近零磁场下灰飞虱转录表达分析稳定性内参基因筛选

doi:10.3864/j.issn.0578-1752.2019.19.006

摘要/Abstract

摘要：

【背景】 地磁场（geomagnetic field,GMF）并不是稳定不变的,其随时间和空间时刻变化。目前,随着对动物磁生物学研究的日益深入,基于实时荧光定量PCR (qRT-PCR)技术开展的磁响应基因转录表达谱研究有力促进了磁响应通路的鉴定和磁感受机制的揭示。【目的】 筛选近零磁场（near-zero magnetic field,NZMF）下短翅型灰飞虱（Laodelphax striatellus）稳定表达的内参基因,使对目的基因的定量分析更加准确。【方法】 迁飞性昆虫灰飞虱采自江苏省农业科学院试验田并在室内使用TN1三叶期稻苗进行扩繁（温度：（25.0±1.0）℃,相对湿度：70%—90%,光周期：14L﹕10D）。采用亥姆霍兹线圈室内模拟近零磁场（NZMF;＜500 nT）和地磁场（GMF;~50 000 nT）,人工模拟磁场强度有效处理空间为直径30 cm的球形空间,试验过程中严格控制除磁场强度外的环境因子（温度：（25.0±1.0）℃,相对湿度：75%,光周期：14L﹕10D）并利用磁通门计每日对人工模拟磁场进行校准和监测,灰飞虱连同TN1三叶期稻苗均置于试管中进行暴露处理,每隔两日与对照磁场中稻苗对调以避免稻苗潜在磁响应对灰飞虱的影响。利用Trizol法分别提取初羽化灰飞虱雌、雄成虫总RNA,检测各生物学重复RNA质量并调至含量一致,反转录为cDNA,利用qRT-PCR技术并结合常用内参筛选分析软件geNorm、NormFinder、BestKeeper以及在线综合分析系统RefFinder对在NZMF和GMF两种磁场强度下灰飞虱体内的内参基因稳定性进行评估筛选,其中,待评估的11个常用内参基因包括Actin1、Tubulin（α1TUB和α2TUB）、Elongation factor 1 alpha（EF-1α）、Glyceraldehyde-3-phosphate dehydrogenase（GAPDH）、Ubiquitin（UBI）、Ribosomal protein S11（RPS11）、Ribosomal protein S15e（RPS15）、Ribosomal protein L8（RPL8）、Ribosomal protein L9（RPL9）和ADP ribosylation factor2（ARF2）。【结果】 不同磁场环境（NZMF vs. GMF）下,灰飞虱短翅雌成虫EF-1α和RPL9表达稳定性在geNorm和NormFinder两种评估方法中都居于前两位,与BestKeeper软件的结果略有差异,进而利用在线工具RefFinder对以上3种方法的评估结果进行稳定性综合排序,结果表明EF-1α稳定性最好,RPL9稳定性次之;灰飞虱短翅雄成虫中,基于geNorm、NormFinder和BestKeeper 3种评估方法,α2TUB和RPL9表达稳定性中均居于前两位,而Actin1表达稳定性虽在NormFinder和BestKeeper中处于前两位,但其在geNorm中稳定性较低,最后,通过在线工具RefFinder综合分析表明,α2TUB稳定性最好,RPL9稳定性次之。【结论】 明确了不同磁场强度（NZMF vs. GMF）下适用于灰飞虱短翅雌、雄成虫中稳定表达的内参基因,其中,若使用双内参系统,雌成虫中可使用EF-1α和RPL9搭配,雄成虫中可使用α2TUB和RPL9搭配,为稳定表达的内参基因系统。此外,RPL9在灰飞虱短翅型雌、雄成虫中均可作为稳定的单一内参基因使用。研究结果确保了对灰飞虱响应磁场强度变化研究中关键目的基因转录表达的准确定量,并为今后开展磁场强度变化下的转录表达谱分析提供了有力保障。

关键词: 灰飞虱, 近零磁场, 磁场强度, 实时荧光定量PCR, 内参基因筛选

Abstract:

