Selection of Stable Internal Reference Genes for Transcript Expression Analyses in Laodelphax striatellus Under Near-Zero Magnetic Field
LIU FanQi1, WAN GuiJun1, ZENG LuYing1, LI ChunXu1, PAN WeiDong2, CHEN FaJun,11 College of Plant Protection, Nanjing Agricultural University, Nanjing 210095 2 Beijing Key Laboratory of Bioelectromagetics, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190
Received:2019-05-5Accepted:2019-06-3Online:2019-10-01 作者简介 About authors 刘凡奇,E-mail:fanqiliu2014@163.com。
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. Keywords:Laodelphax striatellus;near-zero magnetic field;magnetic field intensity;quantitative real-time PCR (qRT-PCR);internal-reference gene selection
PDF (486KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文 本文引用格式 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 doi:10.3864/j.issn.0578-1752.2019.19.006 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 Acricultura Sinica, 2019, 52(19): 3346-3356 doi:10.3864/j.issn.0578-1752.2019.19.006
0 引言
【研究意义】一个世纪前,人类首次提出“磁场地图”的观点[1]。地磁场(geomagnetic field,GMF)为包括昆虫在内的多种动物,如帝王蝶、海龟、龙虾等,提供了定向和位置信息[2]。此外,磁场强度改变可对许多动物的生长发育及新陈代谢产生影响,这些不同强度的磁场可依据强度范围划分为弱磁场(<1 mT)、中等强度磁场(1 mT—1 T)和强磁场(>1 T)[3,4,5,6,7]。地磁场强度由赤道向两极呈现由低到高的渐变态势,其范围从赤道附近的~25 μT到南北磁极的~65 μT[8]。迁飞昆虫多可进行跨纬度长距离迁移[9],这必然涉及其迁出地与迁入地的地磁强度差异[10]。基因表达分析被广泛应用于生命科学领域,在基于磁场强度变化的磁响应研究中发挥重要作用。实时荧光定量PCR(qRT-PCR)作为对基因进行转录水平表达分析的重要技术手段,具有特异性强、覆盖范围广、操作方便、实时性等特点。qRT-PCR包括相对定量和绝对定量,其中相对定量应用更加广泛,可在处理和对照组中均稳定表达的内参基因系统对定量结果的准确性和可重复性至关重要。常用的Actin(ACT)、18S ribosomal RNA(18S RNA)等内参基因是否可在磁场强度差异处理下的动物体内稳定表达,至今尚无验证。因此,筛选出在磁场强度变化环境下能稳定表达的内参基因对于磁响应基因信号通路的准确鉴定具有重要意义,对生物磁感受机制的揭示具有积极促进作用。【前人研究进展】内参基因是维持细胞生命活动所必需的,在所有细胞中均可表达的一类基因,此观点在前基因组时代提出,研究发现,传统内参基因在不同外界因子处理、生物的不同发育历期、不同组织部位、不同生理条件下表达水平均可能存在差异。因此,科研人员需针对不同外界处理、不同研究对象寻找特异性稳定表达的内参基因(系统)。目前已发表的基于磁场强度变化的磁响应研究中,曾选用GAPDH[11]、α-tubulin[12]、ACT1[13]、Ribosomal protein L9(RPL9)[14,15]、Ubiquitin[16]、EF-1α[17]等作为内参基因。虽然曾有研究对强磁场下细胞系内内参基因稳定性进行评估,但由于细胞系和活体动物内细胞外环境存在较大差异,有必要对活体动物体内内参基因的稳定性进行重新评估。另外,强磁场和弱磁场引起磁生物学效应的机制可能不同,前者可能类似于非特异性的胁迫效应,而近零磁场(near-zero magnetic field,NZMF)等弱磁场可能由与动物磁定向类似的磁感受机制介导[18,19],因此选择与自然地磁场强度差异仅为地磁强度水平的近零磁场作为磁场强度变化处理,所评估出的稳定内参基因对后续特异性磁响应机制转录水平研究更具有普适性。目前,利用细胞系开展的生物对磁场强度变化磁响应研究表明,近零磁场处理将显著影响肌动蛋白(Actin)的组装[12],因此参与Actin蛋白表达调控的相关基因(如ACT1)可能受磁场强度变化的影响,从而无法作为不同磁场强度处理实验中的内参基因。