径 Pathway	NR注释 NR_annotation
Acc06866	33.27 b	305.75 b	2 308.34 a	PR1	PTI、SA	病程相关蛋白Pathogenesis-related protein
Acc06865	64.83 b	537.47 b	2 678.93 a	PR1	PTI、SA	病程相关蛋白Pathogenesis-related protein
Acc06864	367.59 b	2 171.17 b	10 865.11 a	PR1	PTI、SA	病程相关蛋白Pathogenesis-related protein
Acc06484	14.20 b	111.05 a	25.56 b	FLS2	PTI	LRR受体样激酶LRR receptor-like serine/threonine-protein kinase
Acc25328	2.92 b	15.19 a	7.70 ab	EIX1_2	PTI	受体样蛋白Receptor-like protein
novel.6186	2.69 b	12.26 a	6.08 ab	EIX1_2	PTI	受体样蛋白Receptor-like protein
novel.36390	2.59 b	11.14 a	6.99 ab	EIX1_2	PTI	受体样蛋白Receptor-like protein
Acc25330	3.16 b	12.70 a	7.51 ab	EIX1_2	PTI	受体样蛋白Receptor-like protein
Acc23527	2.24 b	7.74 a	10.43 ab	CPK	PTI	钙依赖蛋白激酶Calcium-dependent protein kinase
Acc18178	11.18 b	30.83 a	39.39 a	EDS1	ETI	EDS1样蛋白 Protein EDS1B like
Acc07305	28.87 b	86.32 a	89.80 a	CTSF	ETI	半胱氨酸蛋白酶Cysteine protease
Acc11129	78.04 b	159.43 a	305.97 a	CTSF	ETI	半胱氨酸蛋白酶Cysteine protease
Acc02718	10.53 b	32.00 a	31.63 a	RAR1	ETI	富含半胱氨酸和组氨酸结构域蛋白 Cysteine and histidine-rich domain-containing protein
Acc21162	321.06 ab	193.38 b	692.83 a	PR1	SA	病程相关蛋白基本形式Basic form of pathogenesis-related protein like
Acc29710	2.74 b	5.29 ab	11.16 a	4CL	PAL	类4香豆酸辅酶 A 4-coumarate-CoA ligase-like
Acc01331	7.41 b	23.73 a	12.13 ab	4CL	PAL	4香豆酸辅酶 A 4-coumarate-CoA ligase
Acc16561	160.02 b	284.77 ab	449.28 a	POD	PAL	过氧化物酶Peroxidase
Acc20997	1.91 b	1.70 b	10.94 a	POD	PAL	过氧化物酶Peroxidase
Acc00538	5.41 b	28.43 a	37.94 a	POD	PAL	过氧化物酶Peroxidase
Acc32503	362.30 b	390.84 b	777.05 a	POD	PAL	过氧化物酶超家族蛋白Peroxidase superfamily protein
Acc32502	219.54 b	247.40 ab	544.78 a	POD	PAL	过氧化物酶超家族阴性蛋白Peroxidase superfamily negative protein
Acc30435	47.41 b	60.30 b	623.97 a	POD	PAL	木质素阴性过氧化物酶Lignin-forming anionic peroxidase

基因编号 Gene ID	表达量 FPKM（‘21-2-4’）			KO	途径 Pathway	NR注释 NR_annotation
基因编号 Gene ID	0 h	6 h	24 h	KO	途径 Pathway	NR注释 NR_annotation
Acc06866	33.27 b	305.75 b	2 308.34 a	PR1	PTI、SA	病程相关蛋白Pathogenesis-related protein
Acc06865	64.83 b	537.47 b	2 678.93 a	PR1	PTI、SA	病程相关蛋白Pathogenesis-related protein
Acc06864	367.59 b	2 171.17 b	10 865.11 a	PR1	PTI、SA	病程相关蛋白Pathogenesis-related protein
Acc06484	14.20 b	111.05 a	25.56 b	FLS2	PTI	LRR受体样激酶LRR receptor-like serine/threonine-protein kinase
Acc25328	2.92 b	15.19 a	7.70 ab	EIX1_2	PTI	受体样蛋白Receptor-like protein
novel.6186	2.69 b	12.26 a	6.08 ab	EIX1_2	PTI	受体样蛋白Receptor-like protein
novel.36390	2.59 b	11.14 a	6.99 ab	EIX1_2	PTI	受体样蛋白Receptor-like protein
Acc25330	3.16 b	12.70 a	7.51 ab	EIX1_2	PTI	受体样蛋白Receptor-like protein
Acc23527	2.24 b	7.74 a	10.43 ab	CPK	PTI	钙依赖蛋白激酶Calcium-dependent protein kinase
Acc18178	11.18 b	30.83 a	39.39 a	EDS1	ETI	EDS1样蛋白 Protein EDS1B like
Acc07305	28.87 b	86.32 a	89.80 a	CTSF	ETI	半胱氨酸蛋白酶Cysteine protease
Acc11129	78.04 b	159.43 a	305.97 a	CTSF	ETI	半胱氨酸蛋白酶Cysteine protease
Acc02718	10.53 b	32.00 a	31.63 a	RAR1	ETI	富含半胱氨酸和组氨酸结构域蛋白 Cysteine and histidine-rich domain-containing protein
Acc21162	321.06 ab	193.38 b	692.83 a	PR1	SA	病程相关蛋白基本形式Basic form of pathogenesis-related protein like
Acc29710	2.74 b	5.29 ab	11.16 a	4CL	PAL	类4香豆酸辅酶 A 4-coumarate-CoA ligase-like
Acc01331	7.41 b	23.73 a	12.13 ab	4CL	PAL	4香豆酸辅酶 A 4-coumarate-CoA ligase
Acc16561	160.02 b	284.77 ab	449.28 a	POD	PAL	过氧化物酶Peroxidase
Acc20997	1.91 b	1.70 b	10.94 a	POD	PAL	过氧化物酶Peroxidase
Acc00538	5.41 b	28.43 a	37.94 a	POD	PAL	过氧化物酶Peroxidase
Acc32503	362.30 b	390.84 b	777.05 a	POD	PAL	过氧化物酶超家族蛋白Peroxidase superfamily protein
Acc32502	219.54 b	247.40 ab	544.78 a	POD	PAL	过氧化物酶超家族阴性蛋白Peroxidase superfamily negative protein
Acc30435	47.41 b	60.30 b	623.97 a	POD	PAL	木质素阴性过氧化物酶Lignin-forming anionic peroxidase

利用中华猕猴桃杂交后代转录组测序筛选抗溃疡病相关基因

Screening of Kiwifruit Canker Resistance-Related Genes Based on the Transcriptome Sequencing Analysis of Hybrids from Actinidia chinensis

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利用中华猕猴桃杂交后代转录组测序筛选抗溃疡病相关基因

Screening of Kiwifruit Canker Resistance-Related Genes Based on the Transcriptome Sequencing Analysis of Hybrids from Actinidia chinensis

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摘要/Abstract

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图/表 9

参考文献 42

相关文章 15

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