Stability evaluation of reference genes for real-time quantitative PCR normalization in Spodoptera frugiperda(Lepidoptera: Noctuidae)

Real-time quantitative PCR(qPCR) is a reliable and widely used technique for analyzing the expression profiles of target genes in different species, and reference genes with stable expressions have been introduced for the normalization of the data. Therefore, stability evaluation should be considered as the initial step for qPCR experiments. The fall armyworm Spodoptera frugiperda(J. E. Smith)(Lepidoptera: Noctuidae) is a polyphagous pest that consumes many plant species and seriously threatens corn production around the world. However, no studies thus far have examined the stability of reference genes in this pest. In this study, the expression profiles of the eight candidate reference genes of Actin, elongation factor 1 alpha(EF1α), elongation factor 2(EF2), glyceraldehyde-3-phosphate dehydrogenase(GAPDH), ribosomal protein L3(RPL3), ribosomal protein L13(RPL13), alpha-tubulin(α-TUB), and beta-1-tubulin(β-1-TUB) were obtained from S. frugiperda in different samples and the stability was evaluated by ΔCt, BestKeeper, geNorm, NormFinder, and RefFinder methods. The results of pairwise variation(V) calculated by GeNorm indicated two reference genes could be selected for normalization. Therefore, the combinations of the most stable reference genes for different experimental conditions of S. frugiperda were shown as follows: EF2 and RPL13 for developmental stages, RPL3 and β-1-TUB for larval tissue samples, EF2 and EF1α for the larval samples treated with different temperatures, RPL3 and EF1α for the larval samples under starvation stress, and RPL13 and EF1α for all the samples. Our results lay the foundation for the normalization of qPCR analyses in S. frugiperda and could help guarantee the accuracy of subsequent research.     

Selection of reference genes for RT-qPCR analysis of Phenacoccus solenopsis (Hemiptera: Pseudococcidae) sex-dimorphic development

Mealybugs, such as Phenacoccus solenopsis, are highly sexually dimorphic. Winged adult males present such remarkable morphological differences from females that, to the untrained eye, conspecific adults of both sexes of P. solenopsis may be considered as two different insect species. A method to investigate sex-dimorphic mechanisms is by evaluating gene expression using RT-qPCR. However, the accuracy and consistency of this technique depend on the reference gene(s) selected. In this study, we analyzed the expression of 10 candidate reference genes in male and female P. solenopsis at different development stages, using common algorithms including the ?Ct method, NormFinder, geNorm, BestKeeper, and a web-based analysis tool, RefFinder. The results showed that EF1-β, RP-L32 and RP-18 S were selected as the most stable genes by both the ?Ct method and NormFinder; TUB-α was the most stable gene identified by BestKeeper; and RP-L40 and RP-L32 were the most stable genes ranked by geNorm. RefFinder, a comprehensive analysis software, ranked the ten genes and determined EF1-β and RP-L32 as the most suitable reference genes for the various developmental stages in male and female P. solenopsis. Furthermore, the two most suitable reference genes were validated by examining expression of the juvenile hormone acid O-methytransferase(JHAMT) gene. Results of the validation portion of the study showed that JHAMT expression was sex-biased towards males and exhibited a dynamic and classic expression pattern among the P. solenopsis developmental stages. The results can help further our knowledge on the molecular mechanisms underlying sexual dimorphic development in P. solenopsis.     

Reference genes for quantitative real-time PCR analysis and quantitative expression of P5CS in Agropyron mongolicum under drought stress

Reference genes, stably expressing in different tissues and cells, are commonly used as the references in expression analysis. Selecting the optimum reference gene is crucial to the success of experiments. In this study, the expression stabilities of nine common reference genes, including ACT2, 18 S r RNA, APRT, EF-1α, RNA POL II, TUBα, TUBβ, GAPDH and TLF of Agropyron mongolicum, were studied under drought condition. Among them, 18 S r RNA was found to be the most optimum reference gene under drought stress by the analyzing of ge Norm and Norm Finder software. Quantitative expression levels of P5 CS using 18 S r RNA as the reference gene, and proline contents under drought stress in A. mongolicum were further operated, and we found the expression level of P5 CS gene and proline content had a significantly positive relationship(R~2=0.7763, P0.05). This study established and validated 18 S r RNA as the reference genes in A. mongolicum under drought stress, providing a powerful tool for the quantitative expression analysis of drought genes in A. mongolicum.     

