谷子赖氨酸合成通路基因的鉴定及AK基因变异调控作用分析

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

摘要/Abstract

摘要：

目的赖氨酸是人类与牲畜营养中的第一限制性必需氨基酸，其含量低制约谷子营养价值的提升。解析谷子赖氨酸合成代谢的分子机制，可为谷物营养品质改良提供重要的基因资源。方法通过生物信息学鉴定谷子赖氨酸生物合成途径基因，并结合系统发育关系、染色体定位、共线性关系、表达谱分析、候选基因关联分析和单倍型分析，挖掘调控籽粒赖氨酸积累的关键候选基因。结果谷子籽粒赖氨酸含量随发育阶段（青熟期S1‒完熟期S4）呈下降趋势，且自然群体中赖氨酸含量变异系数为36.5%。基于KEGG（kyoto encyclopedia of genes and genomes）数据库注释及谷子基因组信息，共鉴定出15个编码赖氨酸合成相关酶的基因，分属7个基因家族。LBPG（lysine biosynthesis pathway genes）基因表现出组织与发育阶段特异性表达，多数基因在穗部S1‒S4期表达量逐渐下降。候选基因关联分析显示，仅天冬氨酸激酶AK（aspartate kinase）基因的4个家族成员（AK1、AK3、AK4、AK5）存在30个SNP（single nucleotide polymorphism）位点与赖氨酸含量显著关联（P<0.01）。其中，携带AK3基因的单倍型Hap1种质赖氨酸含量比Hap2高17.22%，而携带AK5基因的单倍型Hap1种质比Hap2高12.96%。蛋白互作网络预测及表达相关性分析表明，高丝氨酸激酶HSK（homoserine kinase）基因与AK3存在互作及显著正表达相关（P<0.01），可能通过调控AK3基因表达影响谷子赖氨酸合成。结论谷子籽粒赖氨酸含量随发育时期呈下降趋势，AK家族基因的SNP与赖氨酸含量存在显著关联。AK3和AK5的Hap1单倍型种质赖氨酸含量均显著高于Hap2。此外，高丝氨酸激酶HSK基因与AK3存在显著表达正相关及蛋白互作，可能协同调控谷子籽粒赖氨酸的合成。

关键词: 谷子, 赖氨酸合成通路, 候选基因关联分析, AK基因, 单倍型

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

赵雄伟, 邵莉桃, 李梦晴, 梁逸萱, 张洁如, 曹艳花. 谷子赖氨酸合成通路基因的鉴定及AK基因变异调控作用分析[J]. 生物技术通报, 2026, 42(9): 107-119.

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.

图/表 9

表1 谷子赖氨酸代谢生物合成酶基因及其序列特征

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

表2 谷子AK基因自然变异与籽粒赖氨酸含量的显著关联SNP位点

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

图1 不同穗发育阶段谷子赖氨酸含量及自然群体赖氨酸直方图聚类分析A：谷子籽粒发育时期；B：在4个发育时期的谷子籽粒赖氨酸含量；C：谷子不同品种的籽粒赖氨酸含量聚类图；D：谷子不同亚群的籽粒赖氨酸含量柱形图；S1：青熟期；S2：绿熟期；S3：蜡熟期；S4：完熟期；不同的小写字母表示在P<0.05水平上有显著差异。下同

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

图2 谷子与其他4种植物的赖氨酸合成代谢通路LBPG基因的系统发育树不同颜色的分支线代表不同的进化亚群；枝末端的形状及颜色代表不同的物种来源

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

图3 谷子LBPG的基因结构（A）、保守结构域（B）及与水稻和玉米的共线性分析（C）

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

图4 谷子赖氨酸代谢通路及LBPG基因在不同生长阶段的表达模式热图中的列从左至右依次代表成熟期的根系、茎干和叶片组织，以及籽粒发育过程中青熟期（S1）、绿熟期（S2）、蜡熟期（S3）和完熟期（S4）

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

图5 谷子LBPG基因SNP与籽粒赖氨酸含量关联的曼哈顿图

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

图6 AK基因多态性位点分布（A）及不同单倍型种质赖氨酸含量的显著性差异分析（B）

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

图7 谷子AK3和AK5蛋白与其他蛋白的互作网络预测模型和蛋白质互作复合体预测A：谷子AK3和AK5蛋白与其他蛋白的互作网络；B：谷子籽粒S3时期Si1g37880分别与AK3、AK5基因的表达量相关性；C：Si1g37880与AK3、AK5的蛋白互作复合体模型

