火龙果溃疡病菌实时荧光定量PCR内参基因的筛选

doi:10.13560/j.cnki.biotech.bull.1985.2022-1322

摘要/Abstract

摘要：

火龙果溃疡病是由火龙果溃疡病菌引起的重要真菌病害，近年来在世界火龙果产区的爆发严重影响了火龙果产业的发展。为研究火龙果溃疡病菌响应逆境胁迫的基因表达和功能，需要筛选火龙果溃疡病菌在各种条件下表达稳定的内参基因。以火龙果溃疡病菌菌株LJ02为研究对象，以在马铃薯葡萄糖液体培养基（PDW）培养的菌丝体为对照组，以在LB培养基培养的菌丝体和分别在添加过氧化氢、吡唑醚菌酯和苯醚甲环唑的PDW培养的菌丝体为处理组。利用实时荧光定量PCR技术以及内参基因稳定性评估软件（geNorm、NormFinder、Bestkeeper和RefFinder），评估18 个候选内参基因（CYT1、SDH2_1、TBCC、SUI1、RPL19、PPH、ATP5B、UBE2_16、RPL13、UBE2_2、PRS17、SUCLA2、ATP5A、TUB1_2、ACT1、EFTU、EF1A和GAPDH）的表达稳定性。结果显示，火龙果溃疡病菌的18个候选基因在上述培养条件下的Ct值介于14.80-24.66之间，表达水平适中；稳定性最高的内参基因是CYT1，其次为SUI1，GAPDH和ACT1的稳定性最差；最少使用2个内参基因组合可以提高定量PCR分析结果的准确性，此时SUCLA2和ATP5A是最稳定的内参基因组合。该结果可为研究火龙果溃疡病菌生物学过程中的基因表达提供理论依据。

关键词: 火龙果溃疡病菌, 内参基因, 实时荧光定量PCR, 菌丝生长, 表达分析, 非生物胁迫, 内参基因分析软件

Abstract:

The pitaya canker disease is a serious fungal disease caused by Neoscytalidium dimidiatum. In recent years, the outbreak of the pitaya canker disease in the world's pitaya producing areas has seriously affected the pitaya industry development. In order to study the expression level and function of genes responding to stress in N. dimidiatum, it is necessary to screen the reference genes that express stably when the pathogen grows under different cultural conditions. The N. dimidiatum strain LJ02 mycelia cultured in potato dextrose liquid medium（PDW）were taken as the control group, and the mycelia cultured in LB medium, and the mycelia cultured in PDW medium supplemented with hydrogen peroxide, pyrazolyl ester and difenoconazole, respectively, were taken as the treatment groups. The expression stabilities of 18 candidate reference genes（CYT1, SDH2_1, TBCC, SUI1, RPL19, PPH, ATP5B, UBE2_16, RPL13, UBE2_2, PRS17, SUCLA2, ATP5A, TUB1_2, ACT1, EFTU, EF1A and GAPDH）were evaluated by real-time quantitative PCR（RT-qPCR）and stability evaluation software（geNorm, NormFinder, Bestkeeper and RefFinder）. Based on the results of RT-qPCR and software analysis, it was found that the Ct values of 18 candidate genes were within 14.80-24.66 under different culture conditions, with moderate expression levels. The reference gene with the highest stability was CYT1, followed by SUI1, while GAPDH and ACT1 were with the worst expression stability. Using at least two reference genes may improve analysis accuracy of RT-qPCR. At this time, SUCLA2 and ATP5A is the most stable reference gene combination. The results provide a theoretical basis for gene expression analysis in the biological process of N. dimidiatum.

Key words: Neoscytalidium dimidiatum, reference gene, real-time quantitative PCR, mycelial growth, expression analysis, abiotic stres, reference gene analysis software

姚姿婷, 曹雪颖, 肖雪, 李瑞芳, 韦小妹, 邹承武, 朱桂宁. 火龙果溃疡病菌实时荧光定量PCR内参基因的筛选[J]. 生物技术通报, 2023, 39(5): 92-102.

