谷子抗锈病反应相关MAPKK家族成员的鉴定与分析

doi:10.7668/hbnxb.20194834

华北农学报 ›› 2024, Vol. 39 ›› Issue (5): 193-203. doi: 10.7668/hbnxb.20194834

所属专题： 杂粮作物；植物保护；

• 资源环境·植物保护 • 上一篇下一篇

谷子抗锈病反应相关MAPKK家族成员的鉴定与分析

龚珂珂¹^,², 张梦雅², 李志勇², 刘佳², 马继芳², 董志平², 贾小平¹, 白辉²

¹ 河南科技大学农学院,河南洛阳 471023
² 河北省农林科学院谷子研究所,农业农村部特色杂粮遗传改良与利用重点实验室(部省共建),河北省杂粮研究重点实验室,河北石家庄 050035

收稿日期:2024-04-16 出版日期:2024-10-28
通讯作者:
贾小平(1973-),男,内蒙古包头人,教授,博士,主要从事谷子资源评价与分子育种研究。
贾小平(1973-),男,内蒙古包头人,教授,博士,主要从事谷子资源评价与分子育种研究。
作者简介:
龚珂珂(1996-),女,河南鲁山人,在读硕士,主要从事谷子抗病分子生物学研究。
龚珂珂、张梦雅为同等贡献作者。
基金资助:
河北省农林科学院基本科研业务费试点经费包干制项目(HBNKY-BGZ-02); 河北省自然科学基金(C2024301092); 国家自然科学基金(31872880); 国家现代农业产业技术体系专项(CARS-06-14.5-A25)

Identification and Analysis of MAPKK Family Members Related to Rust Resistance in Foxtail Millet

GONG Keke¹^,², ZHANG Mengya², LI Zhiyong², LIU Jia², MA Jifang², DONG Zhiping², JIA Xiaoping¹, BAI Hui²

¹ College of Agriculture,Henan University of Science and Technology,Luoyang 471023,China
² Instituteof Millet Crops,Hebei Academy of Agriculture and Forestry Sciences,the Key Research Laboratory of Genetic Improvement and Utilization for Featured Coarse Cereals (Co-construction by Ministry and Province),Ministry of Agriculture and Rural Affairs,Key Laboratory of Minor Cereal Crops of Hebei Province,Shijiazhuang 050035,China

Received:2024-04-16 Published:2024-10-28

摘要/Abstract

摘要：

丝裂原活化蛋白激酶(MAPKK或MKK)在植物的生长发育与胁迫响应等过程中发挥着重要作用。为了鉴定谷子抗锈病相关的MAPKK基因,为谷子抗锈病机理研究和抗病分子育种提供候选基因,运用生物信息学方法在全基因组水平上鉴定与分析谷子MAPKK基因家族成员(SiMKKs);利用Real-time PCR技术,检测谷子SiMKKs基因在不同组织部位、谷锈菌胁迫与外源激素处理下的表达水平;利用Excel、MEGA、DnaSP分析70份重测序谷子品种中抗锈病相关SiMKKs基因的变异位点和单倍型,结合表型鉴定抗锈病优异单倍型。结果表明,共鉴定到10个谷子SiMKKs成员,分布于5条染色体上,外显子数1~11个不等,编码蛋白质含有331~523个氨基酸;谷子MAPKK基因家族成员分为4组,A和B组包含S/T-X₅-S/T基序,C和D组的SiMKKs不具有该基序,保守基序Motif 1~Motif 6 存在于所有SiMKKs蛋白中;每个SiMKK基因的启动子区均含有防御和胁迫响应、茉莉酸甲酯(MeJA)响应、水杨酸(SA)响应、激发子激活等1~3种生物胁迫相关顺式作用元件。除SiMKK10-1和SiMKK10-3未检测到表达信号外,其余8个SiMKKs基因在不同组织部位、谷锈菌侵染、SA和MeJA处理下均有不同程度的表达。SiMKK4、SiMKK5和SiMKK10-2在孕穗期的根中表达量较高,SiMKK6-1和SiMKK6-2在孕穗期茎中表达量较高;SiMKK4在接种后24 h的抗病反应中上调表达、感病反应中下调表达,其表达与抗病相关;SiMKK4在SA和MeJA激素处理后的16 h内上调表达,之后下调表达,且在SA处理过程中表现持续的表达变化,其余7个SiMKKs基因在SA和MeJA处理中的表达模式也基本一致。SiMKK4基因编码区共分为7个单倍型,Hap_1为优势单倍型,未发现抗病关键变异位点。综上所述,谷子SiMKK4的表达与抗锈病相关,其可能通过SA和MeJA信号途径参与谷子的早期抗病反应。

