甘薯蔓割病(Fusarium oxysporum f. sp. batatas)相关基因IbMAPKK9的克隆与特性分析

doi:10.3724/SP.J.1006.2023.24284

作物学报 ›› 2023, Vol. 49 ›› Issue (12): 3289-3301.doi: 10.3724/SP.J.1006.2023.24284

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

甘薯蔓割病(Fusarium oxysporum f. sp. batatas)相关基因IbMAPKK9的克隆与特性分析

靖小菁¹^,²(), 杨新笋², 靳晓杰², 刘意³, 雷剑², 王连军², 柴沙沙², 张文英¹^,^*(), 焦春海²^,^*()

¹长江大学农学院, 湖北荆州 434025
²湖北省农业科学院粮食作物研究所, 湖北武汉 430064
³海南大学园艺学院 / 海南省热带园艺作物品质调控重点实验室, 海南海口 570228

收稿日期:2022-12-23 接受日期:2023-05-24 出版日期:2023-12-12 网络出版日期:2023-06-04
通讯作者: * 张文英, E-mail: wyzhang@yangtzeu.edu.cn; 焦春海, E-mail: jiaoch@hotmail.com
作者简介:E-mail: 15172397885@163.com
基金资助:
国家重点研发计划项目(2019YFD1001300);国家重点研发计划项目(2019YFD1001305);财政部和农业农村部国家现代农业产业技术体系建设专项(Sweetpotato, CARS-10)

Cloning and characterization of IbMAPKK9 gene associated with Fusarium oxysporum f. sp. batatas in sweet potato

JING Xiao-Jing¹^,²(), YANG Xin-Sun², JIN Xiao-Jie², LIU Yi³, LEI Jian², WANG Lian-Jun², CHAI Sha-Sha², ZHANG Wen-Ying¹^,^*(), JIAO Chun-Hai²^,^*()

¹College of Agriculture, Yangtze University, Jingzhou 434025, Hubei, China
²Food Crops institute, Hubei Academy of Agricultural Sciences, Wuhan 430064, Hubei, China
³Horticulture College, Hainan University / Key Laboratory for Quality Regulation of Tropical Horticultural Plants of Hainan Province, Haikou 570228, Hainan, China

Received:2022-12-23 Accepted:2023-05-24 Published:2023-12-12 Published online:2023-06-04
Contact: * E-mail: wyzhang@yangtzeu.edu.cn; E-mail: jiaoch@hotmail.com
Supported by:
National Key Research and Development Program of China(2019YFD1001300);National Key Research and Development Program of China(2019YFD1001305);China Agriculture Research System of MOF and MARA(Sweetpotato, CARS-10)

摘要/Abstract

摘要：

促丝裂原活化蛋白激酶(MAPK)级联是生物体内一种重要的信号转导途径, 广泛存在于植物中。MAPKKs位于该级联反应通路中间, 对信号传递起到关键作用, 目前在甘薯中少有报道。基于前期的转录组学分析, 本研究克隆出1个与甘薯抗病相关的基因IbMAPKK9。生物信息学分析表明, IbMAPKK9包含一个987 bp开放阅读框(open reading frame, ORF), 编码328个氨基酸, 具有1个蛋白激酶家族保守结构域(PF00069), IbMAPKK9蛋白以α螺旋和无规则卷曲为主, 启动子区包含多种激素(茉莉酸甲酯、乙烯、脱落酸、赤霉素、水杨酸)相关及胁迫响应元件。进化分析表明, IbMAPKK9蛋白与三叶裂薯、日本牵牛花、番茄和马铃薯亲缘关系较近。亚细胞定位显示IbMAPKK9蛋白定位于细胞核。实时荧光定量PCR发现IbMAPKK9在甘薯根、茎、叶和叶柄中均表达, 并响应甘薯蔓割病侵染。瞬时表达分析结果表明, IbMAPKK9引起5个与水杨酸合成途径及信号转导途径相关的基因在48 h内表达上调, 推测IbMAPKK9通过介导水杨酸信号途径影响植物的抗性。本研究可为进一步解析甘薯IbMAPKK9的生物学功能提供理论依据。

关键词: 甘薯, IbMAPKK9, 蔓割病, 基因克隆, 亚细胞定位, 瞬时表达

Abstract:

Mitogen-activated protein kinase (MAPK) cascades are important signaling modules in all organisms, which widely exist in plants. Mitogen-activated protein kinase kinase (MAPKKs) are located in the middle of the cascade reaction pathway and play the key roles in signal divergence. The disease resistance gene IbMAPKK9 was screened and cloned based on transcriptome data in sweetpotato, which contained a 987 bp open reading frame, encoding 328 amino acids and 1 domain (PF00069). Its protein mainly consisted of α-helixes and random coils. Multiple hormones (methyl jasmonate, ethylene, abscisic acid, gibberellin, and salicylic acid) and stress-related response elements were detected in the promoter region. Homologous protein comparison showed that IbMAPKK9 was closely related to ItMAPKK9-like (XP_031110493.1), InMAPKK9-like (XP_019189439.1), SlMAPKK9 (NP_001234595.1), and StMAPKK9 (XP_006363984.1). Besides, IbMAPKK9 gene encoded nuclear localization protein. The relative expression levels revealed that IbMAPKK9 was expressed in roots, stems, leaves, and petioles in response to Fusarium oxysporum f. sp batatas (Fob) infection. Transient expression analysis showed that IbMAPKK9 induced up-regulation of 5 genes related to salicylic acid synthesis pathway and signal transduction pathway within 48 hours, suggesting that IbMAPKK9 affected plant resistance by mediating salicylic acid signal pathway. This study can provide a theoretical basis for further analysis of the biological function of IbMAPKK9.