YAO Zi-ting, CAO Xue-ying, XIAO Xue, LI Rui-fang, WEI Xiao-mei, ZOU Cheng-wu, ZHU Gui-ning. Screening of Reference Genes for RT-qPCR in Neoscytalidium dimidiatum[J]. Biotechnology Bulletin, 2023, 39(5): 92-102.

图/表 11

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序号 No.	基因ID Gene ID	引物 Primer	引物序列 Primer sequence（5'-3'）	退火温度 Annealing temperature/℃	产物长度 PCR product length/bp	基因名称 Gene name	基因注释 Gene description
Gene1	NEO_001463	gene1-qF	GCGGTTTCCAGGTCTATCGTGAAG	59	139	CYT1	Cytochrome c1
		gene1-qR	GGCTCGGTGTCGTACTCGTTCT				Cytochrome c1
Gene2	NEO_001659	gene2-qF	CGTACTGGTGGAACTCGGAGGA	58.5	151	SDH2_1	Succinate dehydrogenase subunit B
		gene2-qR	TTGAGAATGGTGTGGCAACGGTAA				Succinate dehydrogenase subunit B
Gene3	NEO_002342	gene3-qF	GCTCGCCAATCTCCGTCGTT	61.1	176	TBCC	Tubulin-specific chaperone c
		gene3-qR	TCACCACCAGCACGCTATCCT				Tubulin-specific chaperone c
Gene4	NEO_002585	gene4-qF	AGACACAGGCGAGACCAAGCA	55.8	151	SUI1	Translation initiation factor SUI1
		gene4-qR	GCAGGCAAACTTCTTCTTGATGACC				Translation initiation factor SUI1
Gene5	NEO_003941	gene5-qF	ATGCCGAGCCAGAAGTCTTTCC	57.9	117	RPL39	Ribosomal L39 protein
		gene5-qR	GGCGTTGTACCTGATGGTGTTG				Ribosomal L39 protein
Gene6	NEO_004521	gene6-qF	AAGTCTATGGCTTCTACGACGAGTG	58.4	198	PPH	Serine threonine-protein phosphatase pp2a catalytic subunit
		gene6-qR	GCGGCACCTCTTGTATCCTGTC				Serine threonine-protein phosphatase pp2a catalytic subunit
Gene7	NEO_004849	gene7-qF	GCCACCAAGTTCGCTCCTATCC	58.4	147	ATP5B	ATP synthase F1 beta subunit
		gene7-qR	GGCACCACCGAAGAGACCAATC				ATP synthase F1 beta subunit
Gene8	NEO_006464	gene8-qF	CGGTGTCTTCTTCCTCGCTATCC	58.3	183	UBE2_16	Ubiquitin-conjugating enzyme E2-16 kD
