Cloning and Functional Analysis of the Soybean Enoyl-CoA Reductase ECR14 Gene

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

Abstract

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

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.

Figures/Tables 12

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

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

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

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.

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

Fig. 5 Subcellular localization of GmECR14 in Nicotiana tabacum

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

Fig. 7 Total wax load of soybean hairy roots

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

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

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

Fig. 11 Variations in GmECR14 heterologous expression of Arabidopsis permeability

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