冬瓜实时荧光定量PCR内参基因的筛选与评价

doi:10.16420/j.issn.0513-353x.2023-0343

园艺学报 ›› 2024, Vol. 51 ›› Issue (4): 748-760.doi: 10.16420/j.issn.0513-353x.2023-0343

• 遗传育种·种质资源·分子生物学 • 上一篇下一篇

冬瓜实时荧光定量PCR内参基因的筛选与评价

何长霞¹^,², 罗陈², 闫晋强², 刘文睿², 王敏², 谢大森², 吴智明¹^,^*(), 江彪²^,^*()

¹ 仲恺农业工程学院园艺园林学院，广州 510225
² 广东省农业科学院蔬菜研究所，广东省蔬菜新技术研究重点实验室，广州 510640

收稿日期:2023-07-25 修回日期:2023-12-25 出版日期:2024-04-25 发布日期:2024-04-26
通讯作者:
*E-mail：zhiming_wu521@hotmail.com，jiangbiao@gdaas.cn
基金资助:
广东省自然科学基金青年提升项目(2023A1515030049); 广东省基础与应用基础研究基金-区域联合基金青年基金项目(2021A1515111214); 2022年广东省级乡村振兴战略专项资金种业振兴项目(2022-NPY-00-028); 广东省农业科学院中青年学科带头人项目(R2020PY-JG003); 广东省农业科学院优秀博士项目(R2021YJ-YB2004)

Screening and Evaluation of Reference Genes for Real-time Quantitative PCR in Wax Gourd

HE Changxia¹^,², LUO Chen², YAN Jinqiang², LIU Wenrui², WANG Min², XIE Dasen², WU Zhiming¹^,^*(), JIANG Biao²^,^*()

¹ College of Horticulture and Landscape Architecture，Zhongkai University of Agriculture and Engineering，Guangzhou 510225，China
² Guangdong Key Laboratory for New Technology Research on Vegetables，Vegetable Research Institute，Guangdong Academy of Agricultural Sciences，Guangzhou 510640，China

Received:2023-07-25 Revised:2023-12-25 Published:2024-04-25 Online:2024-04-26
Contact:
*E-mail：zhiming_wu521@hotmail.com，jiangbiao@gdaas.cn

摘要/Abstract

摘要：

运用3种统计学软件GeNorm、NormFinder和BestKeeper分析冬瓜9个内参基因（18SrRNA、ACT、CYP、EF-1α、F-box、GAPDH、TUA、TUB和UBQ）在冬瓜不同组织（根、茎、叶、雄花、雌花和子房）、不同发育时期果实（授粉后0、10和20 d）、生物胁迫（疫霉侵染）和非生物胁迫（盐、高温和低温处理）植物叶片样品中的表达稳定性。结果表明，在不同组织中，EF-1α的表达稳定性最好；在不同发育时期果实和疫霉处理植物叶片中，F-box的表达稳定性最好；在盐胁迫处理植物叶片中，18SrRNA表现出较强的稳定性；在高温胁迫处理植物叶片中，ACT的表达稳定性最好；在低温胁迫处理植物叶片中，TUA的稳定性最好。

关键词: 冬瓜, 实时荧光定量PCR, 内参基因

Abstract:

Three statistical software including GeNorm，NormFinder，and BestKeeper were applied to analyze the expression stability of nine internal reference genes（18SrRNA，ACT，CYP，EF-1α，F-box，GAPDH，TUA，TUB，and UBQ）in wax gourd. The plant samples of plants were taken in different tissues （root，stem，leaf，male flower，female flower，ovary），fruits at different developmental stages including （0 d，10 d，and 20 d after pollination）of wax gourd，and leaves under biotic stress（Phytophthora melonis infection），and abiotic stress（salt，high temperature，and low temperature treatment）. The obtained results showed that the expression stability of EF-1α was the best in different tissues. The expression stability of F-box was the best in the fruits at different developmental stages and the leaves under P. melonis infection. By comparison，18SrRNA showed strong stability in the leaves under salt stress，while the expression stability of ACT was the best in the leaves under high temperature stress and TUA had the best stability in the leaves under low temperature stress.

Key words: wax gourd, real-time quantitative PCR, reference genes

何长霞, 罗陈, 闫晋强, 刘文睿, 王敏, 谢大森, 吴智明, 江彪. 冬瓜实时荧光定量PCR内参基因的筛选与评价[J]. 园艺学报, 2024, 51(4): 748-760.

HE Changxia, LUO Chen, YAN Jinqiang, LIU Wenrui, WANG Min, XIE Dasen, WU Zhiming, JIANG Biao. Screening and Evaluation of Reference Genes for Real-time Quantitative PCR in Wax Gourd[J]. Acta Horticulturae Sinica, 2024, 51(4): 748-760.

