Screening of reference genes for real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) in tomato induced by different hormones

doi:10.3785/j.issn.1008-9209.2022.03.081

Journal of Zhejiang University (Agriculture and Life Sciences)

2023, Vol. 49

Issue (1): 31-44 DOI: 10.3785/j.issn.1008-9209.2022.03.081

Horticultural sciences

Screening of reference genes for real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) in tomato induced by different hormones

Shengyi BAI¹(

),Xiaomin WANG^1,^2,^3,⁴(

),Wenjuan LIU¹,Guoxin CHENG^1,^2,^3,⁴,Meng GUO^1,^2,^3,⁴,Wenkong YAO^1,^2,^3,⁴,Yanming GAO^1,^2,^3,⁴,Jianshe LI^1,^2,^3,⁴

^1.School of Agriculture, Ningxia University, Yinchuan 750021, Ningxia, China
^2.Key Laboratory of Modern Molecular Breeding for Dominant and Special Crops in Ningxia, Yinchuan 750021, Ningxia, China
^3.Ningxia Modern Facility Horticulture Engineering and Technology Research Center, Yinchuan 750021, Ningxia, China
^4.Ningxia Facility Horticulture Technology Innovation Center (Ningxia University), Yinchuan 750021, Ningxia, China

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Abstract

Screening of stable reference genes was significant when the real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to study gene expression. To identify the most stable reference genes in tomato induced by different hormones, the leaves of susceptible tomato ‘Moneymaker’ (MM) and resistant tomato inbred line 62579, which were treated with abscisic acid (ABA), methyl jasmonate (MeJA), and salicylic acid (SA) for 0, 24, 48, and 120 h, respectively, were used for qRT-PCR amplification. In the current study, the expression stabilities of eight tomato candidate reference genes, including EF1α, L33, Act, Ubi, GAPDH, UK, CAC, and TIP41, were analyzed using geNorm, NormFinder, and BestKeeper softwares. The results revealed that the average C_T values of eight candidate reference genes ranged from 26 to 34. Based on the data from these softwares, L33 and Ubi, L33 and EF1α,as well as EF1α and L33 were considered to be the stably expressed reference genesin tomato induced by ABA, MeJA, and SA, respectively. In conclusion, L33 is the most stably expressed gene among all studied candidate reference genes in tomato induced by different hormones. The most stable reference genes screened in this study will provide a calibration basis for the expression analyses of differential genes and the research on molecular mechanisms in tomato response to exogenous hormone treatments in the future.

Key words： tomato hormone induction real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) reference gene

Received: 08 March 2022 Published: 07 March 2023

CLC:

S641.2

Corresponding Authors: Xiaomin WANG E-mail: baishengyi0602@163.com;wangxiaomin_1981@163.com

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	Shengyi BAI
	Xiaomin WANG
	Wenjuan LIU
	Guoxin CHENG
	Meng GUO
	Wenkong YAO
	Yanming GAO
	Jianshe LI

Cite this article:

Shengyi BAI,Xiaomin WANG,Wenjuan LIU,Guoxin CHENG,Meng GUO,Wenkong YAO,Yanming GAO,Jianshe LI. Screening of reference genes for real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) in tomato induced by different hormones. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(1): 31-44.

URL:

https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2022.03.081 OR https://www.zjujournals.com/agr/Y2023/V49/I1/31

不同激素处理下番茄实时荧光定量聚合酶链反应内参基因的筛选

为筛选出在不同激素诱导下番茄中较为稳定的内参基因，本研究以脱落酸（abscisic acid, ABA）、茉莉酸甲酯（methyl jasmonate, MeJA）、水杨酸（salicylic acid, SA）3种外源激素分别诱导处理0、24、48、120 h的感病番茄品种‘Moneymaker’（MM）和抗病番茄自交系62579叶片为试验材料，通过实时荧光定量聚合酶链反应（real-time fluorescent quantitative polymerase chain reaction, qRT-PCR）进行扩增，利用geNorm、NormFinder和BestKeeper 3个软件分析EF1α、L33、Act、Ubi、GAPDH、UK、CAC、TIP41这8个番茄候选内参基因的表达稳定性。结果表明，8个候选内参基因的平均C_T值为26～34。综合3个软件的分析结果发现，在ABA诱导下，番茄中较稳定表达的内参基因为L33和Ubi；在MeJA诱导下，番茄中较稳定表达的内参基因为L33和EF1α；在SA诱导下，番茄中较稳定表达的内参基因为EF1α和L33。综上所述，L33基因是番茄在不同激素诱导下表达最稳定的候选内参基因。本研究筛选的最稳定内参基因将为后续番茄响应外源激素处理的差异基因表达分析和分子机制研究提供校准依据。