RNA Interference-Mediated Downregulation of sAC Gene Inhibits Sperm Hyperactivation in Male Rats （Rattus norvegicus）

Hyperactivation is one of the most critical parts for fertilization, cAMP generated by soluble adenylyl cyclase （sAC） is necessary to activate sperm and is a prerequisite for sperm hyperactivation. The aim of this study is to investigate the function of sAC in hyperactivation in male rats. Four siRNAs of sAC gene were designed and separately transformed into rat sperm using electrotransformation method. Cultured for 12 and 24 h, physiological and biochemical indexes of these sperm were analyzed, and the expressions of some hyperactivation-related genes were detected using real-time PCR. We demonstrated 26.3-30.8% and 49.1-50.5% reduction in sAC at the protein by Western blot and mRNA levels by real-time PCR, respectively. The results showed that two siRNAs, Actb-717 and Actb-4205, were the best RNAi sites for silencing sAC. The VCL （curvilinear velocity） and ALH （amplitude of lateral head displacement） of RNA interference （RNAi）-transfected sperm were reduced, cAMP and protein phosphorylation in RNAi transfected sperm were also decreased. The hyperactivation-related genes, such as CatSper2, LDHC and PKA, were downregulated in the sperm, which sAC was knockdown. These findings demonstrated that sAC might play a critical role in cAMP signaling in the rat sperm hyperactivation, and downregulated sAC gene might prevent the expression of these hyperactivation-ralated genes resulting in sperm dysfunction. These findings suggest that these hyperactivation-ralated genes and sAC are functionally related in sperm hyperactivation and sAC falls into an expanding group of sperm proteins that appear to be promising targets for the development of male contraceptives.     

Expansin Genes Expressed Differentially in Peduncle Elongation of Near- Isogenic Wheat Rht Lines

The introduction of reduced height （Rht） genes Rht-Blb and Rht-Dlb led to impressive increases in wheat （Triticum aestivum L.） yields during the Green Revolution. In the present study, the dynamic elongation of peduncle in a set ofnear-isogenic lines （NILs） carrying different Rht alleles （Rht-Blb, Rht-Dlb, Rht-Blc, Rht-Dlb＋Rht-Blb, and Rht-Dlb＋Rht-Blc） were investigated. The reduction of the final length of peduncle in NILs was dependent mainly on the elongation rate, which was reduced by Rht genes, during rapid elongation phase. Resin sections showed that Rht genes strongly reduced the cell extension in peduncle. The expression of expansin genes, which mediate cell wall loosening and leading to cell expansion, were analysed by using real- time quantitative PCR （qPCR）. Among the 23 possible wheat expansin genes, 17 were expressed in the peduncle. The spatial distribution of expression was further analysed for five expansins that showed high expression levels in the peduncles of Rht lines. Compared to wild type plants, the incorporation of Rht-Dlb allele decreased about 37 and 80% of the expression levels of ExpA 7 and ExpA3 in elongation zone, respectively. The presence Rht-Blc dwarfing genes, however, produced 53% reduction in the expression level of ExpA7, and seriously decreased about 70% of ExpB9 expression. Although the expression levels of five genes exhibited variability among the lines, an expansin gene, ExpB2, showed its expression level highly associated with the cell elongation rate in peduncle of different Rht lines.     

Differential Gene Expression in Response to Drought Stress in Maize Seedling

To provide the useful information for the choice of molecular marker used in marker-assisted selection of drought tolerance, it is necessary to find out more candidate genes and fulfill the information gaps in gene expression regulation under drought stress. In this study, we isolated four differentially expressed cDNA fragments from leaves of a droughttolerant inbred line by suppression subtractive hybridization and reverse Northern hybridization, and validated their differential expression patterns among six inbred lines with different drought tolerance in response to drought stress by quantitative real-time PCR. Sequence similarity analysis indicated that two of four differentially expressed cDNA showed homology to gene DegP encoding trypsin-like serine protease, and gene PGAM-i encoding cofactor-independent phosphoglyceromutase, respectively. Expressions of the genes corresponding to four cDNA fragments was decreased at 6 h after drought stress treatment in most of the six inbred lines, and then returned to the control level with further stress in three of the tolerant inbred lines. The expression of the gene PGAM-i and the genes corresponding to fragments E4 and F4 were increased to a high level in tolerant inbred line 81565. In the two drought-sensitive inbred lines （Dan340 and ES40）, the expression of these genes was still down-regulated. The probable mechanisms of these genes in response to drought stress were discussed. These results indicated that the drought-tolerant inbred lines upregulated the expression of the drought-tolerant candidate genes, in contrast, drought-sensitive inbred lines downregulated the expression of the genes.     