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

参考文献 38

[1]	Trovato M, Funck D, Forlani G, et al. Editorial: amino acids in plants: regulation and functions in development and stress defense [J]. Front Plant Sci, 2021, 12: 772810.
[2]	李暮男, 兰凤英. 小米的营养成分及保健功能研究进展 [J]. 河北北方学院学报: 自然科学版, 2017, 33(7): 56-60.
	Li MN, Lan FY. Research progress on nutritional components and health care functions of millet [J]. J Hebei N Univ Nat Sci Ed, 2017, 33(7): 56-60.
[3]	Amir R, Galili G, Cohen H. The metabolic roles of free amino acids during seed development [J]. Plant Sci, 2018, 275: 11-18.
[4]	Bouchereau J, Schiff M. Inherited disorders of lysine metabolism: a review [J]. J Nutr, 2020, 150: 2556S-2560S.
[5]	Jiang W, Zhao Y, Wu X, et al. Health inequalities of global protein-energy malnutrition from 1990 to 2019 and forecast prevalence for 2044: data from the Global Burden of Disease Study 2019 [J]. Public Health, 2023, 225: 102-109.
[6]	Arruda P, Barreto P. Lysine catabolism through the saccharopine pathway: enzymes and intermediates involved in plant responses to abiotic and biotic stress [J]. Front Plant Sci, 2020, 11: 587.
[7]	Wang WY, Xu MY, Wang GP, et al. New insights into the metabolism of aspartate-family amino acids in plant seeds [J]. Plant Reprod, 2018, 31(3): 203-211.
[8]	Yang QQ, Zhao DS, Liu QQ. Connections between amino acid metabolisms in plants: lysine as an example [J]. Front Plant Sci, 2020, 11: 928.
[9]	Torruella G, Suga H, Riutort M, et al. The evolutionary history of lysine biosynthesis pathways within eukaryotes [J]. J Mol Evol, 2009, 69(3): 240-248.
[10]	Hudson AO, Singh BK, Leustek T, et al. An LL-diaminopimelate aminotransferase defines a novel variant of the lysine biosynthesis pathway in plants [J]. Plant Physiol, 2006, 140(1): 292-301.
[11]	Arruda P, Kemper EL, Papes F, et al. Regulation of lysine catabolism in higher plants [J]. Trends Plant Sci, 2000, 5(8): 324-330.
[12]	Zhu XH, Galili G. Lysine metabolism is concurrently regulated by synthesis and catabolism in both reproductive and vegetative tissues [J]. Plant Physiol, 2004, 135(1): 129-136.
[13]	Reyes AR, Bonin CP, Houmard NM, et al. Genetic manipulation of lysine catabolism in maize kernels [J]. Plant Mol Biol, 2009, 69(1/2): 81-89.
[14]	Yang QQ, Yu WH, Wu HY, et al. Lysine biofortification in rice by modulating feedback inhibition of aspartate kinase and dihydrodipicolinate synthase [J]. Plant Biotechnol J, 2021, 19(3): 490-501.
[15]	舒军, 王啸旗, 郭宁馨, 等. 谷子不同核心种质的小米氨基酸组分积累特性分析及其评价 [J]. 山西农业科学, 2022, 50(12): 1622-1630.
	Shu J, Wang XQ, Guo NX, et al. Analysis and evaluation of the accumulation characteristics of amino acid components in different core germplasms of foxtail millet [J]. J Shanxi Agric Sci, 2022, 50(12): 1622-1630.
[16]	刘美池, 邓歆, 刘新春, 等. 青稞种质资源籽粒赖氨酸含量的评价 [J]. 大麦与谷类科学, 2023, 40(5): 8-17.
	Liu MC, Deng X, Liu XC, et al. Evaluation of lysine content in grains of highland barley germplasm resources [J]. Barley Cereal Sci, 2023, 40(5): 8-17.
[17]	Liu YW, Xie SJ, Yu JJ. Genome-wide analysis of the lysine biosynthesis pathway network during maize seed development [J]. PLoS One, 2016, 11(2): e0148287.
[18]	Bailey TL, Boden M, Buske FA, et al. MEME Suite: tools for motif discovery and searching [J]. Nucleic Acids Res, 2009, 37(): W202-W208.
[19]	Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets [J]. Mol Biol Evol, 2016, 33(7): 1870-1874.
[20]	Chen CJ, Chen H, Zhang Y, et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data [J]. Mol Plant, 2020, 13(8): 1194-1202.
