Genome-wide Identification of the ME Family in Cyperus esculentusis and Functional Analysis of CeNAD-ME2

doi:10.13560/j.cnki.biotech.bull.1985.2025-0473

Abstract

Abstract:

Objective The systematic analysis of the members of the ME gene family in the yellow nutsedge (Cyperus esculentus) and the exploration of ME involved in the oil synthesis of the yellow nutsedge tuber provide a scientific reference for the comprehensive understanding of the oil-rich mechanism of the yellow nutsedge and the cultivation of new crop germplasm with oil-rich nutritional organs. Method Omics tools were employed to identify CeME gene family members and analyze their physicochemical properties. Quantitative PCR (RT-qPCR) was used to detect CeME expression patterns during key tuber developmental stages. Target CeME genes were heterologously expressed in Saccharomyces cerevisiae and Nicotiana tabacum. ME enzyme activity, lipid profiles, and metabolic shifts were analyzed in the transgenic lines. Result Six CeME genes were identified, and distributed across six chromosomes, including four NADP-dependent (CeNADP-ME1-CeNADP-ME4) and two NAD-dependent (CeNAD-ME1-CeNAD-ME2) isoforms. All CeMEs harbored 18-19 introns, with CeNADP-ME1 containing 19 introns. Promoter regions of CeMEs contained multiple cis-acting elements linked to development, hormones, and stress responses. All CeME proteins possessed canonical ME domains and twelve conserved motifs. CeMEs were upregulated during the tuber lipid accumulation phase (80-120 d after sowing), with CeNAD-ME2 exhibiting the highest expression. CeNAD-ME2 was localized to mitochondria. The overexpression of CeNAD-ME2 increased total lipids by 5.6% and palmitoleic acid (C16:1) by 28% compared to the wild-type. Transgenic tobacco lines showed 1.5‒4 fold higher ME activity, with total lipids and oleic acid (C18:1) content elevated by 5.2% and 5.6%, respectively, while soluble sugars and starch decreased by 2% and 5%. Conclusion Six CeME genes are identified in yellow nutsedge, with CeNAD-ME2 playing a pivotal role in tuber lipid biosynthesis. Heterologous expression of CeNAD-ME2 redirects carbon flux toward lipid synthesis, significantly enhancing total lipid and monounsaturated fatty acid content in hosts. These findings provide a foundation for elucidating lipid accumulation mechanisms in yellow nutsedge and engineering oil-enriched crops.

Key words: Cyperus esculentusis L, malic enzyme, genome identification, fatty acid synthesis, genetic transformation of yeast and tobacco

LI Zhan-qian, LI Chen, LI Shu-ting, MA Ju-hua, JING Hai-qing, SUN Yan, ZHOU Ya-li, XUE Jin-ai, LI Run-zhi. Genome-wide Identification of the ME Family in Cyperus esculentusis and Functional Analysis of CeNAD-ME2[J]. Biotechnology Bulletin, 2025, 41(10): 210-221.

Figures/Tables 16

Table 1 Primer sequences

基因 Gene	正向引物 Forward primer （5′-3′）	反向引物 Reverse primer （5′-3′）
CeNADP-ME1	GAGGAGCTGTTGCCATCTGT	TGTGACAGATGGCGAACGG
CeNADP-ME2	CACTGACGGAGGACGGATT	TCTGCGTGCTTGCCTATTACA
CeNADP-ME3	GCATGAGAAGAGCATCCAGG	CTTCTGCGTGCCTGCCTATTA
CeNADP-ME4	CTTCTGCCTCCTGCTATCGTT	TGGTTCCATCTTCAGGCGTC
CeNAD-ME1	TGGTGACAGATGGAAGCAGGA	CTGTATGTCTCCGCTGCTGG
CeNAD-ME2	AGAATGCTCGGCACTGACC	AGTGGTCAAGAAGGTGAAGCC
CeActin	TCGGTGGTATTGGAACTG	AAGCACTGGAGCGTAGCC
pBI121-CeNAD-ME2	aacctgcaggtcgactctagaATGTGGAACATGAGGCAGCTG	ggtttaaacgagctcggtaccTCACTTTTCATGAACCAAGGGG
pYES2.0-CeNAD-ME2	attaagcttggtaccgagctcATGTGGAACATGAGGCAGCTG	tacatgatgcggccctctagaTCACTTTTCATGAACCAAGGGG
1300-CeNAD-ME2	atacaccaaatcgactctagaATGTGGAACATGAGGCA	cgatcggggaaattcgagctcTCACTTTTCATGAACCAAGGGG