平欧杂种榛实时荧光定量PCR内参基因的筛选与体系建立

【目的】构建中国榛属植物主要栽培种平欧杂种榛实时荧光定量PCR内参基因的筛选体系,并筛选稳定的内参基因,为榛属植物的基因表达分析提供内参基因,进而为其植物资源利用和创新育种研究提供理论基础。【方法】利用课题组前期对平欧杂种榛不同亲和性授粉、授粉后不同时间的雌蕊转录组测序数据,结合相关文献搜索,共选取12个候选内参基因;以平欧杂种榛主栽品种‘达维’的盛花期雌蕊、未伸长期雄花序、幼嫩叶片、花粉、一年生枝形成层、嫩茎、根尖、根蘖等8个不同组织器官为研究材料;通过反转录PCR初筛,实时荧光定量PCR检测表达量,并利用ge Norm、Norm Finder、Best Keeper、Delta Ct程序和Ref Finder在线网站评价内参基因的稳定性。【结果】反转录PCR初筛表明,12个候选内参基因引物的特异性良好,Cha STP5和Cha TF在不同组织器官材料中表达存在明显差异,其余候选内参基因在8个组织中均有表达。实时荧光定量PCR的表达谱分析表明,Ch18S r RNA的表达量最高,Cha STP5表达量最低,其余10个候选内参基因均为中等表达量;Cha STP5和Cha TF的稳定性最差,其余10个候选内参基因的稳定性处于中等水平。ge Norm、Norm Finder、Best Keeper和Delta Ct的结果表明,Cha Actin均排名第一,为最稳定的内参基因,Ch18S r RNA的排名均在前五,而其他候选内参基因在不同程序分析结果中的排名存在差异。稳定性综合分析表明,Cha Actin和Ch18S r RNA的稳定性良好,即在8个不同组织器官中表达量均一且在4个稳定性分析程序中均排名靠前,适合作为内参基因;ge Norm程序的变异系数分析则表明,选取6个内参基因便可对RT-q PCR的数据进行精准的标准化处理;相关性分析表明,4个程序均在0.01水平上呈现显著的相关性,Norm Finder和Delta Ct程序的相关性最高,Delta Ct与Best Keeper的相关性最低。【结论】建立了平欧杂种榛实时荧光定量PCR内参基因的筛选体系:以反转录PCR初筛引物,荧光定量PCR分析引物特性及基因表达,4个程序单独评价引物稳定性,Ref Finder综合分析选出最适稳定内参基因,并选出榛属植物8个不同组织器官中最为稳定的2个内参基因Cha Actin和Ch18S r RNA。     

幼龄小鼠小肠组织内参基因的选择

选择B2 M、ACTB、GAPDH、SDHA、HPRT1和ARBP共6个内参基因,研究其在幼龄小鼠小肠组织内的表达情况.结果表明:6个内参基因均可获得特异性扩增产物;经geNorm程序和NormFinder分析,6个内参基因稳定度由高到低为SDHA=HRPT1>ARBP>ACTB>GAPDH>B2 M;基因表达的稳定值分别为0.006(SDHA)、0.006(HRPT1)、0.007(ARBP)、0.018(ACTB)、0.029(GAPDH)、0.111(B2 M),最终筛选出SDHA和HRPT1 2个内参基因适合用于校正目的基因的表达.     