【本研究切入点】灰飞虱(Laodelphax striatellus)广泛分布于东亚、东南亚、北非及欧洲等地区[20,21],其寄主范围广,具有翅的多型现象(长翅型和短翅型),并表现出趋光和迁飞特性[22]。近年来以稻飞虱为模式昆虫开展的基于磁场强度变化的磁生物学效应研究进一步突出了稻飞虱在磁生物学研究领域的潜在模式作用[23,24]。NZMF与自然GMF的磁场强度差异仅为地磁强度水平,既可将其视为屏蔽地磁场的阴性对照,又可利用其探究地磁强度水平磁场强度变化对生物的潜在磁效应,且由于该磁场强度更能反映生物正常生理性磁响应,因此评估筛选可用于灰飞虱对NZMF(vs. GMF)磁响应研究中的稳定内参基因(系统)将是本研究及后续磁感受机制研究的重要切入点。【拟解决的关键问题】利用人工磁场模拟磁场强度变化(NZMF vs. GMF),以灰飞虱初羽化1日龄短翅雌、雄成虫作为研究对象,筛选可在近零磁场和对照地磁场中稳定表达的内参基因,为灰飞虱磁响应研究中关键目标基因的转录表达精确分析提供方法学依据,提高后续动物特异性磁响应基因信号转导通路鉴定的准确性。
Fig. 1Expression levels of 11 candidate internal-reference genes in brachypterous female adults of L. striatellus
Table 2 表2 表2利用geNorm、NormFinder、BestKeeper和RefFinder分析灰飞虱短翅雌成虫中各内参基因稳定性 Table 2The expression stability of the candidate reference genes in brachypterous female adults of L. striatellus calculated by geNorm, NormFinder, BestKeeper and RefFinder
Fig. 3Expression levels of 11 candidate reference genes in brachypterous male adults of L. striatellus
Table 3 表3 表3利用geNorm、NormFinder、BestKeeper和RefFinder分析灰飞虱短翅雄成虫中各内参基因稳定性 Table 3The expression stability of the candidate reference genes in brachypterous male adults of L. striatellus calculated by geNorm, NormFinder, BestKeeper and RefFinder
不同磁场环境(NZMF vs. GMF)下,EF-1α和RPL9在短翅雌成虫体内能稳定表达,可作为理想的双内参基因体系;α2TUB和RPL9在短翅雄成虫体内稳定表达,可作为理想的双内参基因体系;在短翅型雌、雄成虫对近零磁场的磁响应研究中,RPL9均表现出稳定表达的特性,可作为单一内参基因进行基因转录水平表达研究。研究结果明确了不同磁场强度下适用于灰飞虱短翅雌、雄成虫中稳定表达的内参基因,确保了对灰飞虱响应磁场强度变化研究中关键目标基因转录表达水平的准确定量,可为今后开展磁场强度变化下的转录表达谱分析提供有力保障。
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贺静斓, 张明, 刘瑞莹, 万贵钧, 潘卫东, 陈法军 . 近零磁场下干扰磁响应关键基因对褐飞虱寿命的影响 中国农业科学, 2019,52(1):45-55. DOI:10.3864/j.issn.0578-1752.2019.01.005Magsci [本文引用: 1] <p id="C2">【目的】 隐花色素(cryptochrome, Cry)和铁硫簇蛋白IscA(iron-sulfur cluster assembly,即MagR)是生物体内潜在的磁受体蛋白,本研究通过RNA干扰(RNAi)技术,分别敲减褐飞虱(<italic>Nilaparvata lugens</italic>)体内的磁响应关键基因<italic>NlCry1</italic>、<italic>NlCry2</italic>和<italic>NlMagR</italic>,旨在探明近零磁场(near-zero magnetic field,NZMF)环境下,以上3种基因在褐飞虱寿命调节过程中的作用,从而间接探讨这3种基因对磁场的响应情况。【方法】 采用RNAi技术,以实验室正常磁场环境下稳定饲养的短翅初羽化褐飞虱雌雄成虫为材料,通过向其体内注射双链RNA(dsRNA)分别抑制磁响应关键基因<italic>NlCry1</italic>、<italic>NlCry2</italic>和<italic>NlMagR</italic>,随后立即分别放入正常磁场(geomagnetic field,GMF)和近零磁场中,于每日相同时间观察记录试虫寿命。同时于注射后的1、2和3 d通过RNAiso Plus法提取GMF中褐飞虱雌成虫总RNA,反转录合成第一链DNA,后采用实时荧光定量PCR(RT-qPCR)技术检测该基因的表达情况,以确定基因干扰效率。