图/表 9

参考文献 32

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名称 Name	基因 ID Gene ID	基因全称 Gene description	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer	长度/ bp Length	效率/% Efficiency	相关系数（R²） Correlation coefficient
18SrRNA	Bhi08G000009	18S核糖体RNA 18S Ribosomal RNA	GAGGGCTCGAAGACGATCAG	TTCTCATAAGGTGCTGGCGG	105	95	1.00
ACT	Bhi10G001911	肌动蛋白 Actin	CCCCATGCTATCCTCCGTCT	GTCGAGCGCAACATAAGCAA	153	92	0.99
CYP	Bhi05G001495	亲环素 Cyclophilin	TTTCACCGCCGGAAATGGTA	TGGTTCCAGGTCCAGCATTC	125	99	0.95
EF-1α	Bhi06G000576	转录延伸因子-1α Elongation factor 1α	CCAAGAAGCTCTCCCTGGTG	CCAATCTGCCCTGGATGGTT	165	100	0.99
F-box	Bhi04G000968	F-box蛋白 G-box family protein	CCATTCCCACCTCGTGTTCA	TCCCACAGTTTCCTCACAGC	137	92	1.00
GAPDH	Bhi04G001956	甘油醛-3-磷酸脱氢酶Glyceraldehyde-3-phosphate	CAAGCAAGGATGCCCCAATG	CTGGGTGGCAGTGATGGAAT	185	102	0.98
TUA	Bhi11G001304	α微管蛋白 α-Tubulin	CCGAGTGCGAAAATTAGCCG	TCCACAGACAAGCGTTCCAA	120	98	0.99
TUB	Bhi03G001656	β微管蛋白 β-Tubulin	CACGTTCAAGGTGGGCAATG	TCTCCCTGGTACTTTCCGGT	101	112	0.98
UBQ	Bhi10G000739	多聚泛素酶 Ubiquitin extension protein	TCGTATCAAGGAGCGGGTTG	TCAGGGCAAGCACTAGATGG	149	113	1.00

名称 Name	基因 ID Gene ID	基因全称 Gene description	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer	长度/ bp Length	效率/% Efficiency	相关系数（R²） Correlation coefficient
18SrRNA	Bhi08G000009	18S核糖体RNA 18S Ribosomal RNA	GAGGGCTCGAAGACGATCAG	TTCTCATAAGGTGCTGGCGG	105	95	1.00
ACT	Bhi10G001911	肌动蛋白 Actin	CCCCATGCTATCCTCCGTCT	GTCGAGCGCAACATAAGCAA	153	92	0.99
CYP	Bhi05G001495	亲环素 Cyclophilin	TTTCACCGCCGGAAATGGTA	TGGTTCCAGGTCCAGCATTC	125	99	0.95
EF-1α	Bhi06G000576	转录延伸因子-1α Elongation factor 1α	CCAAGAAGCTCTCCCTGGTG	CCAATCTGCCCTGGATGGTT	165	100	0.99
F-box	Bhi04G000968	F-box蛋白 G-box family protein	CCATTCCCACCTCGTGTTCA	TCCCACAGTTTCCTCACAGC	137	92	1.00
GAPDH	Bhi04G001956	甘油醛-3-磷酸脱氢酶Glyceraldehyde-3-phosphate	CAAGCAAGGATGCCCCAATG	CTGGGTGGCAGTGATGGAAT	185	102	0.98
TUA	Bhi11G001304	α微管蛋白 α-Tubulin	CCGAGTGCGAAAATTAGCCG	TCCACAGACAAGCGTTCCAA	120	98	0.99
TUB	Bhi03G001656	β微管蛋白 β-Tubulin	CACGTTCAAGGTGGGCAATG	TCTCCCTGGTACTTTCCGGT	101	112	0.98
UBQ	Bhi10G000739	多聚泛素酶 Ubiquitin extension protein	TCGTATCAAGGAGCGGGTTG	TCAGGGCAAGCACTAGATGG	149	113	1.00

内参基因 Reference gene	不同组织 Different tissues		不同发育时期果实 Fruits at different stages		疫霉处理 Phytophthora melonis infection		盐处理 Salt treatment		高温处理 High temperature treatment		低温处理 Low temperature treatment
内参基因 Reference gene	M	S	M	S	M	S	M	S	M	S	M	S
18SrRNA	1.66	1.47	0.71	0.49	0.05	0.11	0.41	0.11	1.60	2.62	0.95	1.03
ACT	1.08	0.29	0.55	0.20	0.28	0.10	0.12	0.04	0.27	0.09	1.17	1.29
CYP	1.46	1.07	0.37	0.44	0.36	0.45	0.37	0.38	0.75	0.69	0.18	0.37
EF-1α	0.68	0.58	0.48	0.12	0.51	0.38	0.33	0.37	0.85	0.82	0.52	0.40
F-box	1.39	0.88	0.28	0.26	0.05	0.03	0.57	0.50	0.65	0.70	0.18	0.53
GAPDH	0.99	0.78	0.39	0.67	0.62	0.65	0.12	0.04	0.97	0.94	0.43	0.63
TUA	1.29	0.69	0.93	0.85	0.16	0.29	0.24	0.21	0.35	0.13	0.63	0.11
TUB	0.68	0.71	0.28	0.51	0.21	0.30	0.50	0.39	0.27	0.09	0.68	0.11
UBQ	1.23	0.73	0.83	0.67	0.44	0.28	0.46	0.32	0.42	0.10	0.32	0.56

