Identification of the CKX gene family in alfalfa and its responses to abiotic stress

doi:10.11686/cyxb2024517

Abstract

Abstract:

Alfalfa （Medicago sativa） is the most widely planted legume forage in the world， with a high yield， strong stress resistance， and good palatability. Low temperature and drought are important abiotic stress factors affecting the yield， quality， and distribution of alfalfa. Cytokinin oxidase/dehydrogenase （CKX） irreversibly degrades cytokinin， thereby regulating plant growth and development， and it also contributes to the plant response to environmental stress. In this study， the CKX gene family of alfalfa was identified and analyzed by bioinformatics methods at the whole-genome level. In total， 31 CKX genes were identified in the alfalfa genome， distributed on chromosomes 1， 2， 3， 4， 7， and 8. Members of the CKX gene family members encoded polypeptides of 273-545 amino acids， all of which were hydrophilic proteins except for MsCKX30. In a phylogenetic analysis， the CKX gene family grouped into three subfamilies with highly conserved domains， and alfalfa CKX genes showed high homology with those of Medicago truncatula. Analyses of gene transcript levels by quantitative real-time polymerase chain reaction showed that MsCKX2， MsCKX3， MsCKX7， MsCKX8， MsCKX15， MsCKX16， and MsCKX18 were induced under low-temperature stress， MsCKX2 and MsCKX15 were induced under drought stress， and MsCKX15 was induced by both low temperature stress and drought stress. These results provide a reference for further research on the functions of CKX genes in alfalfa.

Key words: alfalfa, CKX gene family, bioinformatics analysis, expression analysis

Lin BIAN, Yan ZHANG, Xiao-wei HUO, Rui DAI, Na GUO, Feng-yan YI, Cui-ping GAO, Zhi-qiang ZHANG. Identification of the CKX gene family in alfalfa and its responses to abiotic stress[J]. Acta Prataculturae Sinica, 2025, 34(11): 125-135.

Figures/Tables 8

References 35

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引物名称Primers name	引物序列Primer sequence （5′-3′）	引物名称Primers name	引物序列Primer sequence （5′-3′）
qRT-PCR-MsCKX2-F1	GGAGGGCTTGGTCAATTTGG	qRT-PCR-MsCKX10-R2	AGGCCATATTGTTCCACCCA
qRT-PCR-MsCKX2-R1	TTGGCTGGCTTTAAGAGGGT	qRT-PCR-MsCKX15-F1	ATAGCAGCCAGAGGACAAGG
qRT-PCR-MsCKX2-F2	AGCACGTCAAGGACTAGTCA	qRT-PCR-MsCKX15-R1	CAGGTGCAAGTCCATGTTCA
qRT-PCR-MsCKX2-R2	AAGAGTGCCTCCAACAGTGA	qRT-PCR-MsCKX15-F2	CTACCACCAGAGCTAGCCAA
qRT-PCR-MsCKX3-F1	GCAACAAGTGGGCTGATAGG	qRT-PCR-MsCKX15-R2	CCTTGTCCTCTGGCTGCTAT
qRT-PCR-MsCKX3-R1	AGTGCCAAAGTGTTCAACCC	qRT-PCR-MsCKX16-F1	CACACGAGGACAAGCTATGG
qRT-PCR-MsCKX7-F2	AATTCCCAGCAGCAGTGTTC	qRT-PCR-MsCKX16-R1	CAGTCCAAGAAACAGGTGCA
qRT-PCR-MsCKX7-R2	TCAACAACAACACCATCACGT	qRT-PCR-MsCKX16-F2	TGGCAACCTCGTACATGAAT
qRT-PCR-MsCKX8-F1	GCTGGTGTAAGTGGACAAGC	qRT-PCR-MsCKX16-R2	ATAGCTTGTCCTCGTGTGGA
qRT-PCR-MsCKX8-R1	TGGAAGGTGCTGGTTCAAGA	qRT-PCR-MsCKX18-F2	AAGCCCCTGATATGGTGAGG
qRT-PCR-MsCKX8-F2	TGTTGCAGCAAGAGGACATG	qRT-PCR-MsCKX18-R2	ATAGAGAACCGGTCCAGCTG
qRT-PCR-MsCKX8-R2	AGAGAGTGCCACCAACTGTT	qRT-PCR-β-actin-F	TTTGAGACTTTCAATGTGCCCGCC
qRT-PCR-MsCKX10-F2	TGAACCGAACAAAGTGGGATG	qRT-PCR-β-actin-R	TAGCATGTGGGAGTGCATAACCCT

