Reference genes selection for quantitative gene expression studies in <Emphasis Type="Italic">Pinus massoniana</Emphasis> L.

Key message

Evaluation and selection of reference genes in Pinus massoniana L. (PM) for gene expression studies of various tissues, floral organ development, and abiotic stress.

Abstract

An important prerequisite for obtaining accurate gene expression results using quantitative real-time PCR is the selection of a reference gene or a group of genes having a highly stable level of expression. Pinus massoniana L. (PM) is the predominant fast-growing timber forest tree species in southern China. In this study of PM, we evaluated various tissues, flowers in different developmental phases, leaves from a cultivar with insect resistance, and leaves from plants under several types of abiotic stresses. Comprehensive Analysis was performed using BestKeeper, Normfinder, geNorm, and RefFinder software to select the most stable reference gene or gene group from among 25 candidate genes in these samples. The results showed that different experimental conditions require the use of different reference genes: ACT1 could be used as a reference gene for all samples in this study; UBI4 was the best gene for various tissues and zinc stress; CYP was the most stable gene for leaves from insect-resistant materials and Pb stress; Fbox and UBI11 were the best reference genes for salt stress; Fbox + RRP8, ARF + TUBA, and EF1B + IDH were the best reference groups for drought stress, low temperature stress, and flowers in different developmental phases, respectively. This study presents a reliable selection of reference genes for Masson pine, and the conclusions are meaningful for improving the accuracy of expression analyses in future molecular biology studies.

     

Evaluation and selection of reliable reference genes for gene expression under abiotic stress in cotton (Gossypium hirsutum L.)

Reference genes are critical for normalization of the gene expression level of target genes. The widely used housekeeping genes may change their expression levels at different tissue under different treatment or stress conditions. Therefore, systematical evaluation on the housekeeping genes is required for gene expression analysis. Up to date, no work was performed to evaluate the housekeeping genes in cotton under stress treatment. In this study, we chose 10 housekeeping genes to systematically assess their expression levels at two different tissues (leaves and roots) under two different abiotic stresses (salt and drought) with three different concentrations. Our results show that there is no best reference gene for all tissues at all stress conditions. The reliable reference gene should be selected based on a specific condition. For example, under salt stress, UBQ7, GAPDH and EF1A8 are better reference genes in leaves; TUA10, UBQ7, CYP1, GAPDH and EF1A8 were better in roots. Under drought stress, UBQ7, EF1A8, TUA10, and GAPDH showed less variety of expression level in leaves and roots. Thus, it is better to identify reliable reference genes first before performing any gene expression analysis. However, using a combination of housekeeping genes as reference gene may provide a new strategy for normalization of gene expression. In this study, we found that combination of four housekeeping genes worked well as reference genes under all the stress conditions.     

Stability of Expression of Reference Genes Among Different Lentil (Lens culinaris) Genotypes Subjected to Cold Stress, White Mold Disease, and Aphanomyces Root Rot

Precise quantification of differences in gene expression between plants requires the use of “reference” genes, which are stably expressed across different lines and treatments and serve as endogenous controls for normalizing gene expression data. The objectives of this study were to determine the expression stability of several reference genes across five different lentil varieties subjected to either cold stress, inoculation with Sclerotinia sclerotiorum, the causal agent of white mold disease, or inoculation with Aphanomyces euteiches, the causal agent of Aphanomyces root rot. Expression stability was examined in the stems and leaves of plants subjected to cold stress or inoculation with S. sclerotiorum and in the roots of plants inoculated with A. euteiches. Real-time PCR assays (SYBR Green) were designed for six different genes: translation initiation factor (TIF), 18S rRNA, actin, β-tubulin-2, β-tubulin-3, and glyceraldehyde 3-phosphate dehydrogenase. TIF, actin, and 18S rRNA tended to be the most stably expressed genes, with expression stability (M) values less than 0.5 during cold stress and inoculation with A. euteiches. Two reference genes were required to normalize data from plants exposed to cold stress or inoculated with A. euteiches. The reference genes exhibited the lowest expression stability in plants inoculated with S. sclerotiorum, for which five reference genes were required to normalize data. The reference genes reported in this study appear to have a promise for examining gene expression in lentil foliar and root tissues in response to diverse abiotic and biotic factors.     

