黍子NF-Y基因家族的鉴定及PmNF-YA8的功能分析

doi:10.3864/j.issn.0578-1752.2026.10.003

Abstract

Abstract:

【Objective】Nuclear factor Y (NF-Y) transcription factors regulate the expression of target genes by specifically binding to the CCAAT element in the promoter region of target genes, and play a role in plant responses to abiotic stresses such as drought and salinity. Broomcorn millet exhibits strong tolerance to drought and barrenness, serving as an indispensable crop for saline-alkali and marginal lands. Systematic elucidation of the functions of the NF-Y gene family in broomcorn millet will provide a theoretical basis for enriching the genetic theory of crop stress tolerance and promoting drought-resistant breeding practices.【Method】Genome data of broomcorn millet and bioinformatics approaches were employed to identify NF-Y family members, physicochemical properties, gene structures, construct phylogenetic trees, and predict conserved domains as well as cis-acting elements in promoters. Reverse Transcription Quantitative PCR (RT-qPCR) was used to determine the expression of these genes in different plant tissues, and the expression characteristics of PmNF-YA8 under drought stress were verified through transgenic functional validation in Arabidopsis thaliana.【Result】Based on the proso millet reference genome, 33 PmNF-Y genes (including 11 PmNF-YA, 12 PmNF-YB, and 10 PmNF-YC) were identified, encoding proteins with lengths of 122-571 amino acids, isoelectric points of 4.74-10.19, and molecular weights of 13.58-59.17 kDa. Chromosomal localization analysis showed that these 33 genes (PmNF-YA1-PmNF-YA11, PmNF-YB1-PmNF-YB12, and PmNF-YC1-PmNF-YC10) were distributed across 15 chromosomes. Two types of drought stress-responsive elements were predicted in the promoter regions of PmNF-Y family members. A total of 3, 47, and 59 NF-Y orthologous gene pairs were identified between broomcorn millet and three other plant species (Arabidopsis thaliana, rice, and maize), respectively. RT-qPCR analysis revealed that PmNF-Y genes were widely expressed in tissues such as roots, stems, and leaves. The expression level of PmNF-YA8 was up-regulated in both XHS and NM9 varieties after drought stress. Transgenic Arabidopsis thaliana lines overexpressing PmNF-YA8 were generated, and phenotypic and physiological analyses showed that under 300 mmol·L^-1 mannitol stress, the average root length of transgenic plants (14.03 mm) was longer than that of wild-type (WT) plants (9.07 mm). Additionally, transgenic plants had a lower MDA increase range (55.23%-57.12%) compared with WT plants (100.78%), with SOD and POD activities increased by 24.28%-27.43% and 163.57%-341.33%, respectively, and Pro content (151.31-175.14 μg·g^-1) higher than that of WT plants (143.78 μg·g^-1).【Conclusion】A total of 33 NF-Y family members were identified in broomcorn millet. The PmNF-YA8 gene was cloned from broomcorn millet and heterologously transformed into Arabidopsis thaliana. Its heterologous expression enhanced the drought stress tolerance of Arabidopsis thaliana plants by regulating the expression of stress-related genes, thus making PmNF-YA8 a key candidate gene for modulating drought tolerance in broomcorn millet.

Key words: broomcorn millet, NF-Y, PmNF-YA8, expression pattern, drought stress

SONG YuZhen, BHEEL Chander Kumar, ZHANG YingXing, WANG Yue, SANTRA Dipak Kumar, CAO XiaoNing, WANG RuiYun. Identification of the NF-Y Gene Family and Functional Analysis of PmNF-YA8 in Broomcorn Millet[J].Scientia Agricultura Sinica, 2026, 59(10): 2088-2108.