引物名称Primers name	引物序列Primer sequence （5′-3′）	引物名称Primers name	引物序列Primer sequence （5′-3′）
qRT-PCR-MsCKX2-F1	GGAGGGCTTGGTCAATTTGG	qRT-PCR-MsCKX10-R2	AGGCCATATTGTTCCACCCA
qRT-PCR-MsCKX2-R1	TTGGCTGGCTTTAAGAGGGT	qRT-PCR-MsCKX15-F1	ATAGCAGCCAGAGGACAAGG
qRT-PCR-MsCKX2-F2	AGCACGTCAAGGACTAGTCA	qRT-PCR-MsCKX15-R1	CAGGTGCAAGTCCATGTTCA
qRT-PCR-MsCKX2-R2	AAGAGTGCCTCCAACAGTGA	qRT-PCR-MsCKX15-F2	CTACCACCAGAGCTAGCCAA
qRT-PCR-MsCKX3-F1	GCAACAAGTGGGCTGATAGG	qRT-PCR-MsCKX15-R2	CCTTGTCCTCTGGCTGCTAT
qRT-PCR-MsCKX3-R1	AGTGCCAAAGTGTTCAACCC	qRT-PCR-MsCKX16-F1	CACACGAGGACAAGCTATGG
qRT-PCR-MsCKX7-F2	AATTCCCAGCAGCAGTGTTC	qRT-PCR-MsCKX16-R1	CAGTCCAAGAAACAGGTGCA
qRT-PCR-MsCKX7-R2	TCAACAACAACACCATCACGT	qRT-PCR-MsCKX16-F2	TGGCAACCTCGTACATGAAT
qRT-PCR-MsCKX8-F1	GCTGGTGTAAGTGGACAAGC	qRT-PCR-MsCKX16-R2	ATAGCTTGTCCTCGTGTGGA
qRT-PCR-MsCKX8-R1	TGGAAGGTGCTGGTTCAAGA	qRT-PCR-MsCKX18-F2	AAGCCCCTGATATGGTGAGG
qRT-PCR-MsCKX8-F2	TGTTGCAGCAAGAGGACATG	qRT-PCR-MsCKX18-R2	ATAGAGAACCGGTCCAGCTG
qRT-PCR-MsCKX8-R2	AGAGAGTGCCACCAACTGTT	qRT-PCR-β-actin-F	TTTGAGACTTTCAATGTGCCCGCC
qRT-PCR-MsCKX10-F2	TGAACCGAACAAAGTGGGATG	qRT-PCR-β-actin-R	TAGCATGTGGGAGTGCATAACCCT

