Differential Display Reverse Transcriptase Polymerase Chain Reaction Research Articles

Differential Display Reverse Transcription Polymerase Chain Reaction (DDRT-PCR) for Grey Oyster Mushroom Samples Grown with Acoustic Sound Treatment

Grey oyster mushroom is the most often farmed and marketed type of mushroom for household consumption in Malaysia. The mushroom cultivation takes a long time due to its complicated mycelium growth. Several treatments were introduced such as acoustic sound treatment to increase the growth rate and quality of the crop, but there is no in-depth study regarding the genetic expression of the genes affected. This study aims to evaluate the Differential Display Reverse Transcriptase-Polymerase Chain Reaction (DDRT-PCR) for grey oyster mushroom samples grown with acoustic sound treatment. The mushroom was subjected to the treatment and the RNA was extracted from the mushroom samples and converted into cDNA before undergoing DDRT-PCR. Ten Differential Expressed Transcripts (DETs) that were successfully identified based on the differences between the intensity and absence of amplicons were sent for gene sequencing and BLAST through the NCBI database to obtain relevant results regarding the possible gene annotation. Seven out of ten DETs hit potential genes encoding for housekeeping and structural and development functions. The results showed that acoustic sound treatment did affect the expression of certain genes differently as captured by DDRT-PCR analyses and offers new ideas for the development of ecological agriculture.

New gene functions are involved in the thermotolerance of the wild wheat relative Aegilops umbellulata

Wheat is one of the most important food crops in the world for human consumption, like all plants it is exposed to environmental stresses including high temperatures. The deleterious effect of high temperatures negatively affects plant growth and development, leading to reduced viability and yield. These effects can be reduced by improvement of thermotolerance through innovative breeding strategies, based on the expansion of the genetic pool available, by exploring important genetic functions from wheat wild progenitors. Improving the genetic thermotolerance characteristics of wheat requires greater understanding of genetic bases of thermotolerance, through identification of high temperature stress related genes. A good source of new useful alleles is given by Aegilops species characterized by thermotolerant habits.In this study we have classified as thermotolerant or thermosensitive, on the basis of physiologic tests, some accessions of wheat wild relative species belonging to Aegilops and Triticum genera. A thermotolerant accession of Aegilops umbellulata (AUM5) was selected, subjected to different thermal treatments and analyzed at transcriptional level. By differential display reverse transcriptase polymerase chain reaction (DDRT-PCR), we investigated modulation of gene expression elicited by heat treatments. This approach allowed the identification of various transcript-derived fragments (TDFs) produced by AUM5 in response to different thermal treatments. The functions of the inducible unique genes in the molecular determination of thermotolerance process are discussed.

Identification and Characterization of the Diverse Stress-Responsive R2R3-RMYB Transcription Factor from Hibiscus sabdariffa L.

Various regulatory proteins play a fundamental role to manage the healthy plant growth under stress conditions. Differential display reverse transcriptase PCR and random amplification of cDNA ends (RACE) was used to explore the osmotic stress-responsive transcripts. We identified and characterized the salt stress-responsive R2R3 type RMYB transcription factor from Hibiscus sabdariffa which has an open reading frame of 690 bp, encoding 229 long chain amino acids. In silico analysis confirmed the conserved R2 and R3 domain as well as an NLS-1 localization site. The deduced amino acids of RMYB shared 83, 81, 80, 79, 72, 71, and 66% homology with Arabidopsis thaliana, Glycine max, Oryza sativa, Zea maize, Malus domestica, Populus tremula × Populus alba, and Medicago sativa specific MYB family, respectively. We observed the gene upregulation in stem, leaf, and root tissue in response to abiotic stress. Furthermore, RMYB gene was cloned into plant expression vector under CaMV35S promoter and transformed to Gossypium hirsutum: a local cotton cultivar. Overexpression of RMYB was observed in transgenic plants under abiotic stresses which further suggests its regulatory role in response to stressful conditions. The RMYB transcription factor-overexpressing in transgenic cotton plants may be used as potential agent for the development of stress tolerant crop cultivars.

