山茶花花香生物合成相关基因的实时荧光定量PCR内参基因的筛选及验证

doi:10.16420/j.issn.0513-353x.2025-0772

摘要/Abstract

摘要：

为研究山茶花花香生物合成相关基因的表达模式，筛选在山茶花中稳定表达的内参基因。利用实时荧光定量PCR（qRT-PCR）技术检测10个候选内参基因（α-TUB、CYP、PP2A、ACTIN1、ACTIN2、GAPDH、UBQ、AQP、UBC和HIS）在不同的组织、开花阶段、品种和混合样品中的表达水平，geNorm、NormFinder、BestKeeper 和RefFinder 4种算法综合评估和鉴定内参基因的表达稳定性。结果显示，10个候选内参基因的引物特异性好，均具有作为内参基因的性能；分析山茶花不同开花阶段基因表达时可选用PP2A和UBC，分析不同组织和不同香型品种基因表达时最理想的内参基因为PP2A和ACTIN1，其中PP2A在山茶花不同的组织、开花阶段、香型品种和混合样品中均能稳定表达。通过qRT-PCR扩增了花香相关的9个靶基因，验证PP2A是山茶花花香相关基因表达研究的最佳内参基因，而以传统管家基因GAPDH为内参时，山茶花芳樟醇/橙花叔醇合酶基因CaLIS/NES1和苯乙醛还原酶基因CaPAR的表达模式出现较大偏差。

关键词: 山茶花, 花香, 内参基因, 实时荧光定量PCR, 表达分析, PP2A

Abstract:

To study the expression patterns of genes related to floral scent biosynthesis in Camellia japonica and screen for stably expressed reference genes. Quantitative real-time PCR（qRT-PCR）technology was used to detect the expression levels of 10 candidate reference genes（α-TUB，CYP，PP2A，ACTIN1，ACTIN2，GAPDH，UBQ，AQP，UBC，and HIS）in different tissues，flowering stages，cultivars and mixed samples. The stability of internal reference genes was comprehensively evaluated and identified using four algorithms：geNorm，NormFinder，BestKeeper，and RefFinder. The results showed that the primer specificity of the 10 candidate reference genes was good，and all of them had the performance as reference genes. PP2A and UBC can be used for analyzing gene expression at different flowering stages. The most ideal reference genes for analyzing gene expression in different tissues and fragrance cultivars were identified as PP2A and ACTIN1. PP2A can be stably expressed in different tissues，flowering stages，fragrance cultivars and mixed samples. Nine floral scent-related target genes were amplified by qRT-PCR，and PP2A is the optimal reference gene for studying the expression of floral scent-related genes in Camellia japonica. However，when the traditional housekeeping gene GAPDH was used as the reference gene，significant deviations were observed in the expression patterns of the linalool/nerolidol synthase gene（CaLIS/NES1）and the phenylacetaldehyde reductase gene（CaPAR）.

Key words: Camellia japonica, floral scent, reference gene, quantitative real-time PCR, expression analysis, PP2A

陈艺荃, 樊荣辉, 林兵, 陈燕, 吴建设, 钟淮钦. 山茶花花香生物合成相关基因的实时荧光定量PCR内参基因的筛选及验证[J]. 园艺学报, 2026, 53(2): 598-612.

CHEN Yiquan, FAN Ronghui, LIN Bing, CHEN Yan, WU Jianshe, ZHONG Huaiqin. Screening and Validation of Reference Genes for qRT-PCR Analysis of Genes Related to Floral Scent Biosynthesis in Camellia japonica[J]. Acta Horticulturae Sinica, 2026, 53(2): 598-612.

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基因（ID） Gene	注释 Annotation	引物序列（5′-3′） Primer sequence
CaHMGR （LOC114310350）	羟基-3-甲基戊二酰辅酶A 原酶基因 3-Hydroxy-3-methylglutaryl-CoA reductase	F：CAAAGAGTACCCAGGATCAAA R：GCAATGGCAGACATCAAGGA
CaDXS1 （LOC114301937）	1-脱氧-d木酮糖-5-磷酸合酶基因1 1-Deoxy-d-xylulose 5-phosphate synthase 1	F：GGGAGAATACTGATGGAAGG R：GATTAAATCTGCATCTAAGGGT
CaDXS2 （LOC114264886）	1-脱氧-d木酮糖-5-磷酸合酶基因2 1-Deoxy-d-xylulose 5-phosphate synthase 2	F：ATGTTGCTCACTTCATGTCCCTA R：TTGCGGCAATATGAGATGGT
CaMDS （LOC114279313）	2-C-甲基-D-赤藓糖醇-2,4-环焦磷酸合酶基因 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase	F：CTCCATCGCCAAACGCTCGTC R：CGCGGTTTCAGCCACCACTA
CaGGPPS（LOC114281224）	香叶基香叶基二磷酸合酶基因 Geranylgeranyl diphosphate synthetase	F：ATGTATTGGGTTGTTGTTTCAGG R：GAAAGCTGTTCCTTCGCCTC
CaLIS/NES1 （LOC114290333）	芳樟醇/橙花叔醇合酶基因1 Linalool/nerolidol synthase 1	F：CCAGGCATTGTGACTTCTAC R：AGATAGGCATTCCTTGTTGA
CaLIS/NES2 （LOC114261430）	芳樟醇/橙花叔醇合酶基因2 Linalool/nerolidol synthase 2	F：GCACTTCTACAAAGCCAATA R：TACAAACTCATCAACCCTCT
CaSAMT （LOC114304780）	水杨酸羧基位甲基转移酶基因 Salicylic acid carboxyl methyltransferas	F：TCCCACAATACACTCCATCC R：CAAGCAATGACTCAGCCACA
CaPAR （LOC114300830）	苯乙醛还原酶基因 Phenylacetaldehyde reductase	F：GGAGGGTCAGGGTACATAGC R：ATCAACCGCAGAATCAAAGG

