丹参ADH基因家族的鉴定与表达模式分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-1233

摘要/Abstract

摘要：

目的乙醇脱氢酶（Alcohol dehydrogenase，ADH）是厌氧条件下植物发酵代谢的关键酶之一，在植物抵抗涝渍胁迫中发挥着重要作用。鉴定丹参SmADH基因家族，为丹参SmADH基因家族功能研究及选育抗涝品种提供理论依据。方法基于丹参基因组数据对基因家族成员进行鉴定，并对其理化性质、序列特征、系统发育、基因表达等进行研究；采用RT-qPCR研究家族成员在不同胁迫下的表达模式。结果共鉴定出7条SmADH基因，分属两个亚家族。SmADH蛋白主要为酸性蛋白，编码氨基酸数目379-438，分子量40.79-46.89 kD。除SmADH5/6定位于叶绿体外，其他成员都定位在细胞质中。基因特征分析显示，同一亚族间的成员在保守基序、内含子数目及分布上都比较保守。染色体定位显示，SmADH1位于1号染色体，SmADH2/3/4位于2号染色体，SmADH5/6位于6号染色体，而SmADH7位于contig030片段上。调控miRNA分析显示，SmADH3/4/5/6等可能受smi-miR167、smi-miRN5和smi-miR482等miRNA家族成员的调控。启动子顺式作用元件分析显示，ADH成员启动子区存在大量的胁迫响应及激素响应相关的元件。转录组数据及RT-qPCR分析表明，SmADH2/5/6在根中表达量最高；涝渍胁迫下，SmADH2/6表达迅速上调；盐胁迫下，SmADH2/6的表达显著上调，而SmADH5的表达受到抑制；PEG处理后，SmADH2/5/6的表达显著上调；此外，SmADH6表达还受到GA3的诱导。结论丹参根部高表达的SmADH2/5/6在多种胁迫下发挥作用，而SmADH2/6在抗涝渍胁迫中发挥着尤为重要的作用。

关键词: 丹参, ADH, 表达分析, 涝渍胁迫, 基因家族

Abstract:

Objective Alcohol dehydrogenase (ADH) is one of the key enzymes in plant fermentation metabolism under anaerobic conditions, and plays important roles in plant resistance to waterlogging stress. This work is aimed to identify the SmADH genes from Salvia miltiorrhiza and provide a theoretical basis for the functional study of the SmADH gene family and the breeding of flood-resistant varieties. Method Bioinformatics methods were used to identify members of the gene family based on the S. miltiorrhiza genome data, and to analyze their physicochemical properties, sequence characteristics, evolutionary, and expression regulation. The RT-qPCR was then used to study the expression patterns of SmADHs under different stresses. Result A total of seven SmADH genes were identified, belonging to two subfamilies. SmADH proteins were mainly acidic proteins. The number of encoded amino acids ranged from 379 to 438, with a molecular weight of 40.79 to 46.89 kD. Except for SmADH5/6 localized in chloroplasts, other members were localized in the cytoplasm. Genetic feature analysis showed that members of the same subfamily of SmADHs possessed conserved motifs, similar intron numbers, and intron distributions. Chromosome localization revealed that SmADH1 was located on chromosome 1, SmADH2/3/4 on chromosome 2, SmADH5/6 on chromosome 6, and SmADH7 on contig030. Regulation by miRNA analysis showed that SmADH3/4/5/6 were regulated by miRNA family members such as smi-miR167, smi-miRN5 and smi-miR482. Promoter cis-acting element analysis showed that the promoter regions of SmADHs contained a large number of stress-responsive and hormone-responsive elements. Transcriptome data and qRT-PCR analysis indicated that SmADH2/5/6 had the highest expressions in S. miltiorrhiza roots. Under waterlogging stress, SmADH2/6 expression was rapidly upregulated. After salt stress, the expression of SmADH2/6 was significantly upregulated, while the expression of SmADH5 was inhibited. The expression of SmADH2/5/6 was significantly upregulated after PEG treatment, while the expression of SmADH6 was induced by GA3. Conclusion SmADH2/5/6, which are highly expressed in the roots of S. miltiorrhiza, play a role under various stresses, and SmADH2/6 play a particularly important role in resisting waterlogging stress.

Key words: Salvia miltiorrhiza, ADH, expression analysis, waterlogging stress, gene family

化文平, 刘菲, 浩佳欣, 陈尘. 丹参ADH基因家族的鉴定与表达模式分析[J]. 生物技术通报, 2025, 41(8): 211-219.