【Background】 The geomagnetic field (GMF) is not constant, it can change with time and space. At present, with the development of research on animal magnetic biology, the study on transcriptional profiling of magnetic response genes with quantitative real-time PCR (qRT-PCR) has greatly promoted the identification of magnetic response pathway and the uncovering of magnetoreception mechanism.【Objective】 The objective of this study is to screen the internal reference genes of the brachypterous small brown planthopper (Laodelphax striatellus) under near-zero magnetic field (NZMF), and to make the quantification of target genes more accurate.【Method】 The population of L. striatellus, a migratory insect, was collected from the experimental fields of Jiangsu Academy of Agricultural Sciences and expanded indoors using TN1 three-leaf rice seedlings (temperature: (25.0±1.0)℃, relative humidity: 70%-90%, photoperiod: 14L﹕10D). Helmholtz coils system was used to simulate the near-zero magnetic field (NZMF; ＜500 nT) vs. geomagnetic field (GMF; ~ 50 000 nT), the artificial simulated magnetic field intensity was homogeneous within a spherical space with a diameter of 30 cm. During the experiment, environmental factors other than the magnetic field intensity were strictly controlled (temperature: (25.0±1.0)℃, relative humidity: 75%, photoperiod: 14L﹕10D) and the artificial simulated magnetic field was calibrated and monitored daily using a fluxgate magnetometer. The L. striatellus and TN1 three-leaf rice seedlings were placed in a test tube for exposure treatment. Every two days, the rice seedlings in control and treatment magnetic fields were swapped to avoid the influence triggered by the potential magnetic response of rice seedlings on L. striatellus. Trizol method was used to extract the total RNA of the female and male adults of L. striatellus, respectively. The quality of total RNA was inspected and adjusted to the same mass, and cDNA was then made by reverse transcription. Using qRT-PCR technique and combined with the common internal reference selection software including geNorm, NormFinder, BestKeeper, and the online integrated analysis system RefFinder, the stability of internal reference genes in L. striatellus under NZMF and GMF was evaluated and screened. Among them, 11 common candidate internal reference genes to be evaluated included Actin1, Tubulin (α1TUB and α2TUB), Elongation factor 1 alpha (EF-1α), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Ubiquitin (UBI), Ribosomal protein S11 (RPS11), Ribosomal protein S15e (RPS15), Ribosomal protein L8 (RPL8), Ribosomal protein L9 (RPL9), and ADP ribosylation factor2 (ARF2). 【Result】 For brachypterous female adults under different magnetic field environments (NZMF vs. GMF), the expression stability of EF-1α and RPL9 ranked as the top two in the two assessment software of geNorm and NormFinder, which was slightly different from the results of BestKeeper software. Furthermore, the stability of the above three methods was sorted by online tool RefFinder. The results showed that the stability of EF-1α was the best, followed by RPL9. For brachypterous male adults under different magnetic field environments (NZMF vs. GMF), based on the three evaluation methods of geNorm, NormFinder, and BestKeeper, the expression stability of α2TUB and RPL9 ranked as the top two. Although the expression stability of Actin1 was in the top two in NormFinder and BestKeeper, its stability was low in geNorm. Finally, through the online tool RefFinder synthesis analysis, it was shown that the stability of α2TUB was the best, followed by RPL9. 【Conclusion】 Stably expressed reference genes in the brachypterous male and female adults of L. striatellus were clarified under different magnetic field intensities (NZMF vs. GMF). For a double reference gene system, the combination of EF-1α and RPL9, and the combination of α2TUB and RPL9 can be used in the brachypterous female and male adults, respectively, which is a stable reference gene system. Also, RPL9 can be used alone as a stable reference gene in both male and female brachypterous adults of L. striatellus. The results of this study ensure the accurate quantification of transcription expression of key target genes in response to changes in magnetic field intensity, and provide a strong guarantee for the future analysis of transcription expression profile under changes in magnetic field intensity.

Key words: Laodelphax striatellus, near-zero magnetic field, magnetic field intensity, quantitative real-time PCR (qRT-PCR), internal-reference gene selection

刘凡奇,万贵钧,曾路影,李春绪,潘卫东,陈法军. 近零磁场下灰飞虱转录表达分析稳定性内参基因筛选[J]. 中国农业科学, 2019, 52(19): 3346-3356.

LIU FanQi,WAN GuiJun,ZENG LuYing,LI ChunXu,PAN WeiDong,CHEN FaJun. Selection of Stable Internal Reference Genes for Transcript Expression Analyses in Laodelphax striatellus Under Near-Zero Magnetic Field[J]. Scientia Agricultura Sinica, 2019, 52(19): 3346-3356.

