油莎豆苹果酸酶（ME）全基因组鉴定及CeNAD-ME2功能分析

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

摘要/Abstract

摘要：

目的系统分析油莎豆苹果酸酶（malic enzyme, ME）基因家族成员、挖掘参与油莎豆块茎油脂合成的ME，为全面解析油莎豆块茎富油机制和培育营养器官富油的作物新种质提供科学参考。方法应用组学工具全基因组鉴定油莎豆CeME基因家族成员、分析CeME蛋白理化特性；运用实时荧光定量PCR（RT-qPCR）检测CeME基因在块茎不同发育时期的表达谱；构建靶标CeME表达载体，对酵母（Saccharomyces cerevisiae）和烟草（Nicotiana tabacum）进行遗传转化；检测转化体的ME酶活性和油脂代谢。结果从油莎豆基因组中共鉴定到6个CeMEs基因家族成员，分布于6条染色体，其中4个NADP类型（CeNADP-ME1-CeNADP-ME4）和2个NAD类型（CeNAD-ME1-CeNAD-ME2）。CeNADP-ME1具有19个内含子，其余5个CeME基因均含有18个内含子。CeME基因启动子含有生长发育、激素和逆境响应等多种顺式作用元件。6个CeME蛋白均具有典型的ME酶蛋白结构域和12个保守基序。RT-qPCR分析显示，CeME基因在油莎豆块茎油脂积累关键时期（播种后80‒120 d）表达上调，其中，NAD-ME类型的CeNAD-ME2表达量最高。CeNAD-ME2编码的酶蛋白定位于线粒体。过表达CeNAD-ME2酵母细胞总脂和棕榈油酸（C16：1）分别比野生型酵母提高5.6%和5%。与野生型烟草相比，转CeNAD-ME2株系ME活性增加1.5‒4倍，烟叶总脂和油酸（C18：1）含量分别提高5.2%和5.6%，而可溶性糖和淀粉含量分别下降2%和5%。结论从油莎豆基因组鉴定到6个CeME成员，CeNAD-ME2参与块茎油脂合成。异源表达CeNAD-ME2可驱动碳源更多地流向油脂合成途径，显著提高宿主总脂和单不饱和脂肪酸含量。

关键词: 油莎豆, 苹果酸酶, 全基因组鉴定, 油脂生物合成, 酵母和烟草遗传转化

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

李粘前, 李琛, 李淑婷, 马菊花, 景海青, 孙岩, 周雅莉, 薛金爱, 李润植. 油莎豆苹果酸酶（ME）全基因组鉴定及CeNAD-ME2功能分析[J]. 生物技术通报, 2025, 41(10): 210-221.

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.

图/表 16

表1 引物序列

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

图1 油莎豆ME家族蛋白氨基酸序列比对（A、B）及与其他植物ME蛋白的进化树分析（C）I‒V：苹果酸酶的5个保守结构域。Ce：油莎豆；At：拟南芥；Zm：玉米；Ta：小麦；Os：水稻；St：马铃薯；Gm：大豆

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)

表2 油莎豆CeME家族蛋白理化性质分析

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

图2 油莎豆ME基因在染色体上的分布

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

图3 油莎豆CeMEs基因家族与4种典型植物（拟南芥、小麦、水稻和大豆）的共线性分析

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

图4 油莎豆CeMEs家族蛋白的保守基序/结构域分析（A）和基因内含子/外显子组成分析（B）

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

图5 油莎豆CeMEs基因的顺式作用元件

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

图6 油莎豆ME家族蛋白三维结果预测

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

图7 油莎豆块茎发育不同时期ME基因表达量的定量分析不同字母表示在P<0.05水平差异显著。下同

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

图8 CeNAD-ME2蛋白的亚细胞定位分析

Fig. 8 Subcellular localization analysis of CeNAD-ME2 protein

图9 转基因酵母总脂肪酸含量和转基因酵母脂肪酸组分INVSc1：野生型对照；EV：空载对照；OE：过表达株系

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

图10 烟草遗传转化过程

Fig. 10 Genetic transformation process in tobacco plants

图11 转CeNAD-ME2烟草植株的DNA分子鉴定（A）、RNA分子鉴定（B）及相对表达量（C）M：DL2000 marker；1‒6：转PBI121-CeNAD-ME2阳性条带；WT：野生对照；EV：空载对照：OE：转PBI121-CeNAD-ME2阳性植株。下同