关键词: 谷子, MAPKK, 锈病, Real-time PCR, 单倍型分析

Abstract:

Mitogen-activated protein kinase kinase (MAPKK or MKK) plays an important role in plant growth,development and stress responses.In order to identify MAPKK genes related to rust resistance in foxtail millet and provide candidate genes for the study of rust resistance mechanism and disease-resistant molecular breeding of foxtail millet,the members of MAPKK gene family (SiMKKs) in foxtail millet were identified and analyzed at the whole genome level by bioinformatics methods.Real-time PCR was used to detect the expression level of SiMKKs gene in different tissues,under the stress of rust fungus and exogenous hormone treatment.Excel,MEGA and DnaSP were used to analyze the variation sites and haplotypes of the SiMKKs gene related to rust resistance in 70 re-sequenced foxtail millet varieties,and the excellent rust-resistant haplotypes were identified based on phenotype analysis.The results showed that a total of 10 SiMKKs were identified in foxtail millet,which were distributed on 5 chromosomes.The number of exons ranged from 1 to 11,and the encoded protein contained 331-523 amino acids.The SiMKKs were divided into 4 groups.Groups A and B contained S/T-X₅-S/T motif,while SiMKKs in groups C and D did not have this motif.Conserved Motif 1-Motif 6 existed in all SiMKK proteins.The promoter region of each SiMKK gene contained 1 to 3 biotic stress-related cis-acting elements,such as defense and stress response,methyl jasmonate(MeJA) response,salicylic acid(SA) response and elicitor activation.Except SiMKK10-1 and SiMKK10-3,the other 8 SiMKK genes were expressed with different degrees in different tissues,and under rust infection,SA and MeJA treatments.The highest expression of SiMKK4,SiMKK5 and SiMKK10-2 were in roots at booting stage,and the highest expression of SiMKK6-1 and SiMKK6-2 were in stems at booting stage.The expression of SiMKK4 was up-regulated in the resistant response and down-regulated in the susceptible response within 24 h after inoculation,and its expression was related to disease resistance.The expression of SiMKK4 was up-regulated within 16 h and then down-regulatedafter SA and MeJA treatments,and showed continuous changes during SA treatment.In addition,the expression patterns of the remaining 7 SiMKK genes in SA and MeJA treatments were also consistent.The coding region of SiMKK4 gene contained 7 haplotypes and Hap_1 was the dominant haplotype,and no key variation sites related to disease resistance were found.In summary,the expression of SiMKK4 is identified to be associated with resistance to rust disease in foxtail millet,and SiMKK4 may participate in the early disease resistance response of foxtail millet through SA and MeJA signaling pathways.

Key words: Foxtail millet, MAPKK, Rust disease, Real-time PCR, Haplotype analysis

龚珂珂, 张梦雅, 李志勇, 刘佳, 马继芳, 董志平, 贾小平, 白辉. 谷子抗锈病反应相关MAPKK家族成员的鉴定与分析[J]. 华北农学报, 2024, 39(5): 193-203. doi: 10.7668/hbnxb.20194834.