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引物 Primer	序列 Sequence (5′ to 3′)	扩增效率 Amplification efficiency (%)	GenBank编号 GenBank number	描述 Description
LSACT1-F	CGTGACTTGACCGACTACCT	101.49	KC683802.1	Actin1
LSACT1-R	GCACAGCTTCTCCTTGATGTCT	101.49	KC683802.1	Actin1
LSARF2-F	AATCAAAATAAAGCATCGCCCAC	100.44	JF728807.1	ADP ribosylation factor2
LSARF2-R	AGCAGCCCCATTATTTAACACGA	100.44	JF728807.1	ADP ribosylation factor2
LSα1TUB-F	TCCGAGAAAGCATACCACGAACA	100.58	AY508717.1	Alpha 1-tubulin
LSα1TUB-R	TCCACGGTACAACATGCAACAAG	100.58	AY508717.1	Alpha 1-tubulin
LSα2TUB-F	GTTCGCCATCTACCCTGCTC	100.02	AY550922.1	Alpha 2-tubulin
LSα2TUB-R	CAGATGTCGTAGATAGCCTCATTGT	100.02	AY550922.1	Alpha 2-tubulin
LSEF-1α-F	CTCTGCTCGCCTTCACACTC	99.98	N/A	Elongation factor 1 alpha
LSEF-1α-R	GCCGGGTTGTAGCCAATCTTC	99.98	N/A	Elongation factor 1 alpha
LSGAPDH-F	GCCAGTGCCCAATGTATCAGTC	100.26	HQ385974.1	Glyceraldehyde-3-phosphate dehydrogenase
LSGAPDH-R	AGACGACCTCTTCCTCGGTG	100.26	HQ385974.1	Glyceraldehyde-3-phosphate dehydrogenase
LSRPL8-F	CAACTATGCCACTGTCATTGCT	100.51	HQ385976.1	Ribosomal protein L8
LSRPL8-R	CGCCACAATGCCAACCATC	100.51	HQ385976.1	Ribosomal protein L8
LSRPL9-F	TGTGACCACCGAAAACAACTCT	100.24	JF728806.1	Ribosomal protein L9
LSRPL9-R	TCGTCCTTCTGCTTTGTCGAGT	100.24	JF728806.1	Ribosomal protein L9
LSRPS11-F	GGCACAAGCTACCGCATTAACAT	100.05	N/A	Ribosomal protein S11
LSRPS11-R	GCCTGTTCCTCTGACAACTACT	100.05	N/A	Ribosomal protein S11
LSRPS15-F	GTTGCCAGCCATTTTATCCAGTC	100.12	N/A	Ribosomal protein S15e
LSRPS15-R	TTGGGTTCTTCGTCTGCCAT	100.12	N/A	Ribosomal protein S15e
LSUBI-F	GTTGTTCCAGTGATTGTGCTGT	100.45	N/A	Ubiquitin
LSUBI-R	ACGGCTCCACCTCCAAC	100.45	N/A	Ubiquitin

排序 Rank	geNorm		NormFinder		BestKeeper		RefFinder
排序 Rank	基因 Gene	稳定值 Stability value	基因 Gene	稳定值 Stability value	基因 Gene	稳定值 Stability value	基因 Gene	稳定值 Stability value
1	EF-1α	0.171	EF-1α	0.046	UBI	1.05	EF-1α	2.11
2	RPL9	0.173	RPL9	0.046	RPS11	1.07	RPL9	2.21
3	ARF2	0.175	ARF2	0.056	α2TUB	1.10	UBI	3.34
4	RPS15	0.176	α1TUB	0.059	EF-1α	1.11	RPS15	3.36
5	UBI	0.188	RPS15	0.061	Actin1	1.11	ARF2	3.95
6	RPS11	0.196	UBI	0.073	RPL9	1.12	RPS11	4.56
7	α2TUB	0.208	RPS11	0.078	α1TUB	1.13	α2TUB	5.66
8	α1TUB	0.223	α2TUB	0.100	RPS15	1.14	α1TUB	7.44
9	GAPDH	0.237	GAPDH	0.128	ARF2	1.16	Actin1	7.75
10	Actin1	0.248	Actin1	0.129	RPL8	1.21	GAPDH	9.72
11	RPL8	0.264	RPL8	0.137	GAPDH	1.22	RPL8	10.74

排序 Rank	geNorm		NormFinder		BestKeeper		RefFinder
排序 Rank	基因 Gene	稳定值 Stability value	基因 Gene	稳定值 Stability value	基因 Gene	稳定值 Stability value	基因 Gene	稳定值 Stability value
1	α2TUB	0.012	α2TUB	0.042	Actin1	1.07	α2TUB	1.32
2	RPL9	0.013	Actin1	0.049	RPL9	1.10	Actin1	2.00
3	α1TUB	0.013	RPL9	0.053	α2TUB	1.10	RPL9	2.38
4	RPS11	0.014	GAPDH	0.058	UBI	1.10	α1TUB	3.41
5	RPS15	0.014	α1TUB	0.069	α1TUB	1.11	UBI	5.60
6	Actin1	0.014	RPL8	0.061	GAPDH	1.14	GAPDH	6.00
7	UBI	0.016	RPS15	0.068	ARF2	1.15	RPS11	6.47
8	GAPDH	0.017	UBI	0.069	RPS15	1.16	RPS15	8.24
9	RPL8	0.019	RPS11	0.103	RPS11	1.16	RPL8	8.74
10	ARF2	0.025	EF-1α	0.125	RPL8	1.16	ARF2	9.82
11	EF-1α	0.026	ARF2	0.154	EF-1α	1.16	EF-1α	10.24