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

图12 野生型和转基因烟草NAD-ME酶的活性检测

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

图13 转基因烟草叶片淀粉、可溶性糖、蛋白、总脂肪酸、叶绿素和脂肪酸组分测定

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

图14 转基因烟草种子油含量、种子萌发率、种子千粒重的测定

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

参考文献 39

[1]	Sánchez-Zapata E, Fernández-López J, Angel Pérez-Alvarez J. Tiger nut (Cyperus esculentus) commercialization: health aspects, composition, properties, and food applications [J]. Compr Rev Food Sci Food Saf, 2012, 11(4): 366-377.
[2]	Bullen JJ, Rogers HJ, Spalding PB, et al. Natural resistance, iron and infection: a challenge for clinical medicine [J]. J Med Microbiol, 2006, 55(Pt 3): 251-258.
[3]	瞿萍梅, 程治英, 龙春林, 等. 油莎豆资源的综合开发利用 [J]. 中国油脂, 2007, 32(9): 61-63.
	Qu PM, Cheng ZY, Long CL, et al. Comprehensive development of chufa (Cyperus esculentus L. var. sativus) [J]. China Oils Fats, 2007, 32(9): 61-63.
[4]	Makareviciene V, Gumbyte M, Yunik A, et al. Opportunities for the use of chufa sedge in biodiesel production [J]. Ind Crops Prod, 2013, 50: 633-637.
[5]	Li T, Sun Y, Chen Y, et al. Characterisation of two novel genes encoding Δ9 fatty acid desaturases (CeSADs) for oleic acid accumulation in the oil-rich tuber of Cyperus esculentus [J]. Plant Sci, 2022, 319: 111243.
[6]	McCullough W, Roberts CF. The role of malic enzyme in Aspergillus nidulans [J]. FEBS Lett, 1974, 41(2): 238-242.
[7]	Boles E, de Jong-Gubbels P, Pronk JT. Identification and characterization of MAE1 the Saccharomyces cerevisiae structural gene encoding mitochondrial malic enzyme [J]. J Bacteriol, 1998, 180(11): 2875-2882.
[8]	Thelen JJ, Ohlrogge JB. Metabolic engineering of fatty acid biosynthesis in plants [J]. Metab Eng, 2002, 4(1): 12-21.
[9]	周滈, 杨传平, 柳参奎. 植物苹果酸酶在抵御逆境中的作用 [J]. 林业科技, 2011, 36(6): 44-47.
	Zhou H, Yang CP, Liu SK. The role of plant malic enzyme in defense against stress environments [J]. For Sci Technol, 2011, 36(6): 44-47.
[10]	Rao XL, Dixon RA. The differences between NAD-ME and NADP-ME subtypes of C₄ photosynthesis: more than decarboxylating enzymes [J]. Front Plant Sci, 2016, 7: 1525.
[11]	Wheeler MC, Tronconi MA, Drincovich MF, et al. A comprehensive analysis of the NADP-malic enzyme gene family of Arabidopsis [J]. Plant Physiol, 2005, 139(1): 39-51.
[12]	Tronconi MA, Fahnenstich H, Gerrard Weehler MC, et al. Arabidopsis NAD-malic enzyme functions as a homodimer and heterodimer and has a major impact on nocturnal metabolism [J]. Plant Physiol, 2008, 146(4): 1540-1552.
[13]	Alvarez CE, Saigo M, Margarit E, et al. Kinetics and functional diversity among the five members of the NADP-malic enzyme family from Zea mays, a C₄ species [J]. Photosynth Res, 2013, 115(1): 65-80.
[14]	Ratledge C. The role of malic enzyme as the provider of NADPH in oleaginous microorganisms: a reappraisal and unsolved problems [J]. Biotechnol Lett, 2014, 36(8): 1557-1568.
[15]	Zhu BH, Zhang RH, Lv NN, et al. The role of malic enzyme on promoting total lipid and fatty acid production in Phaeodactylum tricornutum [J]. Front Plant Sci, 2018, 9: 826.
[16]	Zhang Y, Adams IP, Ratledge C. Malic enzyme: the controlling activity for lipid production overexpression of malic enzyme in Mucor circinelloides leads to a 2.5-fold increase in lipid accumulation [J]. Microbiology, 2007, 153(Pt 7): 2013-2025.
[17]	Wynn JP, Ratledge C. Malic enzyme is a major source of NADPH for lipid accumulation by Aspergillus nidulans [J]. Microbiology, 1997, 143(1): 253-257.
[18]	Gerrard Wheeler MC, Arias CL, Righini S, et al. Differential contribution of malic enzymes during soybean and castor seeds maturation [J]. PLoS One, 2016, 11(6): e0158040.
[19]	Morley SA, Ma FF, Alazem M, et al. Expression of malic enzyme reveals subcellular carbon partitioning for storage reserve production in soybeans [J]. New Phytol, 2023, 239(5): 1834-1851.