GONG Keke, ZHANG Mengya, LI Zhiyong, LIU Jia, MA Jifang, DONG Zhiping, JIA Xiaoping, BAI Hui. Identification and Analysis of MAPKK Family Members Related to Rust Resistance in Foxtail Millet[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(5): 193-203. doi: 10.7668/hbnxb.20194834.

http://www.hbnxb.net/CN/Y2024/V39/I5/193

图/表 13

参考文献 41

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引物 Primer	正向序列 (5'-3') Forward sequence (5'-3')	反向序列 (5'-3') Reverse sequence (5'-3')
SiActin	CGCATATGTGGCTCTTGACT	GGGCACCTAAATCTCTCTGC
SiMKK1	ATCAAAGTGATCGGGAAAGG	TTCAACTCCTTGGCAATCTG
SiMKK3-1	AATCTTAAGGGTGAGGCGAA	TGACCGTGCCTACAAAGGTA
SiMKK3-2	GGCATACTGTTGTGCCTCTC	GTTGCCTCTTCTCCTTCTCG
SiMKK4	CTCCAACCTCCTGATCGACT	GTCCATGGTCTGGTTGAGG
SiMKK5	GACCATCGCCTACATGAGC	GGGAACTTGCCAAGGTAGAA
SiMKK6-1	GCACCTGCAGATCAGTTCTC	GGTCCAAATCCTTACCCTCA
SiMKK6-2	GCTGAAAGTGGAAGATGTGC	CCCTTTAAGGCATACAATGTCC
SiMKK10-1	TGCACCTCGACATCAAGC	GGGAGGACTCTTGCGATG
SiMKK10-2	GACCTGAAGCCGTCGAAC	AAGTCGGCGATCTTGACCT
SiMKK10-3	TGATGTGCGCCATATGCT	CTCGCTTCGTGTAGTCCTTG

引物 Primer	正向序列 (5'-3') Forward sequence (5'-3')	反向序列 (5'-3') Reverse sequence (5'-3')
SiActin	CGCATATGTGGCTCTTGACT	GGGCACCTAAATCTCTCTGC
SiMKK1	ATCAAAGTGATCGGGAAAGG	TTCAACTCCTTGGCAATCTG
SiMKK3-1	AATCTTAAGGGTGAGGCGAA	TGACCGTGCCTACAAAGGTA
SiMKK3-2	GGCATACTGTTGTGCCTCTC	GTTGCCTCTTCTCCTTCTCG
SiMKK4	CTCCAACCTCCTGATCGACT	GTCCATGGTCTGGTTGAGG
SiMKK5	GACCATCGCCTACATGAGC	GGGAACTTGCCAAGGTAGAA
SiMKK6-1	GCACCTGCAGATCAGTTCTC	GGTCCAAATCCTTACCCTCA
SiMKK6-2	GCTGAAAGTGGAAGATGTGC	CCCTTTAAGGCATACAATGTCC
SiMKK10-1	TGCACCTCGACATCAAGC	GGGAGGACTCTTGCGATG
SiMKK10-2	GACCTGAAGCCGTCGAAC	AAGTCGGCGATCTTGACCT
SiMKK10-3	TGATGTGCGCCATATGCT	CTCGCTTCGTGTAGTCCTTG

基因名称 Gene name	基因编号 Gene ID	氨基酸数目 Number of animo acids	分子质量/ku Molecular weight	等电点 Isoelectric point	位置/bp Location	功能域(起-止) Functional domain (Start-end)
SiMKK1	Seita.4G036300	350	39.07	6.36	sca_4:2443690-2447291	Pkinase(69-321)
SiMKK3-1	Seita.1G307500	523	58.52	5.80	sca_1:37073712-37083640	Pkinase(89-343)
SiMKK3-2	Seita.1G307400	523	58.42	5.72	sca_1:37054092-37064828	Pkinase(89-343)
SiMKK4	Seita.1G345400	366	39.56	9.45	sca_1:40030826-40031926	Pkinase(94-350)
SiMKK5	Seita.4G043500	453	49.48	10.02	sca_4:3187777-3196841	Pkinase(183-439)
SiMKK6-1	Seita.3G062300	355	39.96	5.57	sca_3:3943035-3947324	Pkinase(73-330)
SiMKK6-2	Seita.5G301500	355	39.88	5.47	sca_5:35560125-35564666	Pkinase(73-330)
SiMKK10-1	Seita.9G117700	332	35.19	8.76	sca_9:7294995-7295993	Pkinase(59-313)
SiMKK10-2	Seita.9G484700	331	35.18	6.08	sca_9:52473555-52475254	Pkinase(44-307)
SiMKK10-3	Seita.9G117500	334	35.08	8.43	sca_9:7285595-7286599	Pkinase(52-315)