Figures/Tables 15

Table 1

Primers used in this study"

引物名称 Primer name	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)	用途 Usage
PmActin	TGACTGTGCTGTCCTCATCA	GTTGCAGCAGCAAATCATCT	实时荧光定量PCR RT-qPCR
qPmNF-YA1	AATGAAGCGAGTTCGTGGA	GTTGAGGGCGAGTGAGATG
qPmNF-YA2	GGCTGCCACCAAGAGAGG	AATGGTGACTGCAGCGCT
qPmNF-YA3	ATTTCTCCCTATGGCACTCA	CAGGCTCCTCCGATACTTCA
qPmNF-YA4	TGCTATGGCGTGCTTACCAC	GGAGGATGCCTTCGTACTG
qPmNF-YA5	ACAGCAGCCTCAATGGACA	CCCAAAGGACGATACAGTC
qPmNF-YA6	TGGGCACACCATCAGCTG	GTCCACCGTAAGCCGCAT
qPmNF-YA7	ATGCGCCCTCAACGTGAA	GGAGCTGTGGCCTCTGTG
qPmNF-YA8	GGAAGCATAATCAGGACA	ATAAGGAAGACAAGCAAAA
qPmNF-YA9	CTGATTATGTGGCTCCCT	ATGGCATTGTATTGTTTTG
qPmNF-YA10	AGCAAGAAGCCCCAGTTA	GCATCAGTAGGCAAAGGT
qPmNF-YA11	TGAAGGATGCTGGTGGAG	AGATGCTGCTTGGGTTGC
qPmNF-YB1	TACCTGCCGATCGCGAAC	CCTGCACCGTCTCCTTGG
qPmNF-YB2	GAGTGCGTCTCCGAGTT	CGCCGTTGATGGTCTTG
qPmNF-YB3	TACCTGCCGATCGCCAAC	CACTCGCTTCGCTGGTGA
qPmNF-YB4	AAGATCGCCAAGGACGCC	GGCACTTGTCACTCGCCT
qPmNF-YB5	ACCATCCAAGAGTGCGCC	GGCGTAGTGATCGAGGCC
qPmNF-YB6	CAGCCGTTACCAGCAGA	TAGTAGTGGCGCGATCC
qPmNF-YB7	GCAGCCGTTACCAGCAGA	CGTCGGCGTAGAATCCCC
qPmNF-YB8	GTGACTGGCGAGGCGAA	AGGAAGGCGTTGAGGGG
qPmNF-YB9	CAGGAGTGCGTGTCCGAG	CTGAGCGGCTCGACGTAG
qPmNF-YB10	AGGAGTGCGTGTCCGAGT	CGGTAGCGGTGGAGGTAG
qPmNF-YB11	GTGCGTCTCCGAGTTCAT	TCCTTGTGGCACTTGTCG
qPmNF-YB12	GCGCGCCAAGGAGACTAT	ATGGCAGATGTCGTCGCC
qPmNF-YC1	CGCACCACCACCTGCTC	CGCTTGTTCTCCTCGGC
qPmNF-YC2	CACCACCACCTGCTCCAG	TTGAAGTCCGACGCCGAC
qPmNF-YC3	TGCAGATGTTCTGGGCGG	TCTTGATGCGGGCGAGTG
qPmNF-YC4	CCCAGCAATTTCACCAA	CCGCACATCCTCATCAG
qPmNF-YC5	GCCCCACCTGATGCACAT	GCGGTTCCTGCTGTTCCT
qPmNF-YC6	AGGTGCCAGGTGGAGGAA	CAGCCACTTCCACGCAGA
qPmNF-YC7	GCAGATGCCACCAGGAGG	GATCGTTGCTGCTGCTGC
qPmNF-YC8	GAGGCCACCACCGACTTC	ATCATGCGGACGTCCTCG
qPmNF-YC9	GATGATCTCCGGCGAGGC	TCTTCTGCACGGCCATGG
qPmNF-YC10	TGGCGCGCATCAAGAAGA	CGTGAGCTCGGCGATGAA
121PmNF-YA8	GGACTCTAGAGGATCCCC TTGTAATGCCTAAGCTTTTAC	CGATCGGGGAAATTCGAG TCACCTAACCAGCTTTGCT	同源重组 Homologous recombination
35S	GACGCACAATCCCACTATCC		抗性鉴定 Resistance identification