HUA Wen-ping, LIU Fei, HAO Jia-xin, CHEN Chen. Identification and Expression Patterns Analysis of ADH Gene Family in Salvia miltiorrhiza[J]. Biotechnology Bulletin, 2025, 41(8): 211-219.

图/表 8

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基因Gene	引物序列 Primer sequence （5′-3′）
SmActin	F-AGGAACCACCGATCCAGACA
（DQ243702）	R-GGTGCCCTGAGGTCCTGTT
SmADH2	F-GAACCCAAAAGACTACAGCAAGCC R-ACGCCAACAAGAACAGCCACTC
SmADH5	F-GTGTGATGCCAGCAAACGGATT R-TGACGGCGACGGAGTTACCA
SmADH6	F-ATGGGAGGTCTTGCTGAGTTTTGT R-CAATGGCATCACCAGGACGC

基因Gene	引物序列 Primer sequence （5′-3′）
SmActin	F-AGGAACCACCGATCCAGACA
（DQ243702）	R-GGTGCCCTGAGGTCCTGTT
SmADH2	F-GAACCCAAAAGACTACAGCAAGCC R-ACGCCAACAAGAACAGCCACTC
SmADH5	F-GTGTGATGCCAGCAAACGGATT R-TGACGGCGACGGAGTTACCA
SmADH6	F-ATGGGAGGTCTTGCTGAGTTTTGT R-CAATGGCATCACCAGGACGC

基因Gene	基长度 Gene length （bp）	编码序列长度CDs length （bp）	内含子数目 Number of introns	氨基酸数目Number of amino acids	理论等电点pI	分子量Molecular weight/kD	平均亲水性指数 GRAVY	亚细胞定位Subcellular localization
SmADH1	2 392	1 149	9	382	6.38	41.32	-0.014	Cytoplasm
SmADH2	2 279	1 140	8	379	5.98	40.87	0.101	Cytoplasm
SmADH3	8 279	1 149	8	382	6.87	41.55	0.259	Cytoplasm
SmADH4	2 824	1 167	9	388	5.75	41.83	0.137	Cytoplasm
SmADH5	4 670	1 299	7	432	8.67	46.20	-0.059	Chloroplasts
SmADH6	4 849	1 317	7	438	8.74	46.89	-0.056	Chloroplasts
SmADH7	3 827	1 140	8	379	6.37	40.79	0.063	Cytoplasm

基因Gene	基长度 Gene length （bp）	编码序列长度CDs length （bp）	内含子数目 Number of introns	氨基酸数目Number of amino acids	理论等电点pI	分子量Molecular weight/kD	平均亲水性指数 GRAVY	亚细胞定位Subcellular localization
SmADH1	2 392	1 149	9	382	6.38	41.32	-0.014	Cytoplasm
SmADH2	2 279	1 140	8	379	5.98	40.87	0.101	Cytoplasm
SmADH3	8 279	1 149	8	382	6.87	41.55	0.259	Cytoplasm
SmADH4	2 824	1 167	9	388	5.75	41.83	0.137	Cytoplasm
SmADH5	4 670	1 299	7	432	8.67	46.20	-0.059	Chloroplasts
SmADH6	4 849	1 317	7	438	8.74	46.89	-0.056	Chloroplasts
SmADH7	3 827	1 140	8	379	6.37	40.79	0.063	Cytoplasm

miRNA	靶基因 Target gene	期望值Expectation	靶位点Target site （bp）	抑制方式 Inhibition way
smi-miR167a	SmADH3	4	60-80	剪切
smi-miR167b	SmADH3	4	60-80	剪切
smi-miR167c	SmADH3	4	60-80	剪切
smi-miR167e	SmADH3	4	60-80	剪切
smi-miRN43	SmADH4	4.5	1 079-1 099	剪切
smi-miR482c	SmADH5	5	601-622	翻译
smi-miR482d	SmADH5	5	601-622	翻译
smi-miRN18	SmADH6	3.5	377-398	翻译
smi-miR482c	SmADH6	5	619-640	翻译
smi-miR482d	SmADH6	5	619-640	翻译
smi-miRN5a	SmADH7	4.5	5-25	翻译
smi-miRN5b	SmADH7	4.5	5-25	剪切
smi-miR530a	SmADH7	5	535-555	剪切