Mariolina Gullì

Molecular genetics of heat tolerance and heat shock proteins in cereals.

Heat stress is common in most cereal-growing areas of the world. In this paper, we summarize the current knowledge on the molecular and genetic basis of thermotolerance in vegetative and reproductive tissues of cereals. Significance of heat stress response and expression of heat shock proteins (HSPs) in thermotolerance of cereal yield and quality is discussed. Major avenues for increasing thermotolerance in cereals via conventional breeding or genetic modification are outlined.

Methods for detection of GMOs in food and feed

This paper reviews aspects relevant to detection and quantification of genetically modified (GM) material within the feed/food chain. The GM crop regulatory framework at the international level is evaluated with reference to traceability and labelling. Current analytical methods for the detection, identification, and quantification of transgenic DNA in food and feed are reviewed. These methods include quantitative real-time PCR, multiplex PCR, and multiplex real-time PCR. Particular attention is paid to methods able to identify multiple GM events in a single reaction and to the development of microdevices and microsensors, though they have not been fully validated for application.

Location of a gene regulating drought-induced abscisic acid production on the long arm of chromosome 5A of wheat.

The accumulation of abscisic acid (ABA) by detached and partially dehydrated wheat leaves is known to be inherited in a quantitative manner. The location of genes having a major effect on drought-induced ABA accumulation in wheat was determined using a set of single chromosome substitution lines and populations derived from a cross between a high-ABA- and a low-ABA-producing genotype. Examination of a series of chromosome substitution lines of the high-ABA genotype ‘Ciano 67’ into the low-ABA recipient ‘Chinese Spring’ showed that chromosome 5A carries gene(s) that have a major influence on ABA accumulation in a drought test with detached and partially dehydrated leaves (DLT). A similar DLT was used to examine ABA accumulation in a population of F2 plants and doubled haploid (DH) lines derived from the cross between ‘Chinese Spring’ (low-ABA) and ‘SQ1’ (high-ABA) in which the F2 population (139 plants) and DH lines (96 lines) were also mapped partially with molecular markers. Analysis of variance of ABA accumulation between and within marker allele classes in the F2 confirmed the location of a gene(s) regulating ABA accumulation on the long arm of chromosome 5A. MAPMAKERQTL showed the most likely position for the ABA quantitative trait locus (QTL) to be between the loci Xpsr575 and Xpsr426, about 8 cM from Xpsr426. A similar trend for high ABA accumulation was found in DH lines having the ‘SQ1’ allele at marker loci in the same region of chromosome 5AL, but the QTL effect was not significant. The function of the QTL is discussed.

Stable changes in CD4+ T lymphocyte miRNA expression after exposure to HIV-1

MicroRNAs (miRNAs) inhibit HIV-1 expression by either modulating host innate immunity or by directly interfering with viral mRNAs. We evaluated the expression of 377 miRNAs in CD4(+) T cells from HIV-1 élite long-term nonprogressors (éLTNPs), naive patients, and multiply exposed uninfected (MEU) patients, and we observed that the éLTNP patients clustered with naive patients, whereas all MEU subjects grouped together. The discriminatory power of miRNAs showed that 21 miRNAs significantly differentiated éLTNP from MEU patients and 23 miRNAs distinguished naive from MEU patients, whereas only 1 miRNA (miR-155) discriminated éLTNP from naive patients. We proposed that miRNA expression may discriminate between HIV-1-infected and -exposed but negative patients. Analysis of miRNAs expression after exposure of healthy CD4(+) T cells to gp120 in vitro confirmed our hypothesis that a miRNA profile could be the result not only of a productive infection but also of the exposure to HIV-1 products that leave a signature in immune cells. The comparison of normalized Dicer and Drosha expression in ex vivo and in vitro condition revealed that these enzymes did not affect the change of miRNA profiles, supporting the existence of a Dicer-independent biogenesis pathway.

Multiplex real-time PCR using SYBR® GreenER™ for the detection of DNA allergens in food

AbstractWe describe the development of a six-target real-time multiplex PCR assay with the SYBR® GreenER™ fluorescent dye, targeted to genes encoding for allergenic proteins commonly present in many processed food products (patent application pending). The assay was successfully trialled on reconstructed food matrices and on a range of commercial foodstuffs, and is proposed as a ready-to-use analytical tool for food manufacturers to detect the presence or confirm the absence of sequences encoding for important allergenic proteins of plant origin. FigureMultiplex real-time PCR for the detection of DNA allergens in food using ABI Prism® 7000 Sequence detection system

A branched-chain amino acid aminotransferase gene isolated from Hordeum vulgare is differentially regulated by drought stress