【结果】 注射ds<italic>NlCry1</italic>后,褐飞虱雌雄成虫寿命在近零磁场和正常磁场间均无显著差异。注射ds<italic>NlCry2</italic>后,近零磁场中褐飞虱雌雄成虫寿命比正常磁场分别显著延长27.78%和50.04%;此外,与注射ds<italic>GFP</italic>处理相比,正常磁场下注射ds<italic>NlCry2</italic>的雌成虫寿命缩短,而近零磁场下注射ds<italic>NlCry2</italic>的雌成虫寿命延长,但二者差异均不显著;近零磁场和正常磁场下注射ds<italic>NlCry2</italic>的雄成虫寿命均缩短(25.41%和10.73%),且正常磁场下差异显著。近零磁场中,注射ds<italic>NlMagR</italic>的雌成虫寿命较注射ds<italic>GFP</italic>的寿命显著缩短了16.48%,而雄成虫寿命在磁场间、干扰处理间的差异均不显著。【结论】 磁场变化下褐飞虱雌雄成虫体内3种磁响应关键基因对其寿命的调节功能存在差异。其中,<italic>NlCry2</italic>对磁场变化存在敏感响应,表现为敲减该基因与磁场变化的互作显著地影响雌雄成虫寿命,且表现出“性二型性”;<italic>NlMagR</italic>也可对磁场变化产生明显响应,但该响应只存在于雌成虫;此外,<italic>NlCry1</italic>对磁场变化无响应,该基因或与褐飞虱雌雄成虫寿命调节无关。</p> HE JL, ZHANGM, LIU RY, WAN GJ, PAN WD, CHEN FJ . Effects of the interference of key magnetic response genes on the longevity of brown planthopper (Nilaparvata lugens) under near-zero magnetic field Scientia Agricultura Sinica, 2019,52(1):45-55. (in Chinese) DOI:10.3864/j.issn.0578-1752.2019.01.005Magsci [本文引用: 1] <p id="C2">【目的】 隐花色素(cryptochrome, Cry)和铁硫簇蛋白IscA(iron-sulfur cluster assembly,即MagR)是生物体内潜在的磁受体蛋白,本研究通过RNA干扰(RNAi)技术,分别敲减褐飞虱(<italic>Nilaparvata lugens</italic>)体内的磁响应关键基因<italic>NlCry1</italic>、<italic>NlCry2</italic>和<italic>NlMagR</italic>,旨在探明近零磁场(near-zero magnetic field,NZMF)环境下,以上3种基因在褐飞虱寿命调节过程中的作用,从而间接探讨这3种基因对磁场的响应情况。【方法】 采用RNAi技术,以实验室正常磁场环境下稳定饲养的短翅初羽化褐飞虱雌雄成虫为材料,通过向其体内注射双链RNA(dsRNA)分别抑制磁响应关键基因<italic>NlCry1</italic>、<italic>NlCry2</italic>和<italic>NlMagR</italic>,随后立即分别放入正常磁场(geomagnetic field,GMF)和近零磁场中,于每日相同时间观察记录试虫寿命。同时于注射后的1、2和3 d通过RNAiso Plus法提取GMF中褐飞虱雌成虫总RNA,反转录合成第一链DNA,后采用实时荧光定量PCR(RT-qPCR)技术检测该基因的表达情况,以确定基因干扰效率。【结果】 注射ds<italic>NlCry1</italic>后,褐飞虱雌雄成虫寿命在近零磁场和正常磁场间均无显著差异。注射ds<italic>NlCry2</italic>后,近零磁场中褐飞虱雌雄成虫寿命比正常磁场分别显著延长27.78%和50.04%;此外,与注射ds<italic>GFP</italic>处理相比,正常磁场下注射ds<italic>NlCry2</italic>的雌成虫寿命缩短,而近零磁场下注射ds<italic>NlCry2</italic>的雌成虫寿命延长,但二者差异均不显著;近零磁场和正常磁场下注射ds<italic>NlCry2</italic>的雄成虫寿命均缩短(25.41%和10.73%),且正常磁场下差异显著。近零磁场中,注射ds<italic>NlMagR</italic>的雌成虫寿命较注射ds<italic>GFP</italic>的寿命显著缩短了16.48%,而雄成虫寿命在磁场间、干扰处理间的差异均不显著。【结论】 磁场变化下褐飞虱雌雄成虫体内3种磁响应关键基因对其寿命的调节功能存在差异。其中,<italic>NlCry2</italic>对磁场变化存在敏感响应,表现为敲减该基因与磁场变化的互作显著地影响雌雄成虫寿命,且表现出“性二型性”;<italic>NlMagR</italic>也可对磁场变化产生明显响应,但该响应只存在于雌成虫;此外,<italic>NlCry1</italic>对磁场变化无响应,该基因或与褐飞虱雌雄成虫寿命调节无关。</p>
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