Screening of valid reference genes for real-time RT-PCR data normalization in Hevea brasiliensis and expression validation of a sucrose transporter gene HbSUT3

Real-time RT-PCR (RT-qPCR) is a sensitive and precise method of quantifying gene expression, however, suitable reference genes are required. Here, a systematic reference gene screening was performed by RT-qPCR on 22 candidate genes in Hevea brasiliensis. Two ubiquitin-protein ligases (UBC2a and UBC4) were the most stable when all samples were analyzed together. A mitosis protein (YLS8) and a eukaryotic translation initiation factor (eIF1Aa) were the most stable in response to tapping. UBC2b and UBC1 were the most stable among different genotypes. UBC2b and a DEAD box RNA helicase (RH2b) were the most stable across individual trees. YLS8 and RH8 were most stably expressed in hormone-treated samples. Expression of the candidate reference genes varied significantly across different tissues, and at least four genes (RH2b, RH8, UBC2a and eIF2) were needed for expression normalization. In addition, examination of relative expression of a sucrose transporter HbSUT3 in different RNA samples demonstrated the importance of additional reference genes to ensure accurate quantitative expression analysis. Overall, our work serves as a guide for selection of reference genes in RT-qPCR gene expression studies in H. brasiliensis.     

Selection of reference genes for gene expression studies in rats

Selection of the most stable reference gene is critical for a reliable interpretation of gene expression data using RT-PCR. In order so, 17 commonly used genes were analyzed in Wistar rat duodenum, jejunum, ileum and liver following a fat gavage and at two time periods. These reference genes were also tested in liver from Zucker (fa/fa) on a long-term dietary trial. Four strategies were used to select the most suitable reference gene for each tissue: ranking according to biological coefficient of variation and further validation by statistical comparison among groups, geNorm, NormFinder and BestKeeper programs. No agreement was observed among these approaches for a particular gene, nor a common gene for all tissues. Furthermore we demonstrated that normalising using an inadequate reference conveyed into false negative and positive results. The selection of genes provided by BestKeeper resulted in more reliable results than the other statistical packages. According to this program, Tbp, Ubc, Hprt and Rn18s were the best reference genes for duodenum, jejunum, ileum and liver, respectively following a fat gavage in Wistar rats and Rn18s for liver in another rat strain on a long-term dietary intervention. Therefore, BestKeeper is highly recommendable to select the most stable gene to be used as internal standard and the selection of a specific reference expression gene requires a validation for each tissue and experimental design.     

盐胁迫下大豆根组织定量PCR分析中内参基因的选择

实时荧光定量PCR已广泛用于基因表达的分析, 适当的内参基因选择是获得准确分析结果的关键。在大豆(Glycine max)分子生物学研究中, 逆境响应基因和microRNA (miRNA)表达的内参辅助检测基因均有哪些目前尚不清楚。该研究选用不同盐梯度和时间点组合处理的大豆根组织为材料, 对已报道的其它条件下表达相对稳定的内参基因(ACT、ACT2/7、CYP2、ELF1A、ELF1B、F-Box、TUA和UBC2)以及miRNA内参基因(U6、miR1515a、miR1520c、miR1520d、miR171a和miR171b)的表达情况进行了全面检测; 并采用Δ-Ct、Bestkeeper、NormFinder和Genorm四种方法对检测结果进行了综合分析, 发现ELF1B和CYP2适合作为大豆根系盐胁迫响应基因研究的内参基因, miR1515a和U6适合作为盐胁迫下大豆根组织miRNA研究的内参。上述研究结果为大豆盐胁迫响应基因和miRNA表达及其进一步的功能研究奠定了基础。     

Evaluation and Validation of Reference Genes for Normalization of Quantitative Real-Time PCR Based Gene Expression Studies in Peanut

The quantitative real-time PCR (qPCR) based techniques have become essential for gene expression studies and high-throughput molecular characterization of transgenic events. Normalizing to reference gene in relative quantification make results from qPCR more reliable when compared to absolute quantification, but requires robust reference genes. Since, ideal reference gene should be species specific, no single internal control gene is universal for use as a reference gene across various plant developmental stages and diverse growth conditions. Here, we present validation studies of multiple stably expressed reference genes in cultivated peanut with minimal variations in temporal and spatial expression when subjected to various biotic and abiotic stresses. Stability in the expression of eight candidate reference genes including ADH3, ACT11, ATPsyn, CYP2, ELF1B, G6PD, LEC and UBC1 was compared in diverse peanut plant samples. The samples were categorized into distinct experimental sets to check the suitability of candidate genes for accurate and reliable normalization of gene expression using qPCR. Stability in expression of the references genes in eight sets of samples was determined by geNorm and NormFinder methods. While three candidate reference genes including ADH3, G6PD and ELF1B were identified to be stably expressed across experiments, LEC was observed to be the least stable, and hence must be avoided for gene expression studies in peanut. Inclusion of the former two genes gave sufficiently reliable results; nonetheless, the addition of the third reference gene ELF1B may be potentially better in a diverse set of tissue samples of peanut.     