English

No previous study has been reported on the salt-modulated gene(s) of roselle ( Hibiscus sabdariffa L.). Identifying the potentially novel transcripts responsible for salt stress tolerance in roselle will increase knowledge of the molecular mechanism underlying salt stress responses. In this study, differential display reverse transcriptase PCR (DDRT-PCR) was used to compare the overall differences in gene expression between salt-stressed and control plants. A total of 81 primer combinations were used and false positive clones were rejected during a screening and quality control assay. The remaining nine cDNA transcript fragments were extracted from the gel, reamplified, cloned and sequenced. A homology search revealed that four transcripts showed significant homology with known genes. Out of five transcripts, real-time PCR demonstrated that four exhibited high expression in salt-stressed root tissues relative to the control and one transcript was down-regulated. These transcripts may be useful for improving tolerance in salt stress-sensitive plants. Keywords: Roselle, Hibiscus Sabdariffa L., differential display, salt-stress, differentially expressed transcripts, signal transduction. African Journal of Biotechnology , Vol 13(53) 4775-4781

佛手低温胁迫相关基因的差异表达

Low temperature is one of the most common environmental stresses causing severe economic losses of most crops.Plants adapation to low temperature is mediated by differential gene expression,Investigation of the transcriptome profiling stressed by low temperature is important to unveil the mechanism of plant adapation to the stress.Multiple mechanisms are involved this process including differential expression of dehydrins and antifreeze proteins,chaperones and detoxification enzymes,as well as variations of biosynthetic enzymes producing the low molecular weight compounds.Regulatory proteins such as transcription factors,protein kinases and phospholipases are known to be differentially regulated under cold stress.However,few literatures were available on gene express correlated with cold-sensitivity in fingered citron. Fingered citron(Citrus medica L.var.sarcodactylis Swingle)has high values due to its medicinal properties and ornamental potential.However,high sensitivity of fingered citron to low temperature resulted in significant reduction of its production.Fingered citron′s maximum freezing tolerance,considered as the semilethal temperatures(LT50),is-4℃,and 24 h was considered as a critical period for cold stress.It is thus important to understand possible mechanisms involved in cold sensitivity of this plants species,as to promote its tolerance of cold stress. In the present study,the mRNA differential display reverse transcriptase polymerase chain reaction(DDRT-PCR) was used to analyze gene expression patterns involved in the cold sensitivity of Fingered Citrons.The plants were subjected to low temperature(-4℃) for 24 h,and alterations of gene expression patterns were analyzed.One hundred and twenty-one putative differentially expressed DNA fragments were cloned,sequenced,and analyzed.Thirty-eight genes with changed transcript levels were left after the exclusion of repeated sequences,33 of which are genes of known functions and 5 unknown through BLAST analyses.Expression analysis by semiquantitative RT-PCR(sqRT-PCR) showed that twenty-nine genes were up-regulated and five genes were down-regulated under cold stress.No significant variation of remaining four genes were observed,and considered to be false positives. Among the 34 differentially expressed fragments,eight genes were related to plant defense/stress responses,including hepicidin-like precursor,DNA-damage-repair/toleration protein(DRT),26S proteasome regulatory subunit,Retrotransposon-like protein,Avirulence protein(Avr),Ac-like transposase protein,Rhodanese domain protein and β-1,3-glucanase;Eight of nine metabolism-related genes are photosynthesis-related genes includes Abl interactor-like protein-2、3,cytochrome f,P700,plastocyanin-like protein,γ-type carbonic anhydrase protein,ATP synthase CF0 subunit I,Chlorophyllase-1 and phototropins;and 17 other genes are associated with cell wall modification,signal transduction,metabolism,amino acid transport,oxidative damage,transcription and protein synthesis.These data provide a new insight into molecular mechanisms underlying cold regulation of plant defense/stress responses and photosynthesis in Fingered Citrons,which is important to improve the cold tolerance of the citrus species.

