大豆烯酰辅酶A还原酶ECR14基因的克隆与功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0493

摘要/Abstract

摘要：

目的 ECR基因是大豆（Glycine max (L.) Merr.）植物表皮蜡质合成途径中的重要基因，研究大豆GmECR14基因的生物学功能，为进一步探索其分子机制提供理论基础，同时为培育优质、耐逆大豆新品种提供参考。方法利用PCR技术从大豆品种‘Jack’中克隆得到GmECR14基因，结合生物信息学分析其理化性质、蛋白结构以及系统进化关系，通过RT-qPCR技术分析GmECR14基因在干旱及盐胁迫下的表达模式，利用亚细胞定位确定其蛋白表达的位置，构建植物过表达载体和敲除载体，并通过农杆菌介导的遗传转化将GmECR14基因转入大豆毛状根、拟南芥中，分析验证GmECR14基因功能。结果 GmECR14基因编码区全长930 bp，编码309个氨基酸，为典型的跨膜蛋白；启动子分析表明，该基因启动子上存在多种逆境胁迫相关顺式作用元件；亚细胞定位显示其蛋白定位于内质网中。系统进化关系表明大豆GmECR14基因与花生（Arachis hypogaea）AhECR蛋白序列亲缘关系较近。实时荧光定量PCR分析表明，GmECR14基因在干旱和盐胁迫下均受到诱导表达。对转基因大豆毛状根表型、蜡质含量及20% PEG 6000、200 mmol/L NaCl胁迫下进行分析，结果表明过表达植株侧根数较对照明显增多，蜡质含量为对照的24%，耐旱、耐盐性显著提高；而敲除植株中侧根数显著减少，蜡质含量与对照无差异，干旱及盐胁迫后阳性株死亡。在拟南芥中异源表达GmECR14基因，过表达植株蜡质含量较对照增加16.4%，且莲座叶的叶绿素浸出率和水分散失速率均明显下降。结论 GmECR14基因正向调控大豆蜡质合成，提高植株的耐旱性和耐盐性。

关键词: 大豆, 植物表皮蜡质, GmECR14基因, 遗传转化, 盐胁迫, 干旱胁迫

Abstract:

Objective The ECR gene is a key one in the cuticular wax biosynthesis pathway of soybean plants. This study aims to investigate the biological function of the soybean GmECR14 gene, provide a theoretical basis for further exploring its molecular mechanism, and offer references for breeding new soybean varieties with high quality and stress tolerance. Method PCR technology was used to clone the GmECR14 gene the soybean cultivar ‘Jack’ . Bioinformatics analysis was conducted to characterize its physicochemical properties, protein structure, and phylogenetic relationship. RT-qPCR was used to analyze the expression pattern of GmECR14 under drought and salt stress. Subcellular localization was performed to determine the expression site of its encoded protein. Plant overexpression and knockout vectors were constructed, and the GmECR14 gene was introduced into soybean hairy roots and Arabidopsis thaliana via Agrobacterium-mediated genetic transformation to verify its function. Result The coding region of GmECR14 is 930 bp in length, encoding 309 amino acids, and the encoded protein was a typical transmembrane protein. Promoter analysis revealed that the presence of multiple cis-acting elements was related to abiotic stress in the gene promoter. Subcellular localization showed that the protein was localized in the endoplasmic reticulum. Phylogenetic analysis indicated that the soybean GmECR14 gene had a close genetic relationship with the AhECR protein sequence of peanut (Arachis hypogaea). Real-time fluorescent quantitative PCR analysis showed that the expression of GmECR14 was induced under both drought and salt stress. Phenotypic analysis, cuticular wax content determination, and stress tests (20% PEG 6000 and 200 mmol/L NaCl) of transgenic soybean hairy roots showed that: compared with the control, the overexpressing lines had significantly more lateral roots, the cuticular wax content was 0.24 times that of the control, and the tolerances to drought and salt were significantly improved. In contrast, the knockout lines had significantly fewer lateral roots, no difference in cuticular wax content compared with the control, and positive plants died after drought and salt stress. Heterologous expression of GmECR14 in A. thaliana showed that the cuticular wax content of overexpressing plants increased by 16.4% compared with the control, and the chlorophyll leaching rate and water loss rate of rosette leaves significantly decreased. Conclusion The GmECR14 gene positively regulates cuticular wax biosynthesis in soybeans and enhances the drought and salt tolerance of plants.

Key words: soybean, plant cuticular wax, GmECR14 gene, genetic transformation, salt stress, drought stress

张月, 戴月华, 张莹莹, 李奥辉, 李楚慧, 薛金爱, 秦慧彬, 陈妍, 聂萌恩, 张海平. 大豆烯酰辅酶A还原酶ECR14基因的克隆与功能分析[J]. 生物技术通报, 2026, 42(1): 95-104.