[21]	Yang ZR, Zhang HS, Li XK, et al. A mini foxtail millet with an Arabidopsis-like life cycle as a C4 model system [J]. Nat Plants, 2020, 6(9): 1167-1178.
[22]	Li XK, Gao JH, Song JY, et al. Multi-omics analyses of 398 foxtail millet accessions reveal genomic regions associated with domestication, metabolite traits, and anti-inflammatory effects [J]. Mol Plant, 2022, 15(8): 1367-1383.
[23]	Zhao XW, Cao YH, Shao LT, et al. Integration of genome-wide association and genomic prediction for dissecting seed protein and amino acid in foxtail millet [J]. Field Crops Res, 2024, 310: 109344.
[24]	Bradbury PJ, Zhang ZW, Kroon DE, et al. TASSEL: software for association mapping of complex traits in diverse samples [J]. Bioinformatics, 2007, 23(19): 2633-2635.
[25]	Szklarczyk D, Kirsch R, Koutrouli M, et al. The STRING database in 2023: protein-protein association networks and functional enrichment analyses for any sequenced genome of interest [J]. Nucleic Acids Res, 2023, 51(D1): D638-D646.
[26]	Kozakov D, Hall DR, Xia B, et al. The ClusPro web server for protein-protein docking [J]. Nat Protoc, 2017, 12(2): 255-278.
[27]	Saleh ASM, Zhang Q, Chen J, et al. Millet grains: nutritional quality, processing, and potential health benefits [J]. Comp Rev Food Sci Food Safe, 2013, 12(3): 281-295.
[28]	Sachdev N, Goomer S, Singh LR. Foxtail millet: a potential crop to meet future demand scenario for alternative sustainable protein [J]. J Sci Food Agric, 2021, 101(3): 831-842.
[29]	刘慧, 王朝辉, 李富翠, 等. 不同麦区小麦籽粒蛋白质与氨基酸含量及评价 [J]. 作物学报, 2016, 42(5): 768-777.
	Liu H, Wang ZH, Li FC, et al. Contents of protein and amino acids of wheat grain in different wheat production regions and their evaluation [J]. Acta Agron Sin, 2016, 42(5): 768-777.
[30]	Zhong M, Wang LQ, Yuan DJ, et al. Identification of QTL affecting protein and amino acid contents in rice [J]. Rice Sci, 2011, 18(3): 187-195.
[31]	杨建昌, 李超卿, 江贻. 稻米氨基酸含量和组分及其调控 [J]. 作物学报, 2022, 48(5): 1037-1050.
	Yang JC, Li CQ, Jiang Y. Contents and compositions of amino acids in rice grains and their regulation: a review [J]. Acta Agron Sin, 2022, 48(5): 1037-1050.
[32]	Ishimaru T, Parween S, Saito Y, et al. Laser microdissection-based tissue-specific transcriptome analysis reveals a novel regulatory network of genes involved in heat-induced grain chalk in rice endosperm [J]. Plant Cell Physiol, 2019, 60(3): 626-642.
[33]	Huang A, Coutu C, Harrington M, et al. Engineering a feedback inhibition-insensitive plant dihydrodipicolinate synthase to increase lysine content in Camelina sativa seeds [J]. Transgenic Res, 2022, 31(1): 131-148.
[34]	He Q, Wang CC, He Q, et al. A complete reference genome assembly for foxtail millet and Setaria-db, a comprehensive database for Setaria [J]. Mol Plant, 2024, 17(2): 219-222.
[35]	Panchal A, Singh RK, Prasad M. Recent advancements and future perspectives of foxtail millet genomics [J]. Plant Growth Regul, 2023, 99(1): 11-23.
[36]	Galili G, Amir R, Fernie AR. The regulation of essential amino acid synthesis and accumulation in plants [J]. Annu Rev Plant Biol, 2016, 67: 153-178.
[37]	Ufaz S, Shukla V, Soloveichik Y, et al. Transcriptional control of aspartate kinase expression during darkness and sugar depletion in Arabidopsis: involvement of bZIP transcription factors [J]. Planta, 2011, 233(5): 1025-1040.
[38]	Duo H, Chhabra R, Muthusamy V, et al. Assessing sequence variation, haplotype analysis and molecular characterisation of aspartate kinase2 (ask2) gene regulating methionine biosynthesis in diverse maize inbreds [J]. Mol Genet Genomics, 2024, 299: 7.