产蛋前期和产蛋期籽鹅组织内参基因的稳定性

【目的】分析比较使用广泛的7个候选内参基因在产蛋前期和产蛋期籽鹅组织中的表达情况,筛选籽鹅组织基因表达分析中最佳的内参基因及其数目。【方法】应用实时定量反转录PCR技术,分别检测28S rRNA、18S rRNA、GAPDH、ACTB、HPRT1、SDH和TUB在产蛋前期与产蛋期健康籽鹅肝脏、肾脏、心脏、腿肌和卵巢组织中的表达情况,采用绝对定量方法确定基因拷贝数,然后分别使用geNorm和NormFinder程序进行数据分析。【结果】产蛋前期籽鹅各组织中7个内参基因表达稳定度的顺序分别为：GAPDH=HRPT1（0.195）＞TUB（0.244）＞28S rRNA（0.414）＞18S rRNA（0.495）＞ACTB（0.541）＞SDH（0.610）;产蛋期籽鹅各组织中7个内参基因表达稳定度的顺序分别为：GAPDH=28S rRNA（0.128）＞TUB（0.181）＞ACTB（0.192）＞SDH（0.221）＞HRPT1（0.316）＞18S rRNA（0.362）,且7个基因的配对差异分析分别为V2/3（产蛋前期）=0.084、V2/3（产蛋期）=0.069,内参基因的最适数目均为2个。【结论】产蛋前期和产蛋期籽鹅组织目的基因的表达分析中相对适合的内参基因分别为GAPDH和HRPT1、GAPDH和28S rRNA。     

近零磁场下灰飞虱转录表达分析稳定性内参基因筛选

【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。     

仔猪外周血中内参基因的筛选及细胞因子和受体的表达水平

【目的】中国地方猪种在抗病性和免疫力方面优于国外猪种,但是对中外猪种抗病力差异的遗传基础和免疫机理却了解甚少。有推测不同品种猪对疾病的抵抗力不同可能在细胞因子和受体的表达水平上有所反映。本研究以抗病力强的大蒲莲和抗病力相对较弱的长白猪的断奶仔猪为研究对象,对6个具有重要功能的细胞因子（促炎症细胞因子（IL8和TNFα）,抗炎症细胞因子（IL10）,干扰素调节因子（IRF3）和干扰素（IFNA 和IFNG））和1个最具代表性的模式识别受体（TLR4）基因在大蒲莲和长白仔猪群体外周血液中的基因表达量进行检测,并对各基因进行品种和母猪效应分析,为了解中外不同猪种的免疫水平奠定基础。【方法】采集大蒲莲（104头）和长白（171头）35日龄仔猪的外周血,提取总RNA,逆转录为cDNA后,利用荧光定量PCR法,首先对6个常用的候选内参基因（ACTB、GAPDH、B2M、TBP、RPL4和PPIA）利用geNorm 软件进行稳定性评价分析,筛选仔猪外周血RNA基因定量分析中稳定表达的内参基因,然后利用该内参基因进行校正,对上述细胞因子和模式识别受体基因在大蒲莲和长白仔猪群体外周血中的表达量进行定量,并利用最小二乘法对细胞因子和受体在大蒲莲和长白仔猪群体中的表达量进行品种和母猪效应分析。【结果】研究结果归纳为4个方面： 1.外周血样品中内参基因稳定性值M 从大到小依次为：ACTB（0.595）＞GAPDH（0.571）＞PPIA（0.469）＞RPL4（0.466）＞TBP（0.461）＞B2M（0.440）,选择表达稳定性最高的基因B2M作为内参基因对细胞因子和受体基因的表达量进行校正。2.检测的6个细胞因子的基因表达量存在很大的差异,各细胞因子基因表达量ΔCt最大值和最小值之间差值分别为9.23-14.25和5.83-8.73;受体TLR4的ΔCt值的最大和最小值之间相差相对较小,大蒲莲和长白仔猪分别为5.03和3.82。3.大蒲莲和长白两品种间相比较,细胞因子在长白猪群体中的表达量平均值均高于大蒲莲群体,而受体TLR4的基因表达量在长白猪群体中的表达量低于大蒲莲群体;而且,大蒲莲群体内个体间细胞因子和受体表达量变异系数（平均31.43%）要大于长白群体（平均19.10%）,这与大蒲莲选育程度低,群体内个体间的差异程度要大相一致。4. 品种效应分析表明除IL10外,其他6个目的基因的ΔCt值品种间差异达显著（P＜0.05）或极显著（P＜0.01）水平。母猪效应分析表明母猪对各细胞因子和受体的影响均达到了极显著水平（P＜0.01）。【结论】成功筛选了外周血基因表达分析中稳定表达的内参基因（B2M）,对常规饲养管理下大蒲莲和长白仔猪外周血中的主要细胞因子和受体基因的表达进行群体水平分析。本研究结果为了解中外不同猪种的种质特性和抗病品种的选育提供了基础数据。