关键词： 番茄, 激素诱导, 实时荧光定量聚合酶链反应, 内参基因

序号No.	基因名称 Gene name	基因符号 Gene symbol	引物序列（5 $′$ →3 $′$ ） Primer sequence (5 $′$ →3 $′$ )	产物长度 Product length/bp	文献 Reference
1	Elongation factor 1α	EF1α	F: ACAGGCGTTCAGGTAAGGAA	120	[27]
1	Elongation factor 1α	EF1α	R: GAGGGTATTCAGCAAAGGTCTC	120	[27]
2	50S ribosomal protein L33	L33	F: GGGAAGAGGCTGGGATACATC	138	[9]
2	50S ribosomal protein L33	L33	R: AGGAGGCAAATTGGACTTGAAC	138	[9]
3	β-actin	Act	F: GCTCCACCAGAGAGGAAATACAGT	107	[9]
3	β-actin	Act	R: CATACTCTGCCTTTGCAATCCA	107	[9]
4	Ubiquitin	Ubi	F: GGACGGACGTACTCTAGCTGAT	134	[27]
4	Ubiquitin	Ubi	R: AGCTTTCGACCTCAAGGGTA	134	[27]
5	Glyceraldehyde-3-phosphate dehydrogenase	GAPDH	F: ACCACAAATTGCCTTGCTCCCTTG	110	[28]
5	Glyceraldehyde-3-phosphate dehydrogenase	GAPDH	R: ATCAACGGTCTTCTGAGTGGCTGT	110	[28]
6	Uridylate kinase	UK	F: TGGTAAGGGCACCCAATGTGCTAA	114	[29]
6	Uridylate kinase	UK	R: ATCATCGTCCCATTCTCGGAACCA	114	[29]
7	Clathrin adaptor complexes medium subunit	CAC	F: CCTCCGTTGTGATGTAACTGG	173	[28]
7	Clathrin adaptor complexes medium subunit	CAC	R: ATTGGTGGAAAGTAACATCATCG	173	[28]
8	TAP42-interacting protein	TIP41	F: ATGGAGTTTTTGAGTCTTCTGC	235	[23]
8	TAP42-interacting protein	TIP41	R: GCTGCGTTTCTGGCTTAGG	235	[23]

Table 1 Primer information of tomato candidate reference genes

Fig. 1 Testing results of RNA integrity in tomato induced by different hormonesA. Susceptible tomato MM; B. Resistant tomato 62579. M: DL2000 marker.

Fig. 2 Agarose gel electrophoresis analysis of PCR products of eight candidate reference genes in tomatoM: DL2000 marker; 1: EF1α;2: L33;3: Act;4: Ubi;5: GAPDH;6: UK;7: CAC;8: TIP41.

Fig. 3 Distribution of C_T values of eight candidate reference genes in tomato

Fig. 4 Expression stability (M) of eight candidate reference genes analyzed by geNorm softwareA. ABA-induced treatment; B. MeJA-induced treatment; C. SA-induced treatment.

Fig. 5 Pairwise variation (V) of eight candidate reference genes analyzed by geNorm softwareA. ABA-induced treatment; B. MeJA-induced treatment; C. SA-induced treatment.

Table 2 Analysis results from NormFinder software

Table 3 Expression stability of eight candidate reference genes under ABA induction analyzed by BestKeeper software

Table 4 Expression stability of eight candidate reference genes under MeJA induction analyzed by BestKeeper software

Table 5 Expression stability of eight candidate reference genes under SA induction analyzed by BestKeeper software

Table 6 Ranking of eight candidate reference genes stability in tomato induced by ABA

Table 7 Ranking of eight candidate reference genes stability in tomato induced by MeJA

Table 8 Ranking of eight candidate reference genes stability in tomato induced by SA


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