基因（ID） Gene	注释 Annotation	引物序列（5′-3′） Primer sequence
CaHMGR （LOC114310350）	羟基-3-甲基戊二酰辅酶A 原酶基因 3-Hydroxy-3-methylglutaryl-CoA reductase	F：CAAAGAGTACCCAGGATCAAA R：GCAATGGCAGACATCAAGGA
CaDXS1 （LOC114301937）	1-脱氧-d木酮糖-5-磷酸合酶基因1 1-Deoxy-d-xylulose 5-phosphate synthase 1	F：GGGAGAATACTGATGGAAGG R：GATTAAATCTGCATCTAAGGGT
CaDXS2 （LOC114264886）	1-脱氧-d木酮糖-5-磷酸合酶基因2 1-Deoxy-d-xylulose 5-phosphate synthase 2	F：ATGTTGCTCACTTCATGTCCCTA R：TTGCGGCAATATGAGATGGT
CaMDS （LOC114279313）	2-C-甲基-D-赤藓糖醇-2,4-环焦磷酸合酶基因 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase	F：CTCCATCGCCAAACGCTCGTC R：CGCGGTTTCAGCCACCACTA
CaGGPPS（LOC114281224）	香叶基香叶基二磷酸合酶基因 Geranylgeranyl diphosphate synthetase	F：ATGTATTGGGTTGTTGTTTCAGG R：GAAAGCTGTTCCTTCGCCTC
CaLIS/NES1 （LOC114290333）	芳樟醇/橙花叔醇合酶基因1 Linalool/nerolidol synthase 1	F：CCAGGCATTGTGACTTCTAC R：AGATAGGCATTCCTTGTTGA
CaLIS/NES2 （LOC114261430）	芳樟醇/橙花叔醇合酶基因2 Linalool/nerolidol synthase 2	F：GCACTTCTACAAAGCCAATA R：TACAAACTCATCAACCCTCT
CaSAMT （LOC114304780）	水杨酸羧基位甲基转移酶基因 Salicylic acid carboxyl methyltransferas	F：TCCCACAATACACTCCATCC R：CAAGCAATGACTCAGCCACA
CaPAR （LOC114300830）	苯乙醛还原酶基因 Phenylacetaldehyde reductase	F：GGAGGGTCAGGGTACATAGC R：ATCAACCGCAGAATCAAAGG

基因（ID） Gene	注释 Annotation	引物序列（5′-3′） Primer sequence	产物长/bp Product length	斜率 Slope	扩增效率/% PCR efficiency（E）	判定系数 Correlation coefficient（R²）
α-TUB（LOC114256112）	α微管蛋白基因 α-tubulin	F：GCAAGCAGGTATTCAGGTAG R：TAACGGTAGGTTCCAGGTCA	194	-3.2897	101.3627	0.9959
CYP（LOC114311397）	亲环蛋白基因 Cyclophilin	F：CTCTTCCTCGTCGCCTCAAT R：CCTCCTCCACCTCATCTACC	76	-3.2643	102.4624	0.9960
PP2A（LOC114282106）	蛋白磷酸酶2A基因 Protein phosphatase 2A	F：CTGCATCGAAAGACAGGGTA R：CTGACATCAACATCTGGGTC	148	-3.3671	98.1488	0.9996
ACTIN1（LOC114311395）	肌动蛋白基因1 Actin1	F：CGATACGCCGTGTTTGATTT R：AGTAGGGTCGGTGGCTTGGA	186	-3.2286	104.0477	0.9938
ACTIN2（LOC114288971）	肌动蛋白基因2 Actin2	F：GGTATGTCAAGTGCGGTTTC R：TTCTTGTTCCATGAGGGATT	107	-3.1123	109.5587	0.9975
GAPDH（LOC114311438）	甘油醛-3-磷酸脱氢酶基因Glyceraldehyde-3- phosphate dehydrogenase	F：AGGGCAAACTCAAGGGTATT R：TAGCATTCAGCAACAGAGGC	214	-3.4563	94.6824	0.9980
UBQ（LOC114305206）	多聚泛素酶基因 Polyubiquitin	F：GACCCTACACTTGGTGCTGC R：TCTGGTGGAATACCCTCCTT	150	-3.1280	108.7820	0.9921
AQP（LOC114261180）	水通道蛋白基因 Aquaporin	F：AAGAAGGCGGTCTGGGATGA R：CTCAAGATGAACTGGTGGTAGAA	92	-3.4931	93.3208	0.9983
HIS（LOC114293472）	组蛋白H3基因 Histone	F：CACATCGAGGAGCAGCAAGG R：TTTCCAGCCAATTCAAGCAC	162	-3.4854	93.6026	0.9947
UBC（LOC114280527 ）	泛素结合酶基因 Ubiquitin-conjugating enzyme	F：TATCAAAGGTCCTGCTGTCC R：CCTCATATTTAGCCCTATCAGTC	111	-3.4571	94.6524	0.9986