		gene8-qR	GAGCAGATGGAGAGCAGAACCTTC				Ubiquitin-conjugating enzyme E2-16 kD
Gene9	NEO_008113	gene9-qF	TCACGAGTTCGGCTGGAAGTACC	59.7	165	RPL13	Ribosomal L13 protein
		gene9-qR	TCGGCAAGCTGAGTCTTGACCTT				Ribosomal L13 protein
Gene10	NEO_008284	gene10-qF	TCTGCTCAACGACCCGAACAC	56.4	115	UBE2_2	Ubiquitin-conjugating enzyme e2_2
		gene10-qR	CCAACTCTTCTCCACCGTCTCC				Ubiquitin-conjugating enzyme e2_2
Gene11	NEO_008330	gene11-qF	CTGTCAAGAAGTCCGCCAAGGT	56.5	157	RPS17	Ribosomal protein S17e
		gene11-qR	CGCTTCATCAAGTGAGTGGTGTAG				Ribosomal protein S17e
Gene12	NEO_008873	gene12-qF	TGGTGACATTGGCTGCCTGGT	60.4	186	SUCLA2	Succinyl-CoA synthetase beta subunit
		gene12-qR	ATGTTGACGAAGATGGCGGTGAC				Succinyl-CoA synthetase beta subunit
Gene13	NEO_010290	gene13-qF	TCCTCTCATCTTCGCTGGTGTCA	56.2	110	ATP5A	ATP synthase F1 alpha subunit
		gene13-qR	GCTCGGACTCGTTGCTCTTCAG				ATP synthase F1 alpha subunit
Gene14	NEO_010575	gene14-qF	CAAGGAGGATGCCGCCAACAA	59	145	TUB1_2	Tubulin alpha-2 chain
		gene14-qR	ACCGCCGAAGGAGTGGAAGAT				Tubulin alpha-2 chain
Gene15	NEO_011231	gene15-qF	CCAAGTCCAACCGTGAGAAGATGA	56.7	102	ACT1	Actin
		gene15-qR	CGGAAGCGTACAGCGACAGAA				Actin
Gene16	NEO_011865	gene16-qF	AGACCGACAACCGCCACTATGC	60.1	184	EFTU	Translation elongation factor Tu
		gene16-qR	ACAATCTTCTGGACACCGACCTGAC				Translation elongation factor Tu
Gene17	NEO_012815	gene17-qF	CTGGTGAGTTCGAGGCTGGTATCT	58.2	121	EF1A	Translation elongation factor 1-alpha protein
		gene17-qR	CACTTGGTGGTGTCCATCTTGTTGA				Translation elongation factor 1-alpha protein
Gene18	NEO_013111	gene18-qF	CCGTACCGCTGCTACCAACATC	59.8	195	GAPDH	Glyceraldehyde-3-phosphate dehydrogenase
		gene18-qR	GCCTTGATAGTCGCCTTGATCTCAT				Glyceraldehyde-3-phosphate dehydrogenase

