The plant genes involved in cellular signaling and
metabolism have not been fully identified, while the function(s) of many of
those which have are as yet incompletely characterized. Gene expression
analysis allows the identification of genes and the study of their relationship
with cellular processes. There are several options available for studying gene
expression, including the use of cDNA and microarray libraries and techniques
such as suppression subtractive hybridization (SSH), differential display (DD),
RNA fingerprinting by arbitrary primed PCR (RAP), expressed sequence tags
(EST), serial analysis of gene expression (SAGE), representational difference
analysis (RDA), cDNA-amplified fragment length polymorphism (cDNA-AFLP) and RNA
sequencing (RNA-Seq). Focusing on defense-related processes in plants, we
present a brief review and examples of each of these methodologies and their
advantages and limitations regarding the study of plant gene expression.

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Gene Expression: A Review on Methods for the Study of Defense-Related Gene Differential Expression in Plants

Florida hybrid bunch grape (Vitis spp.) cultivars with commercial value are susceptible to anthracnose disease caused by the fungi, Elsinoe ampelina. The Florida grape industry is keen on improving anthracnose tolerance characteristics of these grape cultivars as the cultivation of European grapes is limited in Southeastern United States due to Pierce’s disease incidence. In this connection, a molecular approach was taken to identify and characterize the gene/s differentially expressed upon infestation with E. ampelina in selected Florida hybrid bunch grape cultivars differing in their disease tolerance level. Variation in gene expressions was monitored by employing Differential Display RT-PCR. The results showed significant up-regulation of transcripts ranging between 200 and 800 bp in anthracnose-tolerant (cvs. Blue Lake and Lake Emerald) grape cultivars compared to anthracnose-susceptible (cvs. Suwannee and Blanc du Bois) cultivars. The transcripts that were uniquely induced in tolerant cultivars were identified as chitinase, stilbene synthase, protein/sugar kinases and transcriptional factor. Expression of chitinase and stilbene synthase genes was validated using real time PCR analysis. The results showed that these genes were more rapidly expressed in tolerant cultivars within a day after inoculating with E. ampelina and peaking by day 3, compared to susceptible cultivars. Induction of these anti-fungal genes appears to enable tolerant cultivars to withstand fungal infestation compared to susceptible cultivars. Rapid expression of chitinase and stilbene synthase genes along with others genes may be partly responsible for anthracnose-tolerance in Florida hybrid bunch grapes. Further analysis may shed light on their specific role in anthracnose tolerance.

Differential expression of chitinase and stilbene synthase genes in Florida hybrid bunch grapes to Elsinoë ampelina infection

The current ongoing studies and elucidations of control mechanisms of stress tolerance at molecular level that might facilitate the usage of molecular tools for developing further tolerant floras, is mainly centered on the expression of specific genes related to stress. Differential Display Reverse Transcriptase polymerase chain reaction (DDRT-PCR), a competent, profound and reproducible technology, is more advantageous in numerous ways than other approaches of gene expression analysis. Until 1992, the only and single technique applied for isolation of differentially expressed genes was subtractive hybridization or differential hybridization. In 1992, a different and innovative PCR-based method called Differential Display (DDRT-PCR) was developed by Liang and Pardee. The technological simplicity and wider applicability made this technique very novel. It has successfully utilized in a number of organisms starting from yeast to mammals. This technique was introduced and developed to accelerate the identification of differentially expressed genes to overcome the shortcomings of earlier known methods which were sensitive to error, unresponsive and strenuous. The present review emphasized the multidimensional applications of DDRTPCR in studying the differentially expressed gene under biotic and abiotic stress especially the salinity stress.