Differential display was used to isolate cDNA clones showing differential expression in response to ABA, drought and cold in barley seedling shoots. One drought-regulated cDNA clone (DD12) was further analyzed and found to encode a branched-chain amino acid aminotransferase (HvBCAT-1). A genomic clone was isolated by probing the Morex BAC library with the cDNA clone DD12 and the structure of Hvbcat-1 was elucidated. The coding region is interrupted by six introns and contains a predicted mitochondrial transit peptide. Hvbcat1 was mapped to chromosome 4H. A comparison was made to rice and Arabidopsis genes to identify conserved structural patterns. Complementation of a yeast (Saccharomyces cerevisiae) double knockout strain revealed that HvBCAT-1 can function as the mitochondrial (catabolic) BCATs in vivo. Transcript levels of Hvbcat-1, increased in response to drought stress. As the first enzyme in the branched-chain amino acid (BCAA) catabolic pathway, HvBCAT-1 might have a role in the degradation of BCAA. Degradation of BCAA could serve as a detoxification mechanism that maintains the pool of free branched-chain amino acids at low and non toxic levels, under drought stress conditions.

Four members of the HSP101 gene family are differently regulated in Triticum durum Desf.

Heat shock proteins play an essential role in preventing deleterious effects of high temperatures. In many plants, HSP101 has a central role in heat stress survival. We report the isolation and characterization of four cDNAs corresponding to different members of the durum wheat HSP101 gene family. Expression analysis revealed differences in their induction. Accordingly, durum wheat HSP101 genes are differently regulated, therefore having distinct roles in stress response and thermotolerance acquisition. These findings are important for further dissection of the molecular mechanisms underlying the stress response and for understanding the functions of the HSP101 family members. This information could be important for the exploitation of specific alleles in marker assisted selection for abiotic stress resistance.

Comparison of DNA extraction methods and development of duplex PCR and real-time PCR to detect tomato, carrot, and celery in food.

Traceability is of particular importance for those persons who suffer allergy or intolerance to some food component(s) and need a strict avoidance of the allergenic food. In this paper, methodologies are described to fingerprint the presence of allergenic species such as carrot, tomato, and celery by DNA detection. Three DNA extraction methods were applied on vegetables and foods containing or not containing the allergens, and the results were compared and discussed. Fast SYBR Green DNA melting curve temperature analyses and duplex PCR assays with internal control have been developed for detection of these allergenic vegetables and have been tested on commercial foods. Spiking food experiments were also performed, assessing that limits of detection (LOD) of 1 mg/kg for carrot and tomato DNA and 10 mg/kg for celery DNA have been reached.

Ecogeographic adaptation and genetic variation in wild barley, Application of molecular markers targeted to environmentally regulated genes

Long primer PCR (LP-PCR) markers specifically targeted to sequences involved in the response to abiotic stress were utilised to analyse genetic variation within and among wild barley populations from Israel.Populations were sampled in locations reflecting a wide range of habitats and the potential correlation between genetic variation and ecogeographic parameters was examined. A high level of polymorphism was observed. Most of the variability was due to differences within populations, however a substantial portion of genetic variation (36%) could be attributed to differences among populations, indicating local adaptation of genotypes to microclimatic conditions. Moreover, clustering and ordination techniques showed that genotypes grouped together according to their area of origin and were separated on thebasis of ecogeography, chiefly by a combination of temperature, altitude and rainfall variables. Finally, 28% of the polymorphic PCR fragments were significantly correlated with ecogeographic parameters. Our results demonstrate the effectiveness of PCR-based molecular markers targeted to environmentally regulated genes in detecting ‘useful’ variation andthus in monitoring the impact exerted by adaptation to the environment ongenetic differentiation.

Quantitative detection method for Roundup Ready soybean in food using duplex real-time PCR MGB chemistry

BACKGROUND Methodologies that enable the detection of genetically modified organisms (GMOs) (authorized and non-authorized) in food and feed strongly influence the potential for adequate updating and implementation of legislation together with labeling requirements. Quantitative polymerase chain reaction (qPCR) systems were designed to boost the sensitivity and specificity on the identification of GMOs in highly degraded DNA samples; however, such testing will become economically difficult to cope with due to increasing numbers of approved genetically modified (GM) lines. Multiplexing approaches are therefore in development to provide cost-efficient solution. RESULTS Construct-specific primers and probe were developed for quantitative analysis of Roundup Ready soybean (RRS) event glyphosate-tolerant soybean (GTS) 40-3-2. The lectin gene (Le1) was used as a reference gene, and its specificity was verified. RRS- and Le1-specific quantitative real-time PCR (qRTPCR) were optimized in a duplex platform that has been validated with respect to limit of detection (LOD) and limit of quantification (LOQ), as well as accuracy. The analysis of model processed food samples showed that the degradation of DNA has no adverse or little effects on the performance of quantification assay. CONCLUSION In this study, a duplex qRTPCR using TaqMan minor groove binder-non-fluorescent quencher (MGB-NFQ) chemistry was developed for specific detection and quantification of RRS event GTS 40-3-2 that can be used for practical monitoring in processed food products.