Table 1

Table 2

Basic information of NF-Y family members in broomcorn millet"

基因 Gene	基因编号 Gene ID	氨基酸数量 Number of amino acids	等电点 Isoelectric point	分子量 Molecular weight (kDa)	不稳定指数 Instability index	脂肪系数 Aliphatic index	平均亲水性 Average of hydropathicity	亚细胞定位 Subcellular localization
PmNF-YA1	C2845_PM01G17820	256	9.60	28.31	61.41	56.84	-0.630	细胞核Nucleus
PmNF-YA2	C2845_PM01G33930	349	9.21	37.18	58.84	51.63	-0.677	细胞核Nucleus
PmNF-YA3	C2845_PM02G01810	248	9.93	27.07	50.70	54.80	-0.931	细胞核Nucleus
PmNF-YA4	C2845_PM02G06470	347	9.45	37.10	58.46	56.43	-0.607	细胞核Nucleus
PmNF-YA5	C2845_PM02G23560	298	9.92	32.87	69.12	49.43	-0.893	细胞核Nucleus
PmNF-YA6	C2845_PM03G20210	263	10.19	27.43	56.57	63.04	-0.458	细胞核Nucleus
PmNF-YA7	C2845_PM04G02880	571	6.20	59.17	45.78	67.57	-0.364	细胞核Nucleus
PmNF-YA8	C2845_PM04G32230	270	9.37	29.23	62.03	52.44	-0.942	细胞核Nucleus
PmNF-YA9	C2845_PM05G01850	214	7.99	23.25	68.48	47.99	-1.077	细胞核Nucleus
PmNF-YA10	C2845_PM06G33940	214	6.90	23.41	71.31	49.35	-1.076	细胞核Nucleus
PmNF-YA11	C2845_PM06G34350	331	8.75	35.57	55.26	69.91	-0.442	细胞核Nucleus
PmNF-YB1	C2845_PM03G12450	213	6.52	22.44	46.84	51.41	-0.646	细胞核Nucleus
PmNF-YB2	C2845_PM04G32110	213	6.21	22.38	45.04	52.35	-0.596	细胞核Nucleus
PmNF-YB3	C2845_PM05G28680	140	5.19	15.37	47.18	62.07	-0.736	细胞核Nucleus
PmNF-YB4	C2845_PM06G19180	128	6.59	13.62	54.43	71.02	-0.489	细胞核Nucleus
PmNF-YB5	C2845_PM07G34040	160	6.43	17.87	58.91	64.81	-0.796	细胞核Nucleus
PmNF-YB6	C2845_PM09G10960	312	9.33	34.30	55.77	78.53	-0.123	细胞核Nucleus
PmNF-YB7	C2845_PM09G10970	232	9.75	25.59	73.17	56.16	-0.563	细胞核Nucleus
PmNF-YB8	C2845_PM10G11050	221	9.46	23.92	63.28	57.06	-0.517	细胞核Nucleus
PmNF-YB9	C2845_PM11G27440	267	6.56	28.23	39.53	55.02	-0.535	细胞核Nucleus
PmNF-YB10	C2845_PM12G25720	257	6.48	27.42	39.06	54.09	-0.588	细胞核Nucleus
PmNF-YB11	C2845_PM14G13570	171	6.06	17.96	48.20	46.90	-0.723	细胞核Nucleus
PmNF-YB12	C2845_PM14G13580	161	6.32	18.12	62.00	64.41	-0.869	细胞核Nucleus
PmNF-YC1	C2845_PM01G09450	245	5.33	25.92	64.92	65.59	-0.340	细胞核Nucleus
PmNF-YC2	C2845_PM02G31560	249	5.21	26.20	64.62	64.90	-0.331	细胞核Nucleus
PmNF-YC3	C2845_PM04G19400	185	5.36	19.75	54.25	80.70	-0.068	细胞质Cytoplasm细胞核Nucleus
PmNF-YC4	C2845_PM09G13720	122	5.89	14.56	65.50	85.57	-0.534	细胞质Cytoplasm细胞核Nucleus
PmNF-YC5	C2845_PM09G21000	246	5.13	27.37	77.54	72.32	-0.531	细胞质Cytoplasm细胞核Nucleus
PmNF-YC6	C2845_PM10G18480	144	4.74	16.23	86.95	69.79	-0.599	细胞质Cytoplasm细胞核Nucleus
PmNF-YC7	C2845_PM12G04070	169	4.83	19.24	83.24	69.88	-0.631	细胞质Cytoplasm细胞核Nucleus
PmNF-YC8	C2845_PM13G21210	208	5.61	22.27	54.51	69.33	-0.262	细胞核Nucleus
PmNF-YC9	C2845_PM15G26460	128	6.83	13.58	40.01	76.41	-0.148	细胞质Cytoplasm细胞核Nucleus
PmNF-YC10	C2845_PM16G24450	128	6.83	13.65	37.29	76.41	-0.198	细胞质Cytoplasm细胞核Nucleus