基因登录号 Gene ID	基因名Gene name	染色体号 Chromosome number	氨基酸数目Number of amino acids （aa）	分子质量Molecular weight （MW， Da）	等电点Isoeletric point （pI）	总平均亲水性Grand average of hydropathicity （GRAVY）	不稳定系数Instability index （II）	亚细胞定位 Subcellular localization
MS.gene57642.t1	MsCKX1	Chr8.3	526	59752.51	8.37	-0.193	35.42	叶绿体Chloroplast
MS.gene025106.t1	MsCKX2	Chr2.3	518	58195.70	6.13	-0.105	31.71	叶绿体Chloroplast
MS.gene85633.t1	MsCKX3	Chr2.1	518	58155.64	6.13	-0.098	30.53	叶绿体Chloroplast
MS.gene21966.t1	MsCKX4	Chr2.4	518	57982.41	6.36	-0.090	29.52	叶绿体Chloroplast
MS.gene051863.t1	MsCKX5	Chr8.2	476	53638.81	5.11	-0.192	38.56	叶绿体Chloroplast
MS.gene57641.t1	MsCKX6	Chr8.3	537	61217.66	5.77	-0.288	41.04	细胞质Cytoplasm
MS.gene85629.t1	MsCKX7	Chr2.1	545	62258.97	8.15	-0.293	37.19	叶绿体Chloroplast
MS.gene026082.t1	MsCKX8	Chr2.2	545	62135.90	7.72	-0.267	36.00	叶绿体Chloroplast
MS.gene051862.t1	MsCKX9	Chr8.2	415	47459.29	7.24	-0.267	37.40	细胞质Cytoplasm
MS.gene069617.t1	MsCKX10	Chr3.4	454	51318.47	6.07	-0.205	37.62	细胞质Cytoplasm
MS.gene42561.t1	MsCKX11	Chr7.1	544	61440.09	6.33	-0.144	36.79	内质网Endoplasmic reticulum
MS.gene014082.t1	MsCKX12	Chr7.2	544	61459.14	6.39	-0.147	37.30	内质网Endoplasmic reticulum
MS.gene017841.t1	MsCKX13	Chr7.3	544	61426.02	6.26	-0.144	36.95	内质网Endoplasmic reticulum
MS.gene85631.t1	MsCKX14	Chr2.1	463	51802.53	6.42	-0.087	29.53	细胞质Cytoplasm
MS.gene025468.t1	MsCKX15	Chr4.4	459	52111.26	6.88	-0.260	41.46	细胞质Cytoplasm
MS.gene71156.t1	MsCKX16	Chr4.1	459	52137.21	6.75	-0.263	40.36	细胞质Cytoplasm
MS.gene012275.t1	MsCKX17	Chr8.4	324	37503.00	8.87	-0.399	45.53	细胞核Nucleus
MS.gene016164.t1	MsCKX18	Chr1.2	509	56811.82	6.29	-0.202	34.52	细胞质Cytoplasm
MS.gene57615.t1	MsCKX19	Chr8.3	312	35813.91	8.27	-0.357	42.27	细胞核Nucleus
MS.gene63612.t1	MsCKX20	Chr3.2	406	45391.81	6.68	-0.180	34.28	叶绿体Chloroplast
MS.gene93997.t1	MsCKX21	Chr3.1	352	39525.18	6.12	-0.109	35.72	叶绿体Chloroplast
MS.gene070417.t1	MsCKX22	Chr3.3	352	39425.07	6.23	-0.100	34.43	叶绿体Chloroplast
MS.gene34593.t1	MsCKX23	Chr4.3	370	41484.16	6.49	-0.221	39.43	细胞质Cytoplasm
MS.gene025112.t1	MsCKX24	Chr2.4	297	35130.28	8.98	-0.467	43.14	叶绿体Chloroplast
MS.gene42306.t1	MsCKX25	Che3.2	422	46570.09	5.54	-0.099	27.05	胞外Extracellular
MS.gene051560.t1	MsCKX26	Chr1.3	411	45309.57	5.47	-0.123	32.81	细胞质Cytoplasm
MS.gene52444.t1	MsCKX27	Chr1.4	405	44558.64	5.38	-0.134	32.55	细胞质Cytoplasm
MS.gene33043.t1	MsCKX28	Chr1.1	405	44544.57	5.29	-0.133	32.55	细胞质Cytoplasm
MS.gene35542.t1	MsCKX29	Chr4.2	301	33828.21	5.95	-0.196	39.14	细胞质Cytoplasm
MS.gene014478.t1	MsCKX30	Chr3.1	386	42018.91	5.48	0.042	30.42	细胞膜Plasma membrane
MS.gene064702.t1	MsCKX31	Chr3.4	273	29908.98	5.29	-0.039	32.93	细胞质Cytoplasm