序号 No.	基因ID Gene ID	引物 Primer	引物序列 Primer sequence（5'-3'）	退火温度 Annealing temperature/℃	产物长度 PCR product length/bp	基因名称 Gene name	基因注释 Gene description
Gene1	NEO_001463	gene1-qF	GCGGTTTCCAGGTCTATCGTGAAG	59	139	CYT1	Cytochrome c1
		gene1-qR	GGCTCGGTGTCGTACTCGTTCT				Cytochrome c1
Gene2	NEO_001659	gene2-qF	CGTACTGGTGGAACTCGGAGGA	58.5	151	SDH2_1	Succinate dehydrogenase subunit B
		gene2-qR	TTGAGAATGGTGTGGCAACGGTAA				Succinate dehydrogenase subunit B
Gene3	NEO_002342	gene3-qF	GCTCGCCAATCTCCGTCGTT	61.1	176	TBCC	Tubulin-specific chaperone c
		gene3-qR	TCACCACCAGCACGCTATCCT				Tubulin-specific chaperone c
Gene4	NEO_002585	gene4-qF	AGACACAGGCGAGACCAAGCA	55.8	151	SUI1	Translation initiation factor SUI1
		gene4-qR	GCAGGCAAACTTCTTCTTGATGACC				Translation initiation factor SUI1
Gene5	NEO_003941	gene5-qF	ATGCCGAGCCAGAAGTCTTTCC	57.9	117	RPL39	Ribosomal L39 protein
		gene5-qR	GGCGTTGTACCTGATGGTGTTG				Ribosomal L39 protein
Gene6	NEO_004521	gene6-qF	AAGTCTATGGCTTCTACGACGAGTG	58.4	198	PPH	Serine threonine-protein phosphatase pp2a catalytic subunit
		gene6-qR	GCGGCACCTCTTGTATCCTGTC				Serine threonine-protein phosphatase pp2a catalytic subunit
Gene7	NEO_004849	gene7-qF	GCCACCAAGTTCGCTCCTATCC	58.4	147	ATP5B	ATP synthase F1 beta subunit
		gene7-qR	GGCACCACCGAAGAGACCAATC				ATP synthase F1 beta subunit
Gene8	NEO_006464	gene8-qF	CGGTGTCTTCTTCCTCGCTATCC	58.3	183	UBE2_16	Ubiquitin-conjugating enzyme E2-16 kD
		gene8-qR	GAGCAGATGGAGAGCAGAACCTTC				Ubiquitin-conjugating enzyme E2-16 kD
Gene9	NEO_008113	gene9-qF	TCACGAGTTCGGCTGGAAGTACC	59.7	165	RPL13	Ribosomal L13 protein
		gene9-qR	TCGGCAAGCTGAGTCTTGACCTT				Ribosomal L13 protein
Gene10	NEO_008284	gene10-qF	TCTGCTCAACGACCCGAACAC	56.4	115	UBE2_2	Ubiquitin-conjugating enzyme e2_2
		gene10-qR	CCAACTCTTCTCCACCGTCTCC				Ubiquitin-conjugating enzyme e2_2
Gene11	NEO_008330	gene11-qF	CTGTCAAGAAGTCCGCCAAGGT	56.5	157	RPS17	Ribosomal protein S17e
		gene11-qR	CGCTTCATCAAGTGAGTGGTGTAG				Ribosomal protein S17e
Gene12	NEO_008873	gene12-qF	TGGTGACATTGGCTGCCTGGT	60.4	186	SUCLA2	Succinyl-CoA synthetase beta subunit
		gene12-qR	ATGTTGACGAAGATGGCGGTGAC				Succinyl-CoA synthetase beta subunit
Gene13	NEO_010290	gene13-qF	TCCTCTCATCTTCGCTGGTGTCA	56.2	110	ATP5A	ATP synthase F1 alpha subunit
		gene13-qR	GCTCGGACTCGTTGCTCTTCAG				ATP synthase F1 alpha subunit
Gene14	NEO_010575	gene14-qF	CAAGGAGGATGCCGCCAACAA	59	145	TUB1_2	Tubulin alpha-2 chain
		gene14-qR	ACCGCCGAAGGAGTGGAAGAT				Tubulin alpha-2 chain
Gene15	NEO_011231	gene15-qF	CCAAGTCCAACCGTGAGAAGATGA	56.7	102	ACT1	Actin
		gene15-qR	CGGAAGCGTACAGCGACAGAA				Actin
Gene16	NEO_011865	gene16-qF	AGACCGACAACCGCCACTATGC	60.1	184	EFTU	Translation elongation factor Tu
		gene16-qR	ACAATCTTCTGGACACCGACCTGAC				Translation elongation factor Tu
Gene17	NEO_012815	gene17-qF	CTGGTGAGTTCGAGGCTGGTATCT	58.2	121	EF1A	Translation elongation factor 1-alpha protein
		gene17-qR	CACTTGGTGGTGTCCATCTTGTTGA				Translation elongation factor 1-alpha protein
Gene18	NEO_013111	gene18-qF	CCGTACCGCTGCTACCAACATC	59.8	195	GAPDH	Glyceraldehyde-3-phosphate dehydrogenase
		gene18-qR	GCCTTGATAGTCGCCTTGATCTCAT				Glyceraldehyde-3-phosphate dehydrogenase