基因（ID） Gene	注释 Annotation	引物序列（5′-3′） Primer sequence	产物长/bp Product length	斜率 Slope	扩增效率/% PCR efficiency（E）	判定系数 Correlation coefficient（R²）
α-TUB（LOC114256112）	α微管蛋白基因 α-tubulin	F：GCAAGCAGGTATTCAGGTAG R：TAACGGTAGGTTCCAGGTCA	194	-3.2897	101.3627	0.9959
CYP（LOC114311397）	亲环蛋白基因 Cyclophilin	F：CTCTTCCTCGTCGCCTCAAT R：CCTCCTCCACCTCATCTACC	76	-3.2643	102.4624	0.9960
PP2A（LOC114282106）	蛋白磷酸酶2A基因 Protein phosphatase 2A	F：CTGCATCGAAAGACAGGGTA R：CTGACATCAACATCTGGGTC	148	-3.3671	98.1488	0.9996
ACTIN1（LOC114311395）	肌动蛋白基因1 Actin1	F：CGATACGCCGTGTTTGATTT R：AGTAGGGTCGGTGGCTTGGA	186	-3.2286	104.0477	0.9938
ACTIN2（LOC114288971）	肌动蛋白基因2 Actin2	F：GGTATGTCAAGTGCGGTTTC R：TTCTTGTTCCATGAGGGATT	107	-3.1123	109.5587	0.9975
GAPDH（LOC114311438）	甘油醛-3-磷酸脱氢酶基因Glyceraldehyde-3- phosphate dehydrogenase	F：AGGGCAAACTCAAGGGTATT R：TAGCATTCAGCAACAGAGGC	214	-3.4563	94.6824	0.9980
UBQ（LOC114305206）	多聚泛素酶基因 Polyubiquitin	F：GACCCTACACTTGGTGCTGC R：TCTGGTGGAATACCCTCCTT	150	-3.1280	108.7820	0.9921
AQP（LOC114261180）	水通道蛋白基因 Aquaporin	F：AAGAAGGCGGTCTGGGATGA R：CTCAAGATGAACTGGTGGTAGAA	92	-3.4931	93.3208	0.9983
HIS（LOC114293472）	组蛋白H3基因 Histone	F：CACATCGAGGAGCAGCAAGG R：TTTCCAGCCAATTCAAGCAC	162	-3.4854	93.6026	0.9947
UBC（LOC114280527 ）	泛素结合酶基因 Ubiquitin-conjugating enzyme	F：TATCAAAGGTCCTGCTGTCC R：CCTCATATTTAGCCCTATCAGTC	111	-3.4571	94.6524	0.9986

基因 Gene	所有样品 All samples		不同组织 Different tissues		不同开花阶段 Different flowering stages		不同香型品种 Different fragrance cultivars
基因 Gene	SD	CV/%	SD	CV/%	SD	CV/%	SD	CV/%
α-TUB	0.631	2.330	0.578	2.155	0.628	2.343	0.531	1.910
CYP	1.197	3.946	0.771	2.595	1.383	4.579	1.017	3.266
PP2A	0.414	1.534	0.603	2.267	0.188	0.685	0.175	0.646
ACTIN1	0.563	2.037	0.662	2.435	0.567	2.028	0.301	1.077
ACTIN2	1.390	4.057	1.246	3.661	1.200	3.551	1.606	4.601
GAPDH	1.306	5.414	1.983	7.985	0.789	3.394	1.062	4.342
UBQ	1.282	6.059	0.784	3.605	1.866	8.920	1.648	7.741
AQP	1.116	4.308	1.300	5.091	0.863	3.332	0.742	2.799
HIS	1.411	6.061	1.422	6.126	1.240	5.322	1.269	5.447
UBC	1.132	4.878	1.340	6.034	0.302	1.280	1.494	6.343