基因登录号 Gene ID	基因名Gene name	染色体号 Chromosome number	氨基酸数目Number of amino acids （aa）	分子质量Molecular weight （MW， Da）	等电点Isoeletric point （pI）	总平均亲水性Grand average of hydropathicity （GRAVY）	不稳定系数Instability index （II）	亚细胞定位 Subcellular localization
MS.gene57642.t1	MsCKX1	Chr8.3	526	59752.51	8.37	-0.193	35.42	叶绿体Chloroplast
MS.gene025106.t1	MsCKX2	Chr2.3	518	58195.70	6.13	-0.105	31.71	叶绿体Chloroplast
MS.gene85633.t1	MsCKX3	Chr2.1	518	58155.64	6.13	-0.098	30.53	叶绿体Chloroplast
MS.gene21966.t1	MsCKX4	Chr2.4	518	57982.41	6.36	-0.090	29.52	叶绿体Chloroplast
MS.gene051863.t1	MsCKX5	Chr8.2	476	53638.81	5.11	-0.192	38.56	叶绿体Chloroplast
MS.gene57641.t1	MsCKX6	Chr8.3	537	61217.66	5.77	-0.288	41.04	细胞质Cytoplasm
MS.gene85629.t1	MsCKX7	Chr2.1	545	62258.97	8.15	-0.293	37.19	叶绿体Chloroplast
MS.gene026082.t1	MsCKX8	Chr2.2	545	62135.90	7.72	-0.267	36.00	叶绿体Chloroplast
MS.gene051862.t1	MsCKX9	Chr8.2	415	47459.29	7.24	-0.267	37.40	细胞质Cytoplasm
MS.gene069617.t1	MsCKX10	Chr3.4	454	51318.47	6.07	-0.205	37.62	细胞质Cytoplasm
MS.gene42561.t1	MsCKX11	Chr7.1	544	61440.09	6.33	-0.144	36.79	内质网Endoplasmic reticulum
MS.gene014082.t1	MsCKX12	Chr7.2	544	61459.14	6.39	-0.147	37.30	内质网Endoplasmic reticulum
MS.gene017841.t1	MsCKX13	Chr7.3	544	61426.02	6.26	-0.144	36.95	内质网Endoplasmic reticulum
MS.gene85631.t1	MsCKX14	Chr2.1	463	51802.53	6.42	-0.087	29.53	细胞质Cytoplasm
MS.gene025468.t1	MsCKX15	Chr4.4	459	52111.26	6.88	-0.260	41.46	细胞质Cytoplasm
MS.gene71156.t1	MsCKX16	Chr4.1	459	52137.21	6.75	-0.263	40.36	细胞质Cytoplasm
MS.gene012275.t1	MsCKX17	Chr8.4	324	37503.00	8.87	-0.399	45.53	细胞核Nucleus
MS.gene016164.t1	MsCKX18	Chr1.2	509	56811.82	6.29	-0.202	34.52	细胞质Cytoplasm
MS.gene57615.t1	MsCKX19	Chr8.3	312	35813.91	8.27	-0.357	42.27	细胞核Nucleus
MS.gene63612.t1	MsCKX20	Chr3.2	406	45391.81	6.68	-0.180	34.28	叶绿体Chloroplast
MS.gene93997.t1	MsCKX21	Chr3.1	352	39525.18	6.12	-0.109	35.72	叶绿体Chloroplast
MS.gene070417.t1	MsCKX22	Chr3.3	352	39425.07	6.23	-0.100	34.43	叶绿体Chloroplast
MS.gene34593.t1	MsCKX23	Chr4.3	370	41484.16	6.49	-0.221	39.43	细胞质Cytoplasm
MS.gene025112.t1	MsCKX24	Chr2.4	297	35130.28	8.98	-0.467	43.14	叶绿体Chloroplast
MS.gene42306.t1	MsCKX25	Che3.2	422	46570.09	5.54	-0.099	27.05	胞外Extracellular
MS.gene051560.t1	MsCKX26	Chr1.3	411	45309.57	5.47	-0.123	32.81	细胞质Cytoplasm
MS.gene52444.t1	MsCKX27	Chr1.4	405	44558.64	5.38	-0.134	32.55	细胞质Cytoplasm
MS.gene33043.t1	MsCKX28	Chr1.1	405	44544.57	5.29	-0.133	32.55	细胞质Cytoplasm
MS.gene35542.t1	MsCKX29	Chr4.2	301	33828.21	5.95	-0.196	39.14	细胞质Cytoplasm
MS.gene014478.t1	MsCKX30	Chr3.1	386	42018.91	5.48	0.042	30.42	细胞膜Plasma membrane
MS.gene064702.t1	MsCKX31	Chr3.4	273	29908.98	5.29	-0.039	32.93	细胞质Cytoplasm

