Identification of the AsparagusCYP51 Gene Family and the Response to Abiotic Stress

doi:10.13560/j.cnki.biotech.bull.1985.2025-0469

Abstract

Abstract:

Objective Sterol 14α-demethylase (CYP51), a key enzyme in the plant sterol biosynthesis pathway, plays an important role in plant responses to abiotic stress. This study is aimed to identify CYP51 gene family members genome-wide in Asparagus, analyze their molecular characteristics, and validate the function of AtaCYP51G2 from Asparagus taliensis in response to abiotic stress, thereby providing a basis for understanding the molecular mechanisms of stress resistance in Asparagus plants. Method Bioinformatic methods were used to identify CYP51 family members in A. taliensis, A. officinalis, and A. setaceus. Their physicochemical properties,chromosomal localization, gene duplication, phylogenetic relationships, gene structures, and cis-acting elements in promoter regions were analyzed. Transgenic Arabidopsis thaliana lines overexpressing AtaCYP51G2 were generated, and phenotypic observations and physiological/biochemical measurements were performed under drought, salt, and osmotic stress conditions. Real-time quantitative PCR was employed to detect the expression levels of the target gene in transgenic lines and the changes in AtaCYP51G2 expression under stress treatments. Result Four CYP51 gene family members were identified in Asparagus species. Promoter analysis revealed the presence of hormone- and stress-responsive cis-acting elements, which were particularly abundant in the AtaCYP51G2 promoter. Subcellular localization experiments indicated that the AtaCYP51G2 protein is localized to the endomembrane system. The overexpression of AtaCYP51G2 enhanced the tolerance of transgenic Arabidopsis to drought and osmotic stress and partially alleviated oxidative damage under salt stress. Conclusion The AsparagusCYP51 gene family has a small number of members and is highly conserved in terms of gene structure and protein sequence. The overexpression of AtaCYP51G2 enhances the tolerance of transgenic Arabidopsis to drought, salt, and osmotic stress, providing a theoretical basis and potential genetic resources for improving stress resistance in Asparagus plants.

Key words: Asparagus, CYP51, Asparagus taliensis, abiotic stress, gene family, transgenic

ZENG Liang-qin, DONG Chen-wen-hua, LIN Chun, LIU Zheng-jie, MAO Zi-chao. Identification of the AsparagusCYP51 Gene Family and the Response to Abiotic Stress[J]. Biotechnology Bulletin, 2025, 41(12): 1-14.

Figures/Tables 13

Table 1 Primers used in this study

引物编号 Primer number	引物序列 Primer sequence（5'-3'）	目的 Purpose
AtaCYP51G2-1F	ATGGATTTAACAGAGAACAAGTTCTTG	基因克隆
AtaCYP51G2-1R	TTATTCAACCGAAAGCTTCCTCC	基因克隆
AtaCYP51G2-2F	TGGTCGACGTACTAGATGGATTTAACAGAGAACAAGTTCTTGAGCA	载体构建
AtaCYP51G2-2R	GCTCACCATCACTAGTTCAACCGAAAGCTTCCTCCTCTTGT	载体构建
AtaCYP51G2-qF	TCCCGTAAATGCTCAGGTCG	RT-qPCR
AtaCYP51G2-qR	GTTGAGGTGATCGAGCTGGT	RT-qPCR
ACTIN-qF	TGACTACGAGCAGGAGATGGAA	RT-qPCR
ACTIN-qR	AAACGAGGGCTGGAACAAGA	RT-qPCR

Table 2 Physicochemical characteristics of CYP51 gene family in Asparagus species

物种 Species	基因名称 Gene name	基因编号 Gene ID	蛋白长度 Protein length （aa）	分子量 Molecular weight （kD）	等电点 Isoelectric point	脂肪指数 Aliphatic index	平均亲水性 Grand average of ydropathicity	不稳定指数 Instability index	亚细胞定位预测 Predict subcellular localization
大理天门冬 A. taliensis	AtaCYP51G1	Ata0G001960.1	488	55.4	8.7	93.3	-0.116	35.7	质膜 Plasma membrane
大理天门冬 A. taliensis	AtaCYP51G2	Ata09G008170.1	488	55.4	8.69	94.1	-0.101	34.05	内膜 Inner membrane
芦笋 A. officinalis	AoCYP51G1	AsparagusV1_Unassigned.240	510	58.0	8.94	93.84	-0.106	34.85	质膜 Plasma membrane
文竹 A. setaceus	AsCYP51G1	Ase07G1939-RA	488	55.5	8.55	91.68	-0.14	36.22	质膜 Plasma membrane

Fig. 1 Chromosomal localization of the CYP51 gene family in Asparagus speciesA-C indicate the chromosomal localization of the CYP51 gene family in A. taliensis, A. officinalis, and A. setaceus, respectively. The CYP51 genes in Asparagus species are marked in red. Gray lines indicate intraspecific collinear gene pairs

Fig. 2 Phylogenetic tree of CYP51 proteinsThe CYP51 genes in Asparagus species are marked in red. Ata:A. taliensis;Ao:A. officinalis;As:A. setaceus;At:A. thaliana;Os:O. sativa;Mt:Mycobacterium tuberculosis

Fig. 3 Gene structure, conserved motifs, and conserved domain analysis of the CYP51 gene family in Asparagus speciesA: Phylogenetic tree of Asparagus CYP51 protein. B: Gene structure. C: Conserved motifs. D: Conserved domains. E: Conserved motif annotations

Fig. 4 Cis-acting element analysis of the CYP51 gene family in Asparagus speciesA: Distribution of cis-acting elements. B: Number of cis-acting elements. C: Types and quantities of cis-acting elements

Fig. 5 Primary sequence alignment, tertiary structure prediction and alignment of CYP51 proteins in Asparagus speciesA: Predicting tertiary structure of proteins. B: Alignment of tertiary structures. C: Multiple-sequence alignment of primary structures

Fig. 6 Gene expressions of CYP51 genes in different tissues of A. taliensis

Fig. 7 Identification of AtaCYP51G2 transgenic Arabidopsis linesA: Genomic PCR analysis of T2 plants. Lane 1-6: line OE-1; lane 7-12: line OE-2. B: Relative expression of AtaCYP51G2 in wild type (WT), vector control (VC), and transgenic lines (OE-1 and OE-2), determined by qRT-PCR. Data are presented as mean±SD (n=3). Different letters indicate statistically significant differences (P<0.05)

Fig. 8 Subcellular localization of AtaCYP51G2 proteinA: Construction of the expression vector. B: Subcellular localization in tobacco protoplasts

Fig. 9 Evaluation of seed germination and root length in transgenic Arabidopsis under salt and osmotic stressA-C: Phenotypic observation of Arabidopsis grown on solid medium. D: Measurement of Arabidopsis seed germination rate. E: Measurement of Arabidopsis primary root length

Fig. 10 Phenotype and physiological and biochemical measurements of AtaCYP51G2-overexpressing Arabidopsis under drought stressA: Phenotype of wild-type (WT) and AtaCYP51G2-overexpressing (OE) Arabidopsis lines under drought stress; B-F: indiate the physiological and biochemical indicators of WT and OE Arabidopsis plants under drought stress, respectively: total chlorophyll content, malondialdehyde content, proline content, catalase activity, and peroxidase activity. The same below. G: Relative gene expressions

Fig. 11 Physiological measurements of AtaCYP51G2-overexpressing Arabidopsis under salt and osmotic stressA-F: Physiological and biochemical parameters and the relative transcript levels of AtaCYP51G2 overexpression lines under salt stress, whereas panels G-L display the corresponding data under osmotic stress

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