Key words: sweet potato, IbMAPKK9, Fusarium wilt, gene clone, subcellular localization, transient expression

靖小菁, 杨新笋, 靳晓杰, 刘意, 雷剑, 王连军, 柴沙沙, 张文英, 焦春海. 甘薯蔓割病(Fusarium oxysporum f. sp. batatas)相关基因IbMAPKK9的克隆与特性分析[J]. 作物学报, 2023, 49(12): 3289-3301.

JING Xiao-Jing, YANG Xin-Sun, JIN Xiao-Jie, LIU Yi, LEI Jian, WANG Lian-Jun, CHAI Sha-Sha, ZHANG Wen-Ying, JIAO Chun-Hai. Cloning and characterization of IbMAPKK9 gene associated with Fusarium oxysporum f. sp. batatas in sweet potato[J]. Acta Agronomica Sinica, 2023, 49(12): 3289-3301.

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引物名称 Primer name	序列 Primer sequence (5′-3′)
IbMAPKK9-CDS-F	ATGGCACTTGTTCGTGAACGT
IbMAPKK9-CDS-R	TTAATTTTGTGAATTAATTTGGGATTTG
IbMAPKK9-PRO-F	CTGCCAATGTGTATCACATGTTAT
IbMAPKK9-PRO-R	GATGAGAAGGTAGAAGGGGAGT
IbMAPKK9-qPCR-F	GCGGTCTACGCCCTCAAAG
IbMAPKK9-qPCR-R	GAATATCGGAGGTGCCGTGAC
β-Actin-F	AGCAGCATGAAGATTAAGGTTGTAGCAC
β-Actin-R	TGGAAAATTAGAAGCACTTCCTGTGAAC
NtPAL4-F	CATTGCCACATTCAGCAACA
NtPAL4-R	GGGTGGTGCTTCAACTTGTG
NtICS1-F	GAGGGTGCGGAAAAAAATA
NtICS1-R	CCTTGATAAGCATCGGGTT
NtNPR1-F	TTCTGTATCTCTTGCTATG
NtNPR1-R	ATCTACTGTTGTCCTCTGT
NtNPR3-F	GGGGCTACTTACCTA
NtNPR3-R	TCCCTGTCTGATTCA
NtNPR5-F	AAGGATGGATGGGAT
NtNPR5-R	AGTTGGGTTGTTTGC

位点名称 Site name	序列 Sequence (5′-3′)	功能 Function
ABRE	ACGTG	脱落酸反应的顺式作用元件 Cis-acting element involved in abscisic acid response
ARE	AAACCA	厌氧诱导反应的顺式作用调节元件 Cis-acting regulatory element essential for the anaerobic induction
ERE	ATTTTAAA	乙烯响应元件 Ethylene responsive element
G-box	CACGTG	参与光反应的顺式作用元件 Light responsive element
P-box	CCTTTTG	赤霉素反应元件 Gibberellin-responsive element
MYB	TAACCA	干旱诱导相关元件 MYB binding site involved in drought-inducibility
MYC	CATGTG	逆境胁迫相关顺式调控元件 Stress related cis-regulatory elements
G-box	CACGTG	茉莉酸甲酯应答元件 Cis-acting regulatory element involved in the MeJA-responsiveness

名称 Protein name	编号 Protein ID	注释 Protein annotation	蛋白长度 Protein length (aa)	置信度 Confidence
MPK3.0	A0A1J6KJD9	丝裂原活化蛋白激酶MAPK3 Mitogen-activated protein kinase 3	471	0.999
MPK3.1	A0A1J6IKA5	丝裂原活化蛋白激酶MAPK3 Mitogen-activated protein kinase 3	392	0.999
MPK3.2	A5H2L1	丝裂原活化蛋白激酶(Ser/Thr激酶家族) Mitogen activated protein kinases (Ser/Thr protein kinase family)	375	0.999
NTF4.0	A0A314KXT0	丝裂原活化蛋白激酶(Ser/Thr激酶家族) Mitogen activated protein kinases(Ser/Thr protein kinase family)	394	0.988
NTF4.1	A5H2L0	丝裂原活化蛋白激酶(Ser/Thr激酶家族) Mitogen activated protein kinases (Ser/Thr protein kinase family)	393	0.993
MEKK1.0	A0A314L1U5	丝裂原活化蛋白激酶激酶激酶MAPKKK1 Mitogen-activated protein kinase kinase kinase 1	637	0.980
MEKK1.1	A0A1J6IHU2	丝裂原活化蛋白激酶激酶激酶MAPKKK1 Mitogen-activated protein kinase kinase kinase 1	561	0.980
NPK1.6	A0A314L7I4	丝裂原活化蛋白激酶激酶 Mitogen-activated protein kinase kinase	668	0.938
CTR1.1	A0A314KLJ0	丝氨酸蛋白激酶 Serine kinase	161	0.936
CTR1.8	A0A1J6IUT7	丝氨酸蛋白激酶 Serine kinase	827	0.936

网站 Software	细胞质 Chloroplast	细胞核 Nuclear	线粒体 Mitochondrion	细胞骨架 Cytoskelrtal
WoLF PSORT	—	9	1	3
PSORT II	4.3%	69.6%	21.7%	4.3%

甘薯蔓割病(Fusarium oxysporum f. sp. batatas)相关基因IbMAPKK9的克隆与特性分析

Cloning and characterization of IbMAPKK9 gene associated with Fusarium oxysporum f. sp. batatas in sweet potato

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