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

doi:10.7668/hbnxb.20194834

Abstract

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

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.

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Figures/Tables 13

References 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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