天门冬属CYP51基因家族鉴定及在非生物胁迫中的响应

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

摘要/Abstract

摘要：

目的甾醇14α-去甲基化酶（sterol 14α-demethylase, CYP51）是植物甾醇生物合成途径中的关键酶，在植物应对非生物胁迫中发挥重要作用。从天门冬属全基因组范围内鉴定CYP51基因家族成员，分析其分子特征及验证大理天门冬AtaCYP51G2基因在非生物胁迫的响应功能，为理解天门冬属植物的抗逆分子机制提供依据。方法利用生物信息学方法，鉴定了天门冬属大理天门冬、芦笋和文竹的CYP51基因家族成员，分析其理化性质、染色体定位、基因复制、系统进化、基因结构和启动子顺式作用元件等。通过构建AtaCYP51G2转基因拟南芥株系，在干旱、盐和渗透胁迫条件下，对其进行表型观察和生理生化指标测定。利用实时荧光定量PCR检测转基因株系中基因的表达水平及胁迫处理下AtaCYP51G2的表达变化。结果共鉴定出天门冬属植物4个CYP51基因家族成员。启动子分析显示存在激素和胁迫响应元件，在AtaCYP51G2中富集。亚细胞定位实验表明AtaCYP51G2定位于内膜系统。过表达AtaCYP51G2增强了转基因拟南芥对干旱和渗透胁迫的耐受性，对盐胁迫下的氧化损伤也有一定的缓解作用。结论天门冬属CYP51基因家族成员数量少，在基因结构和蛋白序列上高度保守。过表达AtaCYP51G2增强了转基因拟南芥对干旱、盐和渗透胁迫的耐受性，为改良天门冬属植物的抗逆性提供了理论基础和潜在基因资源。

关键词: 天门冬属, CYP51, 大理天门冬, 非生物胁迫, 基因家族, 转基因

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

曾梁秦, 董陈文华, 林春, 刘正杰, 毛自朝. 天门冬属CYP51基因家族鉴定及在非生物胁迫中的响应[J]. 生物技术通报, 2025, 41(12): 254-266.

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): 254-266.

图/表 13

表1 本研究所用引物

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

表2 天门冬属植物CYP51基因家族成员的理化性质

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 hydropathicity	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

图1 天门冬属CYP51基因家族的染色体定位A-C分别是大理天门冬、芦笋、文竹的CYP51基因家族的染色体定位；标注为红色的是天门冬属CYP51基因；灰色线条是种内共线基因对

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

图2 CYP51蛋白系统进化树红色为天门冬属CYP51基因家族成员。Ata：大理天门冬；Ao：芦笋；As：文竹；At：拟南芥；Os：水稻；Mt：结核杆菌

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

图3 天门冬属植物CYP51基因家族基因结构、保守基序和保守结构域分析A：天门冬属CYP51蛋白系统进化树；B：基因结构；C：保守基序；D：保守结构域；E：保守基序标识

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

图4 天门冬属植物CYP51基因家族顺式作用元件分析A：顺式作用元件分布；B：顺式作用元件数量；C：顺式作用元件类型及数量

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

图5 天门冬属植物CYP51蛋白的一级结构多序列比对、三级结构预测及比对A：蛋白三级结构预测；B：蛋白三级结构比对；C：蛋白一级结构多序列比对

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

图6 大理天门冬不同组织中CYP51基因的相对表达量*P<0.05， **P<0.01， ***P<0.01， ****P<0.001； the same below

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

图7 AtaCYP51G2转基因拟南芥株系的鉴定A：T2代株系的基因组PCR检测。泳道1-6：OE-1株系；泳道7-12：OE-2株系。B：AtaCYP51G2基因在野生型（WT）、空载体对照（VC）、转基因株系（OE-1、OE-2）中的相对表达量。数据表示平均值±标准差（n=3），不同字母表示显著差异（P<0.05）

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)

图8 AtaCYP51G2蛋白亚细胞定位A：表达载体构建；B：在烟草原生质体中的亚细胞定位

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

图9 盐胁迫和渗透胁下转基因拟南芥种子萌发和根长的测定A-C：固体培养基中拟南芥表型观察；D：拟南芥种子萌发率测定；E：拟南芥主根长测定

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

图10 过表达AtaCYP51G2拟南芥在干旱胁迫下的表型及生理生化指标测定A：干旱胁迫下WT及OE拟南芥株系的表型；B-F：分别是干旱胁迫下WT及OE拟南芥的生理生化指标：总叶绿素、丙二醛、脯氨酸含量及过氧化氢酶活性、过氧化物酶活性；下同；G：相对基因表达水平

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: 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

图11 过表达AtaCYP51G2拟南芥在盐胁迫和渗透胁迫下的生理生化指标测定A-F：盐胁迫下过表达AtaCYP51G2的生理生化指标值和基因相对表达量测定值；G-L：渗透胁迫下对应的生理生化指标值和基因相对表达量测定值

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 overexpressing lines under salt stress, whereas panel G-L display the corresponding data under osmotic stress

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