Table 2

Fig. 1

Fig. 2

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Fig. 4

Fig. 5

Fig. 6

Table 3

Fig. 7

Fig. 8

Fig. 9

Fig. 10

Fig. 11

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重复基因对 Duplicated gene pairs	非同义替换率 Ka	同义替换率 Ks	Ka/Ks	选择类型 Selective type	分化时间 Differentiation time (Mya)
PmNF-YA2/PmNF-YA4	0.0410	0.0766	0.5356	纯化选择Purifying selection	5.89
PmNF-YC1/PmNF-YC2	0.0091	0.0686	0.1328	纯化选择Purifying selection	5.28
PmNF-YA1/PmNF-YA5	0.0336	0.1395	0.2407	纯化选择Purifying selection	10.73
PmNF-YA2/PmNF-YA6	0.5211	1.1394	0.4574	纯化选择Purifying selection	87.64
PmNF-YA1/PmNF-YA8	0.3022	0.9316	0.3243	纯化选择Purifying selection	71.66
PmNF-YA4/PmNF-YA6	0.5262	1.2798	0.4112	纯化选择Purifying selection	98.44
PmNF-YA5/PmNF-YA8	0.3351	1.0048	0.3335	纯化选择Purifying selection	77.29
PmNF-YA4/PmNF-YA7	0.4815	1.3161	0.3659	纯化选择Purifying selection	101.24
PmNF-YA6/PmNF-YA7	0.0578	0.0769	0.7513	纯化选择Purifying selection	5.91
PmNF-YB1/PmNF-YB2	0.0213	0.0548	0.3890	纯化选择Purifying selection	4.22
PmNF-YC3/PmNF-YC8	0.0759	0.5250	0.1445	纯化选择Purifying selection	40.38
PmNF-YA9/PmNF-YA10	0.0123	0.0338	0.3643	纯化选择Purifying selection	2.60
PmNF-YB5/PmNF-YB11	0.4304	0.7383	0.5830	纯化选择Purifying selection	56.79
PmNF-YC5/PmNF-YC6	0.0122	0.1303	0.0933	纯化选择Purifying selection	10.02
PmNF-YB6/PmNF-YB8	0.0618	0.1902	0.3248	纯化选择Purifying selection	14.63
PmNF-YC5/PmNF-YC7	0.0919	0.9224	0.0996	纯化选择Purifying selection	70.96
PmNF-YC6/PmNF-YC7	0.1018	1.1412	0.0892	纯化选择Purifying selection	87.79
PmNF-YC9/PmNF-YC10	0.0141	0.0756	0.1863	纯化选择Purifying selection	5.82

Identification of the NF-Y Gene Family and Functional Analysis of PmNF-YA8 in Broomcorn Millet

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