基因登录号 Gene ID	基因名 Gene name	蛋白质 Protein	α-螺旋 Alpha helix	β-转角 Beta turn	延伸链 Extended strand	无规则卷曲 Random coil
MS.gene57642.t1	MsCKX1	MsCKX1	33.46	5.70	18.63	42.21
MS.gene025106.t1	MsCKX2	MsCKX2	35.71	5.41	19.69	39.19
MS.gene85633.t1	MsCKX3	MsCKX3	33.98	5.79	20.08	40.15
MS.gene21966.t1	MsCKX4	MsCKX4	34.94	6.18	20.46	38.42
MS.gene051863.t1	MsCKX5	MsCKX5	32.35	6.72	19.75	41.18
MS.gene57641.t1	MsCKX6	MsCKX6	37.43	5.59	16.95	40.04
MS.gene85629.t1	MsCKX7	MsCKX7	31.93	6.24	17.43	44.40
MS.gene026082.t1	MsCKX8	MsCKX8	33.39	5.69	17.98	42.94
MS.gene051862.t1	MsCKX9	MsCKX9	32.53	7.71	20.00	39.76
MS.gene069617.t1	MsCKX10	MsCKX10	33.26	6.17	19.82	40.75
MS.gene42561.t1	MsCKX11	MsCKX11	31.07	5.51	19.30	44.12
MS.gene014082.t1	MsCKX12	MsCKX12	29.04	6.99	20.77	43.20
MS.gene017841.t1	MsCKX13	MsCKX13	31.07	6.07	19.85	43.01
MS.gene85631.t1	MsCKX14	MsCKX14	33.05	5.62	20.52	40.82
MS.gene025468.t1	MsCKX15	MsCKX15	32.24	6.54	19.61	41.61
MS.gene71156.t1	MsCKX16	MsCKX16	32.24	6.10	20.70	40.96
MS.gene012275.t1	MsCKX17	MsCKX17	37.35	5.86	17.59	39.20
MS.gene016164.t1	MsCKX18	MsCKX18	31.83	6.29	20.04	41.85
MS.gene57615.t1	MsCKX19	MsCKX19	38.46	5.45	17.63	38.46
MS.gene63612.t1	MsCKX20	MsCKX20	36.70	5.91	19.95	37.44
MS.gene93997.t1	MsCKX21	MsCKX21	31.53	5.40	20.74	42.33
MS.gene070417.t1	MsCKX22	MsCKX22	31.82	6.25	20.45	41.48
MS.gene34593.t1	MsCKX23	MsCKX23	30.81	5.68	19.46	44.05
MS.gene025112.t1	MsCKX24	MsCKX24	39.39	5.39	16.50	38.72
MS.gene42306.t1	MsCKX25	MsCKX25	37.44	5.45	17.06	40.05
MS.gene051560.t1	MsCKX26	MsCKX26	30.90	5.60	19.46	44.04
MS.gene52444.t1	MsCKX27	MsCKX27	31.60	7.16	18.52	42.72
MS.gene33043.t1	MsCKX28	MsCKX28	33.09	6.91	19.51	40.49
MS.gene35542.t1	MsCKX29	MsCKX29	32.89	7.31	22.59	37.21
MS.gene014478.t1	MsCKX30	MsCKX30	36.27	7.25	20.98	35.49
MS.gene064702.t1	MsCKX31	MsCKX31	34.07	6.59	20.51	38.83