内参基因 Reference gene	不同组织 Different tissues		不同发育时期果实 Fruits at different stages		疫霉处理 Phytophthora melonis infection		盐处理 Salt treatment		高温处理 High temperature treatment		低温处理 Low temperature treatment
内参基因 Reference gene	M	S	M	S	M	S	M	S	M	S	M	S
18SrRNA	1.66	1.47	0.71	0.49	0.05	0.11	0.41	0.11	1.60	2.62	0.95	1.03
ACT	1.08	0.29	0.55	0.20	0.28	0.10	0.12	0.04	0.27	0.09	1.17	1.29
CYP	1.46	1.07	0.37	0.44	0.36	0.45	0.37	0.38	0.75	0.69	0.18	0.37
EF-1α	0.68	0.58	0.48	0.12	0.51	0.38	0.33	0.37	0.85	0.82	0.52	0.40
F-box	1.39	0.88	0.28	0.26	0.05	0.03	0.57	0.50	0.65	0.70	0.18	0.53
GAPDH	0.99	0.78	0.39	0.67	0.62	0.65	0.12	0.04	0.97	0.94	0.43	0.63
TUA	1.29	0.69	0.93	0.85	0.16	0.29	0.24	0.21	0.35	0.13	0.63	0.11
TUB	0.68	0.71	0.28	0.51	0.21	0.30	0.50	0.39	0.27	0.09	0.68	0.11
UBQ	1.23	0.73	0.83	0.67	0.44	0.28	0.46	0.32	0.42	0.10	0.32	0.56

内参基因 Reference gene	不同组织 Different tissues		不同发育时期果实 Fruits at different stages		疫霉处理 Phytophthora melonis infection		盐处理 Salt treatment		高温处理 High temperature treatment		低温处理 Low temperature treatment
内参基因 Reference gene	SD	r	SD	r	SD	r	SD	r	SD	r	SD	r
18SrRNA	1.12	0.01	0.38	0.48	0.59	0.96	0.86	0.85	3.80	0.87	0.78	-0.03
ACT	1.02	0.93	0.35	0.80	0.42	0.83	1.16	0.75	0.92	0.85	0.71	-0.09
CYP	1.66	0.74	0.74	0.74	0.98	0.88	1.09	0.71	1.20	0.58	1.57	0.91
EF-1α	0.98	0.69	0.44	0.12	0.49	0.45	1.15	0.91	0.40	0.34	0.89	0.84
F-box	1.42	0.89	0.60	0.65	0.54	0.96	1.60	0.90	0.82	0.44	1.57	0.92
GAPDH	1.57	0.90	1.07	0.92	0.47	0.01	1.28	0.80	0.79	0.43	1.59	0.94
TUA	0.54	0.42	0.65	-0.65	0.83	0.91	1.18	0.80	1.22	0.89	0.81	0.78
TUB	1.41	0.74	0.73	0.67	0.73	0.92	0.90	0.80	1.16	0.85	0.91	0.91
UBQ	0.76	0.35	0.51	-0.53	0.27	0.75	0.76	0.83	0.93	0.62	1.49	0.94

冬瓜实时荧光定量PCR内参基因的筛选与评价

Screening and Evaluation of Reference Genes for Real-time Quantitative PCR in Wax Gourd

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V_n/V_n+1	不同组织 Different tissues	不同发育时期果实 Fruits at different stages	疫霉处理 Phytophthora melonis infection	盐处理 Salt treatment	高温处理 High temperature treatment	低温处理 Low temperature treatment
V₂/V₃	0.37	0.13	0.07	0.10	0.12	0.13
V₃/V₄	0.26	0.09	0.06	0.10	0.12	0.13
V₄/V₅	0.26	0.12	0.08	0.08	0.20	0.12
V₅/V₆	0.21	0.10	0.08	0.07	0.14	0.13
V₆/V₇	0.20	0.16	0.09	0.08	0.14	0.10
V₇/V₈	0.18	0.15	0.08	0.07	0.15	0.21
V₈/V₉	0.24	0.14	0.11	0.08	0.42	0.21

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冬瓜实时荧光定量PCR内参基因的筛选与评价

Screening and Evaluation of Reference Genes for Real-time Quantitative PCR in Wax Gourd

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