[20]	Ji HY, Liu DT, Yang ZL. High oil accumulation in tuber of yellow nutsedge compared to purple nutsedge is associated with more abundant expression of genes involved in fatty acid synthesis and triacylglycerol storage [J]. Biotechnol Biofuels, 2021, 14(1): 54.
[21]	Gao Y, Sun Y, Gao HL, et al. Correction to: Ectopic overexpression of a type-II DGAT (CeDGAT2-2) derived from oil-rich tuber of Cyperus esculentus enhances accumulation of oil and oleic acid in tobacco leaves [J]. Biotechnol Biofuels, 2021, 14(1): 139.
[22]	李秀峰, 张欣欣, 高野哲夫, 等. 水稻（Oryza sativa L.）苹果酸酶（OsNADP-ME3）基因在逆境下的表达特性研究 [J]. 基因组学与应用生物学, 2012, 31(4): 327-332.
	Li XF, Zhang XX, Takano T, et al. Expression characteristics of rice (Oryza sativa L.) malic enzyme (OsNADP-ME3) gene under environmental stress [J]. Genom Appl Biol, 2012, 31(4): 327-332.
[23]	刘增辉. 小麦NADP-苹果酸酶与逆境关系的初步研究 [D]. 青岛: 青岛科技大学, 2010.
	Liu ZH. Preliminary study on the relationg of NADP-malic enzyme and stresses [D]. Qingdao: Qingdao University of Science & Technology, 2010.
[24]	Kawaoka A, Kawamoto T, Sekine M, et al. A cis-acting element and a trans-acting factor involved in the wound-induced expression of a horseradish peroxidase gene [J]. Plant J, 1994, 6(1): 87-97.
[25]	Ni M, Tepperman JM, Quail PH. PIF3, a phytochrome-interacting factor necessary for normal photoinduced signal transduction, is a novel basic helix-loop-helix protein [J]. Cell, 1998, 95(5): 657-667.
[26]	张玉慧, 王玉兰, 夏春兰, 等. 松叶猪毛菜NADP-苹果酸酶基因家族及启动子克隆分析 [J]. 西北植物学报, 2022, 42(5): 760-769.
	Zhang YH, Wang YL, Xia CL, et al. Cloning and analysis of NADP-ME gene family and promoters in Salsola laricifolia [J]. Acta Bot Boreali Occidentalia Sin, 2022, 42(5): 760-769.
[27]	史先飞, 高宇, 黄旭升, 等. 油莎豆丙酮酸激酶基因的鉴定及其在块茎发芽和幼苗形态建成时期的表达分析 [J]. 激光生物学报, 2022, 31(6): 533-541.
	Shi XF, Gao Y, Huang XS, et al. Identification of pyruvate kinase genes and their expression analysis during Cyperus esculentus tuber germination and seedling establishment [J]. Acta Laser Biol Sin, 2022, 31(6): 533-541.
[28]	Dao O, Kuhnert F, Weber APM, et al. Physiological functions of malate shuttles in plants and algae [J]. Trends Plant Sci, 2022, 27(5): 488-501.
[29]	Wedding RT. Malic enzymes of higher plants: characteristics, regulation, and physiological function [J]. Plant Physiol, 1989, 90(2): 367-371.
[30]	Bonnerot C, Galle AM, Jolliot A, et al. Purification and properties of plant cytochrome b5 [J]. Biochem J, 1985, 226(1): 331-334.
[31]	Kumar R, Tran LP, Neelakandan AK, et al. Higher plant cytochrome b5 polypeptides modulate fatty acid desaturation [J]. PLoS One, 2012, 7(2): e31370.
[32]	Schultz CJ, Coruzzi GM. The aspartate aminotransferase gene family of Arabidopsis encodes isoenzymes localized to three distinct subcellular compartments [J]. Plant J, 1995, 7(1): 61-75.
[33]	Allen DK, Young JD. Carbon and nitrogen provisions alter the metabolic flux in developing soybean embryos [J]. Plant Physiol, 2013, 161(3): 1458-1475.
[34]	Eastmond PJ, Dennis DT, Rawsthorne S. Evidence that a malate/inorganic phosphate exchange translocator imports carbon across the leucoplast envelope for fatty acid synthesis in developing castor seed endosperm [J]. Plant Physiol, 1997, 114(3): 851-856.
[35]	李秀婷. 现代啤酒生产工艺 [M]. 北京: 中国农业大学出版社, 2013.
	Li XT. Modern technology of beer production [M]. Beijing: China Agricultural University Press, 2013.
[36]	荆美玲. 油莎豆油脂合成基因CePDAT3基因的克隆及功能研究 [D]. 长春: 吉林农业大学, 2022.
	Jing ML. Cloning and functional study of CePDAT3 gene for oil synthesis fromcyperus esculentusis [D]. Changchun: Jilin Agricultural University, 2022.
[37]	Cramer CL, Weissenborn DL, Oishi KK, et al. Bioproduction of human enzymes in transgenic tobacco [J]. Ann N Y Acad Sci, 1996, 792(1): 62-71.
[38]	吉夏洁. 过表达蓖麻RcWRI1提高烟叶油脂积累 [D]. 太谷: 山西农业大学, 2018.
	Ji XJ. Overexpression of Ricinus communis RcWRI1 improves oil accumulation in tobacco leaves [D]. Taigu: Shanxi Agricultural University, 2018.
[39]	Wilson RS, Thelen JJ. In vivo quantitative monitoring of subunit stoichiometry for metabolic complexes [J]. J Proteome Res, 2018, 17(5): 1773-1783.