Validation of reference genes for RT-qPCR analysis of CYP4T expression in crucian carp

Reference genes are commonly used for normalization of target gene expression during RT-qPCR analysis. However, no housekeeping genes or reference genes have been identified to be stable across different tissue types or under different experimental conditions. To identify the most suitable reference genes for RT-qPCR analysis of target gene expression in the hepatopancreas of crucian carp (Carassius auratus) under various conditions (sex, age, water temperature, and drug treatments), seven reference genes, including beta actin (ACTB), beta-2 microglobulin (B2M), embryonic elongation factor-1 alpha (EEF1A), glyceraldehyde phosphate dehydrogenase (GAPDH), alpha tubulin (TUBA), ribosomal protein l8 (RPL8) and glucose-6-phosphate dehydrogenase (G6PDH), were evaluated in this study. The stability and ranking of gene expression were analyzed using three different statistical programs: GeNorm, Normfinder and Bestkeeper. The expression errors associated with selection of the genes were assessed by the relative quantity of CYP4T. The results indicated that all the seven genes exhibited variability under the experimental conditions of this research, and the combination of ACTB/TUBA/EEF1A or of ACTB/EEF1A was the best candidate that raised the accuracy of quantitative analysis of gene expression. The findings highlighted the importance of validation of housekeeping genes for research on gene expression under different conditions of experiment and species.     

Selection of candidate reference genes for real-time PCR studies in lettuce under abiotic stresses

The process of selection and validation of reference genes is the first step in studies of gene expression by real-time quantitative polymerase chain reaction (RT-qPCR). The genome of lettuce, the most popular leaf vegetable cultivated worldwide, has recently been sequenced; therefore, suitable reference genes for reliable results in RT-qPCR analyses are required. In the present study, 17 candidate reference genes were selected, and their expression stability in lettuce leaves under drought, salt, heavy metal, and UV-C irradiation conditions and under the application of abscisic acid (ABA) was evaluated using geNorm and NormFinder software. The candidate reference genes included protein-coding traditional and novel reference genes and microRNAs (miRNAs). The results indicate that the expression stability is dependent on the experimental conditions. The novel protein-coding reference genes were more suitable than the traditional reference genes under drought, UV-C irradiation, and heavy metal conditions and under the application of ABA. Only under salinity conditions were the traditional protein-coding reference genes more stable than the novel genes. In addition, the miRNAs, mainly MIR169, MIR171/170 and MIR172, were stably expressed under the abiotic stresses evaluated, representing a suitable alternative approach for gene expression data normalization. The expression of phenylalanine ammonia lyase (PAL) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) was used to further confirm the validated protein-coding reference genes, and the expression of MIR172 and MIR398 was used to confirm the validated miRNA genes, showing that the use of an inappropriate reference gene induces erroneous results. This work is the first survey of the stability of reference genes in lettuce and provides guidelines to obtain more accurate RT-qPCR results in lettuce studies.     

A multiplex RT-PCR approach to detect aflatoxigenic strains of Aspergillus flavus

AIMS: To develop a multiplex reverse transciption-polymerase chain reaction (RT-PCR) protocol to discriminate aflatoxin-producing from aflatoxin-nonproducing strains of Aspergillus flavus. METHODS AND RESULTS: The protocol was first optimized on a set of strains obtained from laboratory collections and then validated on A. flavus strains isolated from corn grains collected in the fields of the Po Valley (Italy). Five genes of the aflatoxin gene cluster of A. flavus, two regulatory (aflR and aflS) and three structural (aflD, aflO and aflQ), were targeted with specific primers to highlight their expression in mycelia cultivated under inducing conditions for aflatoxins production. 48-h-old cultures expressed the complete set of the genes analysed here whereas 24-h-old ones did not. Genomic PCR (quadruplex PCR) was also performed in parallel using chromosomal DNA extracted from the same set of strains to correlate the integrity of the genes with their expression. CONCLUSIONS: We show that a good correlation exists between gene expression of the aflatoxin genes, here analysed by multipex RT-PCR, and aflatoxin production, except for one strain that apparently transcribed all the relevant genes but did not produce aflatoxin in the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first example of the application of a combination of multiplex PCR and RT-PCR approaches to screen a population of A. flavus for the presence of aflatoxigenic and nonaflatoxigenic strains. The proposed protocol will be helpful in evaluating the risk posed by A. flavus in natural environments and might also be a useful tool to monitor its presence during the processing steps of food and feed commodities.     

Aflatoxin biosynthesis cluster gene cypA is required for G aflatoxin formation

Aspergillus flavus isolates produce only aflatoxins B1 and B2, while Aspergillus parasiticus and Aspergillus nomius produce aflatoxins B1, B2, G1, and G2. Sequence comparison of the aflatoxin biosynthesis pathway gene cluster upstream from the polyketide synthase gene, pksA, revealed that A. flavus isolates are missing portions of genes (cypA and norB) predicted to encode, respectively, a cytochrome P450 monooxygenase and an aryl alcohol dehydrogenase. Insertional disruption of cypA in A. parasiticus yielded transformants that lack the ability to produce G aflatoxins but not B aflatoxins. The enzyme encoded by cypA has highest amino acid identity to Gibberella zeae Tri4 (38%), a P450 monooxygenase previously shown to be involved in trichodiene epoxidation. The substrate for CypA may be an intermediate formed by oxidative cleavage of the A ring of O-methylsterigmatocystin by OrdA, the P450 monooxygenase required for formation of aflatoxins B1 and B2.     