/pdf/analysis-of-differential-gene-expression-under-2iogbui5a9.pdf

Analysis of Differential Gene Expression under Salinitythrough Differential Display Reverse Transcription PolymeraseChain Reaction (DDRTPCR) Technique: A Review

The DD-RTPCR technique is becoming more efficient nowadays to isolate and characterize genes differentially expressed among cells, tissues or individuals, being used here, for the first time in the study of mandibular glands of minina and media workers and soldiers of the ant species Atta sexdens rubropilosa. The expression profile of bands (transcripts), obtained in the present study, permitted the grouping of gene fragments into three distinctive classes. Class I fragments corresponded with those called constitutive genes, which could be associated with the metabolic processes of the mandibular glands. Class II could be the ones that would be codifying more important products in more specific stages than in others, related to the tasks performed in the colony by individuals of different castes. Class III gene fragments (transcripts 1, 2 and 3) must be caste-specific and they were found in the mandibular glands of soldiers (T1), media workers (T2) and minima workers (T3), respectively. Therefore the present study, besides having highlighted the differential gene expression in the mandibular glands of individuals of three castes of A. s. rubropilosa, demonstrated that the DD-RTPCR technique can be one more tool to be used in gene expression analysis, including establishing intra and inter-caste relations in insects.

/pdf/analysis-of-the-differential-gene-expression-by-the-dd-rtpcr-6wrr40h6r5.pdf

Analysis of the Differential Gene Expression, by the DD-RTPCR Technique, Of Workers Atta sexdens rubropilosa Mandibular Glands (Hymenoptera: Formicidae)

......................................................................................................... 5 1. INTRODUÇÃO................................................................................................ 8 2. OBJETIVOS..................................................................................................... 15 3. MATERIAL E MÉTODOS............................................................................. 18 3.1 MATERIAL................................................................................................... 18 3.2 MÉTODOS.................................................................................................... 18 3.2.1 MORFOLOGIA...................................................................................... 18 3.2.2 HISTOLOGIA......................................................................................... 19 3.2.3 MICROSCOPIA ELETRÔNICA DE VARREDURA........................... 19 3.2.4 MICROSCOPIA ELETRÔNICA DE TRANSMISSÃO........................ 19 3.2.5 HISTOQUÍMICA.................................................................................... 20 3.2.5.1 Detecção de DNA/RNA.................................................................... 20 3.2.5.2 Detecção de Polissacarídeos Ácidos e Neutros................................. 21 3.2.5.3 Detecção de Proteínas Totais............................................................. 21 3.2.5.4 Detecção de Lipídios Ácidos............................................................. 22 3.2.6 DOSAGEM DE PROTEÍNA TOTAL.................................................... 22 3.2.6.1 Preparação do Extrato........................................................................ 22 3.2.6.2 Dosagem Protéica........................................................................... 22 3.2.7 ELETROFORESE DE PROTEÍNAS...................................................... 23 3.2.7.1 Aplicação das amostras no gel de poliacrilamida e eletroforese em condições desnaturantes (SDS-PAGE)................................................................... 24 3.2.8 CITOQUÍMICA ULTRA-ESTRUTURAL............................................. 25 3.2.8.1 Detecção de Proteínas Básicas.......................................................... 25 3.2.8.2 Detecção de Polissacarídeos.............................................................. 25 3.2.8.3 Detecção de Lipídios......................................................................... 26 3.2.9 IDENTIFICAÇÃO DOS GENES EXPRESSOS (DDRT-PCR)............. 27 3.2.9.1 Dosagem de RNA total..................................................... 27 3.2.9.1.1 Coleta do material para extração de RNA total.......................... 27

Glândulas mandibulares de operárias de formigas Atta sexdens rubropilosa (Forel, 1908): morfologia e expressão gênica (Hymenoptera: Formicidae)

Differential Display (DDRT-PCR) is a powerful technique for analyzing differences in gene expression. DDRT-PCR has the following essential steps: total RNA isolation and purification, cDNA synthesis from mRNAs, PCR amplification of cDNAs, visualization of PCR products, reamplification and cloning of the differentially expressed PCR products, confirmation by northern blot, sequencing the confirmed clones, and finally cDNA library screening to isolate the genes of interest. After its invention in 1992, a number of modifications have been introduced to optimize the technique and specifically to reduce the major problem of "false positives". Since understanding of specific gene expression patterns that regulate developmental and stress responses is a major concern of molecular biology, DDRT-PCR has become a very popular molecular technique during the past decade. Additional index terms: flooding, gene expression, mRNA differential display.

José Donizeti Alves

Papers

Differential display: a novel pcr-based method for gene isolation and cloning 1