基因名称 Gene name	基因编号 Gene ID	氨基酸数目 Number of animo acids	分子质量/ku Molecular weight	等电点 Isoelectric point	位置/bp Location	功能域(起-止) Functional domain (Start-end)
SiMKK1	Seita.4G036300	350	39.07	6.36	sca_4:2443690-2447291	Pkinase(69-321)
SiMKK3-1	Seita.1G307500	523	58.52	5.80	sca_1:37073712-37083640	Pkinase(89-343)
SiMKK3-2	Seita.1G307400	523	58.42	5.72	sca_1:37054092-37064828	Pkinase(89-343)
SiMKK4	Seita.1G345400	366	39.56	9.45	sca_1:40030826-40031926	Pkinase(94-350)
SiMKK5	Seita.4G043500	453	49.48	10.02	sca_4:3187777-3196841	Pkinase(183-439)
SiMKK6-1	Seita.3G062300	355	39.96	5.57	sca_3:3943035-3947324	Pkinase(73-330)
SiMKK6-2	Seita.5G301500	355	39.88	5.47	sca_5:35560125-35564666	Pkinase(73-330)
SiMKK10-1	Seita.9G117700	332	35.19	8.76	sca_9:7294995-7295993	Pkinase(59-313)
SiMKK10-2	Seita.9G484700	331	35.18	6.08	sca_9:52473555-52475254	Pkinase(44-307)
SiMKK10-3	Seita.9G117500	334	35.08	8.43	sca_9:7285595-7286599	Pkinase(52-315)

基序 Motif	序列 Sequence
1	HRDIKPSNLLVNAKGEVKIADFGVSAVLSRTMDPCATFVGTAAYMSPERI
2	PSAPPDAASPEFCSFISACLQKDPAKRPSAAZLLNHPFVAG
3	DPYAADIWSLGLTILECATGRFPYLPNEG
4	SLDDLERIKVIGKGSGGTVYLVRHRSTGALYALKVLYMND
5	RKTIPEPYLAEVAKQVLSGLAYLHARRHV
6	FPASGEIAJLLEYMDGGSLAD
7	QPDWAALMCAICFGE
8	EDSVRRQIVREJKILRATQHP
9	VCGDGVKAEGMPSLDELSIDIPSKRVGQFREQFIMQPGNLMSCYYISKQD
10	STFSFSGKVYVGQNDIFDSLSNIRKKLKGDRPREKIVHVVEKLHCRANGD

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谷子抗锈病反应相关MAPKK家族成员的鉴定与分析

Identification and Analysis of MAPKK Family Members Related to Rust Resistance in Foxtail Millet

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基因 Gene	防御和胁迫响应 Defense and stress responsiveness	水杨酸响应 Salicylic acid responsiveness		茉莉酸甲酯响应 MeJA-responsiveness		激发子激活 Elicitor-mediated activation
基因 Gene	TC-rich repeats	TCA-element	SARE	CGTCA-motif	TGACG-motif	AT-rich sequence
SiMKK1	0	0	0	8	8	0
SiMKK3-1	0	0	0	2	2	0
SiMKK3-2	0	1	0	2	2	0
SiMKK4	1	0	1	5	5	0
SiMKK5	0	2	0	2	2	0
SiMKK6-1	0	1	0	2	2	0
SiMKK6-2	2	0	0	2	2	0
SiMKK10-1	0	1	0	5	5	1
SiMKK10-2	1	0	0	1	1	0
SiMKK10-3	0	3	0	4	4	0

单倍型 Haplotype	高抗 High resistance	抗 Resistance	中抗 Middle resistance	感 Susceptible	高感 Highly susceptible	合计 Total
Hap_1	12	12	25	2	10	61
Hap_2	0	0	2	0	0	2
Hap_3	0	0	0	0	1	1
Hap_4	0	0	1	0	0	1
Hap_5	0	0	1	0	1	2
Hap_6	0	2	0	0	0	2
Hap_7	0	1	0	0	0	1
合计Total	12	15	29	2	12	70

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谷子抗锈病反应相关MAPKK家族成员的鉴定与分析

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