Fig. 1 Amino acid sequence alignment of the CeME family proteins (A, B) and the evolutionary tree analysis (C) of CeMEs with ME proteins from other plantsI‒V: Five conserved domains of malic enzyme. Ce: Cyperus esculentusis; At: Arabidopsis thaliana; Zm: Zea mays (maize). Ta: Triticum aestivum (wheat); Os: Oryza sativa (rice); St: Solanum tuberosum (potato); Gm: Glycine max (soybean)

Table 2 Analysis of the physiochemical properties of the ME family proteins in C. yperus esculentusis L.

基因 Gene	基因 ID Gene ID	氨基酸数 Number of amino acids	相对分子质量 Molecular weight (kD)	理论等电点 pI	不稳定系数 Instability factor	亲水性 Hydrophilicity	亚细胞定位预测 Prediction of subcellu-lar location
CeNADP-ME1	CESC_03761	650	71.11	7.00	37.79	-0.206	叶绿体
CeNADP-ME2	CESC_08481	577	64.01	5.97	41.44	-0.112	叶绿体
CeNADP-ME3	CESC_16067	641	71.43	6.73	44.48	-0.153	叶绿体
CeNADP-ME4	CESC_16827	662	73.62	6.78	49.33	-0.121	叶绿体
CeNAD-ME1	CESC_15235	623	68.87	5.59	43.08	-0.112	线粒体
CeNAD-ME2	CESC_17435	609	67.51	7.98	36.58	-0.119	线粒体

Fig. 2 Distribution of CeMEs genes on the chromosomes of C. esculentusis L.

Fig. 3 Collinear analysis of CeMEs gene family with 4 other plants (Arabidopsis thaliana, wheat, rice and soybean)

Fig. 4 Analysis of the conserved motifs and domains among CeMEs family (A)and the inron/exon organization of CeME genes (B) in C. esculentusis

Fig. 5 Cis-acting elements analysis of CeMEs genes in C. esculentusis L.

Fig. 6 3D prediction of the ME family proteins in C. esculentusis L.

Fig. 7 Quantitative analysis of ME gene expression in different stages of C. esculentusis tuber developmentDifferent letters indicate significant differences at P<0.05. The same below

Fig. 8 Subcellular localization analysis of CeNAD-ME2 protein

Fig. 9 Total fatty acid content and fatty acid profiles of the transgenic yeastINVSc1: Wild contro; EV: empty vector control; OE: overexpressed line

Fig. 10 Genetic transformation process in tobacco plants

Fig. 11 DNA molecular identification (A), RNA molecule identification (B) and relative expression (C) of CeNAD-ME2-transgenic tobacco plantsM: DL2000 marker; 1‒6: transfer PBI121-CeNAD-ME2 positive band; WT: wild control; EV: empty vector control; OE: transgenic PBI121-CeNAD-ME2 positive plants. The same below

Fig. 12 Activity detection of NAD-dependent malic enzyme in the wild and transgenic tobacco plants

Fig. 13 Contents of starch, soluble sugar, protein, total fatty acid, chlorophyll,and fatty acid profiles of the transgenic tobacco leaves

Fig. 14 Determination of oil content, seed germination rate, and thousand-seed weight of transgenic tobacco seeds

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