Identification and Validation of Reference Genes for Quantification of Target Gene Expression with Quantitative Real-time PCR for Tall Fescue under Four Abiotic Stresses

Tall fescue (Festuca arundinacea Schreb.) is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41) was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species.     

Multiple insulin degrading enzyme variants alter in vitro reporter gene expression

The insulin degrading enzyme (IDE) variant, v311 (rs6583817), is associated with increased post-mortem cerebellar IDE mRNA, decreased plasma β-amyloid (Aβ), decreased risk for Alzheimer''s disease (AD) and increased reporter gene expression, suggesting that it is a functional variant driving increased IDE expression. To identify other functional IDE variants, we have tested v685, rs11187061 (associated with decreased cerebellar IDE mRNA) and variants on H6, the haplotype tagged by v311 (v10; rs4646958, v315; rs7895832, v687; rs17107734 and v154; rs4646957), for altered in vitro reporter gene expression. The reporter gene expression levels associated with the second most common haplotype (H2) successfully replicated the post-mortem findings in hepatocytoma (0.89 fold-change, p = 0.04) but not neuroblastoma cells. Successful in vitro replication was achieved for H6 in neuroblastoma cells when the sequence was cloned 5′ to the promoter (1.18 fold-change, p = 0.006) and 3′ to the reporter gene (1.29 fold change, p = 0.003), an effect contributed to by four variants (v10, v315, v154 and v311). Since IDE mediates Aβ degradation, variants that regulate IDE expression could represent good therapeutic targets for AD.     

Validation of Reference Genes for Accurate Normalization of Gene Expression in Lilium davidii var. unicolor for Real Time Quantitative PCR

Lilium is an important commercial market flower bulb. qRT-PCR is an extremely important technique to track gene expression levels. The requirement of suitable reference genes for normalization has become increasingly significant and exigent. The expression of internal control genes in living organisms varies considerably under different experimental conditions. For economically important Lilium, only a limited number of reference genes applied in qRT-PCR have been reported to date. In this study, the expression stability of 12 candidate genes including α-TUB, β-TUB, ACT, eIF, GAPDH, UBQ, UBC, 18S, 60S, AP4, FP, and RH2, in a diverse set of 29 samples representing different developmental processes, three stress treatments (cold, heat, and salt) and different organs, has been evaluated. For different organs, the combination of ACT, GAPDH, and UBQ is appropriate whereas ACT together with AP4, or ACT along with GAPDH is suitable for normalization of leaves and scales at different developmental stages, respectively. In leaves, scales and roots under stress treatments, FP, ACT and AP4, respectively showed the most stable expression. This study provides a guide for the selection of a reference gene under different experimental conditions, and will benefit future research on more accurate gene expression studies in a wide variety of Lilium genotypes.     

Identification and Validation of Reference Genes and Their Impact on Normalized Gene Expression Studies across Cultivated and Wild Cicer Species

Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses.     

Growth,ionic response,and gene expression of shoots and roots of perennial ryegrass under salinity stress

Growth, ionic responses, and expression of candidate genes to salinity stress were examined in two perennial ryegrass accessions differing in salinity tolerance. The salinity tolerant (PI265349) and sensitive accessions (PI231595) were subjected to 75-mM NaCl for 14 days in a growth chamber. Across two accessions, salinity stress increased shoot dry weight and concentrations of malondialdehyde (MDA) and Na⁺ in the shoots and roots, but decreased shoot Ca²⁺ and root K⁺ concentrations. Salinity stress also increased root expressions of SOS1, PIP1, and TIP1. Plant height and chlorophyll content were unaffected by salinity stress in the tolerant accession but significantly decreased in the sensitive accession. Shoot MDA content did not change in the tolerant accession but increased in the sensitive accession. A more dramatic increase in Na⁺ was found in the roots of the sensitive accession. Relative to the control, salinity stress reduced expression of SOS1, NHX1, PIP1, and TIP1 in the shoots but increased expression of these genes in the roots of the tolerant accession. Expression levels of SOS1 increased in the roots and expression of NHX1 increased in the shoots but decreased in the roots of the sensitive accession under salinity stress. A decline in PIP1 expression in the shoots and dramatic increases in TIP expression in both shoots and roots were found in the sensitive accession under salinity stress. The results suggested maintenance of plant growth and leaf chlorophyll content, lesser Na⁺ accumulation in the roots, and lower lipid peroxidation in the shoots which could be associated with salinity tolerance. The decreased expressions of SOS1, NHX1, and TIP1 in the shoots, and increased expressions of NHX1 and PIP1 in the roots might also be related to salinity tolerance in perennial ryegrass.