Identification of Lysine Synthesis Pathway Genes and Analysis of the Regulation of AK Gene Variations in Foxtail Millet

doi:10.13560/j.cnki.biotech.bull.1985.2025-1203

Abstract

Abstract:

Objective Lysine is the first limiting essential amino acid for humans and livestock, and its low content restricts the improvement of nutritional quality in foxtail millet (Setaria italica L.). Elucidating the molecular mechanisms of lysine biosynthesis and metabolism in foxtail millet is essential for providing genetic resources for improving grain nutritional quality. Method The genes involved in the lysine biosynthesis pathway (LBPG) were identified via bioinformatics. Key candidate genes regulating grain lysine accumulation were explored by combining phylogenetic analysis, chromosomal localization, collinearity analysis, expression profiling, candidate gene association analysis, and haplotype analysis. Result The lysine content in foxtail millet grains declined during grain development (S1‒S4), and the variation coefficient of lysine content in natural populations was 36.5%. Based on Kyoto encyclopedia of genes and genomes (KEGG) database annotation and foxtail millet genome information, a total of 15 genes encoding lysine synthesis-related enzymes were identified, belonging to 7 gene families. LBPGs (lysine biosynthesis pathway genes) exhibited tissue-specific and stage-specific expression, with most genes showing down-regulation in the panicle from the S1 to S4 stages. Candidate gene association analysis showed that 30 single nucleotide polymorphisms (SNPs) within four members of the aspartate kinase (AK) family (AK1, AK3, AK4, and AK5) were significantly associated with lysine content (P<0.01). Specifically, accessions carrying the AK3 Hap1 and AK5 Hap1 haplotypes exhibited 17.22% and 12.96% higher lysine content, respectively, than those carrying the Hap2 haplotypes. Protein interaction network and expression correlation analyses indicated that the homoserine kinase (HSK) gene interacted with AK3 and had a significantly positive expression correction (P<0.01). And it may modulate lysine synthesis by regulating the expression of AK3. Conclusion Lysine content in foxtail millet grains decreases as the grain matures. SNPs in AK family genes are key factors influencing lysine variation. Hap1 of AK3 and AK5 are superior haplotypes for high-lysine breeding. Furthermore, the HSK gene likely acts in coordination with AK3 to regulate lysine biosynthesis in foxtail millet grains.

Key words: Setaria italica, lysine synthesis pathway, candidate gene association analysis, AK gene, haplotype

ZHAO Xiong-wei, SHAO Li-tao, LI Meng-qing, LIANG Yi-xuan, ZHANG Jie-ru, CAO Yan-hua. Identification of Lysine Synthesis Pathway Genes and Analysis of the Regulation of AK Gene Variations in Foxtail Millet[J]. Biotechnology Bulletin, 2026, 42(9): 107-119.

Figures/Tables 9

Table 1 Lysine biosynthesis pathway enzyme genes in foxtail millet and their sequence characteristics

基因家族 Gene family	基因名称 Gene name	EC 编号 EC number	基因ID Gene ID	氨基酸 Amino acid （aa）	编码区长度 CDS length （bp）	等电点 Isoelectric point
AK	AK1	2.7.2.4	Si6g12370	939	2 820	7.07
	AK2		Si5g43640	554	1 665	6.13
	AK3		Si2g11670	568	1 707	6.62
	AK4		Si9g02030	565	1 698	6.54
	AK5		Si2g17120	918	2 757	6.35
ASD	ASD	1.2.1.11	Si9g08540	372	1 119	7.66
DapB	DapB1	1.17.1.8	Si9g46060	326	981	5.55
DapB	DapB2	1.17.1.8	Si1g15210	342	1 029	6.21
DapF	DapF	5.1.1.7	Si3g32590	357	1 074	5.95
DHDPS	DHDPS1	4.3.3.7	Si4g14890	368	1 107	6.87
DHDPS	DHDPS2	4.3.3.7	Si7g21820	377	1 134	6.27
LL-DAP	LL-DAP1-AT	2.6.1.83	Si9g42730	463	1 392	7.57
	LL-DAP2-AT		Si9g49300	436	1 311	6.49
	LL-DAP3-AT		Si2g38880	464	1 395	6.20
LysA	LysA	4.1.1 .20	Si3g36730	493	1 482	6.29

Table 2 Significantly associated SNP loci from natural variations in AK genes for lysine content in foxtail millet

基因 Gene	染色体 Chromosome	基因长度 Gene length （bp）	SNP总数 Total SNPs	关联性状 Associated trait	显著SNP数 No. of sig. SNP	-log₁₀ (P)	R² （%）
AK1	6	12 423	26	DT_2019	7	2.29	6.08
AK3	2	6 027	93	JC_2019， DT_2019， TG_2019， TG_2020	3	2.67	9.03
AK4	9	4 486	45	JC_2019， DT_2019	6	2.23	6.96
AK5	2	10 811	31	DT_2019， TG_2019	14	2.25	7.06

Fig. 1 Lysine content in foxtail millet at different panicle developmental stages and cluster analysis with a histogram of lysine content in a natural populationA: Developmental stages of foxtail millet grains. B: Lysine content in seeds of foxtail millet at four developmental stages. C: Cluster diagram of seed lysine content across different varieties of foxtail millet. D: Bar chart of grain lysine content in different subgroups. S1: Green ripe stage. S2: Mature green stage. S3: Wax ripe stage. S4: Full ripe stage. The different lowercase letters indicate significant differences at P<0.05 level. The same below

Fig. 2 Phylogenetic tree of lysine metabolic pathway LBPG genes in foxtail millet and four other plant speciesDifferent colored branch lines represent distinct phylogenetic subgroups. The shapes and colors at the tips of the branches indicate different species

Fig. 3 Gene structure (A), conserved domains (B), and collinearity analysis with rice and maize (C) of LBPG in foxtail millet

Fig. 4 Lysine metabolism pathway and expression patterns of LBPG genes in foxtail millet at different growth stagesFrom left to right, the columns in the heatmaps represent roots, stems, and leaves at the mature stage, followed by the green ripe stage (S1), mature green stage (S2), wax ripe stage (S3), and full ripe stage (S4) during grain development

Fig. 5 Manhattan plot of the association between SNPs of LBPG genes and seed lysine content in foxtail millet

Fig. 6 Distribution of polymorphic sites in AK genes (A) and significant difference analysis of lysine content in germplasms with different haplotypes (B)

Fig. 7 Prediction of interaction networks of AK3 and AK5 proteins and their protein interaction complexes in foxtail milletA: Interaction network of AK3 and AK5 proteins with other proteins in foxtail millet. B: Expression correlations of Si1g37880 with AK3 and AK5 genes in foxtail millet grains at the S3 stage. C: Model of the protein interaction complex formed by Si1g37880 with AK3 and AK5

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