序号 No.	内参基因 Reference gene	扩增效率 RT-qPCR efficiency/%	相关系数 Correlation coefficient R²	序号 No.	内参基因 Reference gene	扩增效率 RT-qPCR efficiency/%	相关系数 Correlation coefficient R²
Gene 1	CYT1	106.4	0.998 5	Gene 10	UBE2_2	96.5	0.999 9
Gene 2	SDH2_1	106.2	0.986 5	Gene 11	RPS17	96.7	0.996 6
Gene 3	TBCC	104.3	0.989 4	Gene 12	SUCLA2	103.6	0.996 6
Gene 4	SUI1	100.1	0.999 5	Gene 13	ATP5A	101.9	0.996 9
Gene 5	RPL39	108.3	0.998 8	Gene 14	TUB1_2	93.6	0.999 6
Gene 6	PPH	108.9	0.999 8	Gene 15	ACT1	91.6	0.999 9
Gene 7	ATP5B	104.7	0.997 4	Gene 16	EFTU	102.4	0.994 1
Gene 8	UBE2_16	104.2	0.999 9	Gene 17	EF1A	94.8	0.996 8
Gene 9	RPL13	100.9	0.999 9	Gene 18	GAPDH	90.9	0.995 0

序号 No.	内参基因 Reference gene	扩增效率 RT-qPCR efficiency/%	相关系数 Correlation coefficient R²	序号 No.	内参基因 Reference gene	扩增效率 RT-qPCR efficiency/%	相关系数 Correlation coefficient R²
Gene 1	CYT1	106.4	0.998 5	Gene 10	UBE2_2	96.5	0.999 9
Gene 2	SDH2_1	106.2	0.986 5	Gene 11	RPS17	96.7	0.996 6
Gene 3	TBCC	104.3	0.989 4	Gene 12	SUCLA2	103.6	0.996 6
Gene 4	SUI1	100.1	0.999 5	Gene 13	ATP5A	101.9	0.996 9
Gene 5	RPL39	108.3	0.998 8	Gene 14	TUB1_2	93.6	0.999 6
Gene 6	PPH	108.9	0.999 8	Gene 15	ACT1	91.6	0.999 9
Gene 7	ATP5B	104.7	0.997 4	Gene 16	EFTU	102.4	0.994 1
Gene 8	UBE2_16	104.2	0.999 9	Gene 17	EF1A	94.8	0.996 8
Gene 9	RPL13	100.9	0.999 9	Gene 18	GAPDH	90.9	0.995 0

基因序号 No.	基因名称 Gene name	几何平均值 Geo mean	算术平均值 AR mean	最小值 Min	最大值 Max	相关系数 Correlation coefficient（r）	标准偏差 Std dev	变异系数 CV
Gene1	CYT1	19.94	19.94	19.24	20.68	0.960	0.31	1.53
Gene2	SDH2_1	19.69	19.69	19.06	20.16	0.754	0.30	1.54
Gene3	TBCC	22.90	22.92	21.50	24.66	0.915	0.83	3.63
Gene4	SUI1	17.65	17.66	16.58	18.49	0.952	0.47	2.66
Gene5	RPL39	19.71	19.72	18.77	20.67	0.851	0.63	3.21
Gene6	PPH	21.46	21.47	20.20	22.48	0.936	0.72	3.38
Gene7	ATP5B	18.21	18.22	17.97	18.74	0.521	0.20	1.11
Gene8	UBE2_16	18.16	18.17	17.48	19.18	0.912	0.44	2.40
Gene9	RPL13	19.97	19.98	18.81	20.69	0.761	0.56	2.79
Gene10	UBE2_2	20.47	20.49	19.63	21.76	0.963	0.53	2.6
Gene11	RPS17	19.82	19.83	18.73	20.78	0.803	0.61	3.09
Gene12	SUCLA2	20.78	20.79	20.15	21.48	0.825	0.38	1.81
Gene13	ATP5A	19.49	19.50	18.88	20.03	0.87	0.33	1.67
Gene14	TUB1_2	22.06	22.07	21.31	23.66	0.892	0.55	2.50
Gene15	ACT1	21.30	21.33	19.76	22.72	0.835	1.13	5.29
Gene16	EFTU	21.48	21.49	20.66	22.36	0.577	0.65	3.00
Gene17	EF1A	16.04	16.05	15.35	16.48	0.893	0.31	1.96
Gene18	GAPDH	15.97	16.00	14.80	17.62	0.929	0.89	5.56