斑地锦RT-qPCR内参基因的筛选

实时荧光定量PCR(RT-qPCR)的前提条件之一是具有合适的内参基因。为筛选斑地锦(Euphorbia maculata)合适的RT-qPCR内参基因,该文利用同源克隆法克隆斑地锦GAPDH、EF-1α、act、UBQ、TUB-α、eIF-4A、CYP等基因片段,RT-qPCR检测7个候选内参基因在斑地锦不同生长期根、茎、叶和果实中的表达情况,并用geNorm、NormFinder和BestKeeper等生物学软件对各候选基因表达稳定性进行评价。结果表明:(1)克隆的GAPDH、EF-1α、act、UBQ、TUB-α、eIF-4A、CYP基因片段为729、808、753、422、233、656、313 bp,分别编码242、269、250、140、77、218、103个氨基酸,与其他植物相应氨基酸序列的最高同源性均在85%以上。(2)综合3个分析软件分析内参基因表达稳定性得出,表达稳定性排名为UBQ>EF-1α>TUB-α>eIF-4A>GAPDH>CYP>act。因此,可以选取UBQ作为斑地锦RT-qPCR分析的内参基因,用于不同生长期基因组织特异性表达研究。     

Divergent regulation of aflatoxin production at acidic pH by two <Emphasis Type="Italic">Aspergillus</Emphasis> strains

Production of aflatoxins (AF) by Aspergillus flavus and A. parasiticus is known to occur only at acidic pH. Although typical A. flavus isolates produced more AF as the external pH became increasingly acidic, an atypical strain from West Africa produced less. The lower AF production was not well correlated with decreases in expression of the aflatoxin pathway regulatory gene, aflR, or of two other biosynthesis genes.     

Evaluation of reference genes for real-time quantitative PCR studies in

ABSTRACT: BACKGROUND: The selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The present study aimed to identify reference genes as internal controls for gene expression studies by RT-qPCR in azole-stimulated Candida glabrata. RESULTS: The expression stability of 16 reference genes under fluconazole stress was evaluated using fold change and standard deviation computations with the hkgFinder tool. Our data revealed that the mRNA expression levels of three ribosomal RNAs (RDN5.8, RDN18, and RDN25) remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNAs showed only approximately 2.9-, 3.0-, and 2.5-fold induction by azole, respectively. By contrast, mRNA levels of the other 10 reference genes (ACT1, EF1a, GAPDH, PPIA, RPL2A, RPL10, RPL13A, SDHA, TUB1, and UBC4) were dramatically increased in C. glabrata following antifungal treatment, exhibiting changes ranging from 4.5- to 32.7-fold. We also assessed the expression stability of these reference genes using the 2-[increment][increment]CT method and three other software packages. The stability rankings of the reference genes by geNorm and the 2-[increment][increment]CT method were identical to those by hkgFinder, whereas the stability rankings by BestKeeper and NormFinder were notably different. We then validated the suitability of six candidate reference genes (ACT1, PGK1, RDN5.8, RDN18, UBC7, and UBC13) as internal controls for ten target genes in this system using the comparative CT method. Our validation experiments passed for all six reference genes analyzed except RDN18, where the amplification efficiency of RDN18 was different from that of the ten target genes. Finally, we demonstrated that the relative quantification of target gene expression varied according to the endogenous control used, highlighting the importance of the choice of internal controls in such experiments. CONCLUSIONS: We recommend the use of RDN5.8, UBC13, and PGK1 alone or the combination of RDN5.8 plus UBC13 or PGK1 as reference genes for RT-qPCR analysis of gene expression in C. glabrata following azole treatment. In contrast, we show that ACT1 and other commonly used reference genes (GAPDH, PPIA, RPL13A, TUB1, etc.) were not validated as good internal controls in the current model.     

The phylogenetics of mycotoxin and sclerotium production in Aspergillus flavus and Aspergillus oryzae

Aspergillus flavus is a common filamentous fungus that produces aflatoxins and presents a major threat to agriculture and human health. Previous phylogenetic studies of A. flavus have shown that it consists of two subgroups, called groups I and II, and morphological studies indicated that it consists of two morphological groups based on sclerotium size, called "S" and "L." The industrially important non-aflatoxin-producing fungus A. oryzae is nested within group I. Three different gene regions, including part of a gene involved in aflatoxin biosynthesis (omt12), were sequenced in 33 S and L strains of A. flavus collected from various regions around the world, along with three isolates of A. oryzae and two isolates of A. parasiticus that were used as outgroups. The production of B and G aflatoxins and cyclopiazonic acid was analyzed in the A. flavus isolates, and each isolate was identified as "S" or "L" based on sclerotium size. Phylogenetic analysis of all three genes confirmed the inference that group I and group II represent a deep divergence within A. flavus. Most group I strains produced B aflatoxins to some degree, and none produced G aflatoxins. Four of six group II strains produced both B and G aflatoxins. All group II isolates were of the "S" sclerotium phenotype, whereas group I strains consisted of both "S" and "L" isolates. Based on the omt12 gene region, phylogenetic structure in sclerotium phenotype and aflatoxin production was evident within group I. Some non-aflatoxin-producing isolates of group I had an omt12 allele that was identical to that found in isolates of A. oryzae.