ZHANG Yue, DAI Yue-hua, ZHANG Ying-ying, LI Ao-hui, LI Chu-hui, XUE Jin-ai, QIN Hui-bin, CHEN Yan, NIE Meng-en, ZHANG Hai-ping. Cloning and Functional Analysis of the Soybean Enoyl-CoA Reductase ECR14 Gene[J]. Biotechnology Bulletin, 2026, 42(1): 95-104.

图/表 12

表1 GmECR14基因顺式作用元件统计

Table 1 Statistics of cis-acting elements of GmECR14 gene

顺式元件类型 Cis-acting element	生物学功能 Biological function	核心序列 Core sequence
LTR	胁迫响应 Stress responsive	CCGAAA
ARE	胁迫响应 Stress responsive	AAACCA
TCT-motif	光响应 Light responsive	TCTTAC
GATA-motif	光响应 Light responsive	GATA
MRE	光响应 Light responsive	AACCTAA
GT1-motif	光响应 Light responsive	GGTTAA
Box 4	光响应 Light responsive	ATTAAT
O²-site	生长发育 Growth and development	GATGACATGG
MBS	干旱诱导性 Drought inducibility	CAACTG
TCA-element	水杨酸 Salicylic acid	CCATCTTTTT

图1 GmECR14蛋白的理化性质分析A：GmECR14蛋白亲水性预测；B：GmECR14蛋白跨膜结构域预测

Fig. 1 Analysis of the GmECR14 protein's physicochemical characteristicsA: Forecasting the hydrophilicity profile for the GmECR14 protein. B: Prediction of the GmECR14 protein’s transmembrane domain

图2 GmECR14蛋白的二级结构（左）和三级结构（右）

Fig. 2 Secondary structure (left) and tertiary structure (right) of soybean GmECR14 proteins

图3 GmECR14与其他物种ECR蛋白序列的系统进化树Gm：大豆；At：拟南芥；Bc：油菜；Cs：脐橙；Si：谷子；Mi：芒果；Sb：高粱；Md：苹果；Cm：葫芦；Qr：板栗；Gh：棉花；Rc：蓖麻；Pa：樱桃；As：花生；Ta：小麦；Vv：葡萄；Cxc：柑橘；Ntc：烟草

Fig. 3 Phylogenetic tree of GmECR 14 and ECR proteins of other plant speciesGm: Glycine max. At: Arabidopsis thaliana. Bc: Brassica napus L. Cs: Citrus sinensis (L) Osbeck. Si: Setaria italica. Mi: Mangifera indica L. Sb: Sorghum bicolor L. Md: Malus pumila Mill. Cm: Lagenaria Siceraria. Qr: Castanea mollissima Blume. Gh: Gossypium hirsutum. Rc: Ricinus communis L. Pa: Prunus pseudocerasus. As: Arachis hypogaea L. Ta: Triticum aestivum L. Vv: Vitis vinifera L. Cxc: Citrus reticulate Blanco. Ntc: Nicotiana tabacum L.

图4 GmECR14表达模式分析误差线为标准误，图中不同字母表示差异显著（P<0.05），下同

Fig. 4 Analysis of expression pattern of GmECR14Error bars indicate standard errors. The different letters in the figure indicate significant differences (P<0.05). The same below

图5 GmECR14在烟草中的亚细胞定位

Fig. 5 Subcellular localization of GmECR14 in Nicotiana tabacum

图6 大豆毛状根表型A：不同处理下大豆毛状根侧根数差异图；B：对照毛状根；C：敲除阳性毛状根；D：不同处理下大豆毛状根侧根对比图。**：P<0.01

Fig. 6 Soybean hairy root phenotypeA: Difference in the number of lateral roots of soybean hairy roots under different treatments. B: Control hairy roots. C: Knockout-positive soybean hairy roots. D: Comparison of lateral roots of soybean hairy roots under different treatments

图7 大豆毛状根总蜡量

Fig. 7 Total wax load of soybean hairy roots

图8 干旱和盐处理下大豆毛状根的表型

Fig. 8 Phenotypes of soybean hairy roots under drought and salt treatments

图9 干旱和盐处理下大豆毛状根GmECR14基因的相对表达水平

Fig. 9 Relative expressions of GmECR14 gene in the soybean hairy roots under drought and salt treatments

图10 野生型和GmECR14转基因株系叶片总蜡量

Fig. 10 Total wax load in the leaves of wild-type and GmECR14-transgenec lines

图11 GmECR14异源表达拟南芥通透性变化

Fig. 11 Variations in GmECR14 heterologous expression of Arabidopsis permeability

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