Differential expression analysis of defense-related genes responsive to Tilletia indica infection in wheat

Karnal bunt caused by Tilletia indica is a quarantine disease responsible for qualitative and quantitative losses in wheat. The present study aimed to determine the expression of defense-related genes involved in early stages of infection in response to T. indica by using the differential display reverse transcriptase-PCR method (DDRT-PCR) in susceptible (WL 711) and resistant (HD 29) cultivars of wheat. The DDRT-PCR banding profiles generated by the random primers in combination with oligo-dT primers of resistant and susceptible wheat plants were evaluated at 0 h, 12 h, 24 h, and 48 h after inoculation. Out of 9 sets of primers, 7 sets of primers produced remarkable induced transcripts. The highest up-regulated transcripts were observed in the resistant cultivar (HD 29), whereas the down-regulated transcripts were found in the susceptible (WL 711) cultivar. Several sets of primers were designed from the defense-related gene sequences available in the GenBank database, and of these, 4 primer sets amplified the desired specific bands. Based on the putative functions, 3 defense-related genes, namely puroindoline protein PINB (AT_-_ KB2), \Beta-1,4-glucanase (AT_-_ KB3), and chitinase (AT_-_ KB7), and 1 housekeeping gene, actin (AT_-_ KB1), were characterized.

Differential Display of Abundantly Expressed Genes of Trichoderma harzianum During Colonization of Tomato-Germinating Seeds and Roots

The identification of Trichoderma genes whose expression is altered during early stages of interaction with developing roots of germinated seeds is an important step toward understanding the rhizosphere competency of Trichoderma spp. The potential of 13 Trichoderma strains to colonize tomato root and promote plant growth has been evaluated. All used strains successfully propagated in spermosphere and continued their growth in rhizoplane simultaneously root enlargement while the strains T6 and T7 were the most abundant in the apical segment of roots. Root colonization in most strains associated with promoting the roots and shoots growth while they significantly increased up to 43 and 40% roots and shoots dry weights, respectively. Differential display reverse transcriptase-PCR (DDRT-PCR) has been developed to detect differentially expressed genes in the previously selected strain, Trichoderma harzianum T7, during colonization stages of tomato-germinating seeds and roots. Amplified DDRT-PCR products were analyzed on gel agarose and 62 differential bands excised, purified, cloned, and sequenced. Obtained ESTs were submit-queried to NCBI database by BLASTx search and gene ontology hierarchy. Most of transcripts (29 EST) corresponds to known and hypothetical proteins such as secretion-related small GTPase, 40S ribosomal protein S3a, 3-hydroxybutyryl-CoA dehydrogenase, DNA repair protein rad50, lipid phosphate phosphatase-related protein type 3, nuclear essential protein, phospholipase A2, fatty acid desaturase, nuclear pore complex subunit Nup133, ubiquitin-activating enzyme, and 60S ribosomal protein L40. Also, 13 of these sequences showed no homology (E>0.05) with public databases and considered as novel genes. Some of these ESTs corresponded to genes encodes enzymes potentially involved in nutritional support of microorganisms which have obvious importance in the establishment of Trichoderma in spermosphere and rhizosphere, via potentially functioning in acquisition of nutrients from energy-rich carbon compounds leaked from the germinating seeds and roots.

Differential display analysis of cDNA fragments potentially involved in Nostoc flagelliforme response to osmotic stress

Nostoc flagelliforme is a terrestrial, highly drought-tolerant cyanobacterium. However, the molecular mechanism of its drought adaptation still remains little known. In this paper, we attempted to isolate those osmotic stress-regulated cDNA fragments by traditional differential display reverse transcriptase–polymerase chain reaction (DDRT-PCR). The PCR primers were chosen and evaluated from the reported HIP1D sequence-based primers and Ea/Es primers. The physiologically recovered samples were treated with 0.7 M sorbitol for 3 h. A total of 21 cDNA fragments were obtained by DDRT-PCR, and among them, 16 are up-regulated by osmotic stress. BlastX analyses showed that these up-regulated cDNA fragments are most related to ATPases, methyltransferases, transporters, glutathione synthase, signal transduction-associated components, and other unknown proteins. They are potentially important genes for N. flagelliforme to cope with drought stress since they are distinctly up-regulated by osmotic stress. In addition, five cDNA fragments are down-regulated by osmotic stress, such as the cDNA fragment of PSII protein. They may represent those genes involved in normal physiological processes and thus were inhibited by the osmotic stress. Taken together, the interpretation of the functional clues of these osmotic stress-responsive cDNA fragments has provided us an insight into the molecular mechanism of N. flagelliforme coping with the early phase of drought stress.

Identification of salt stress-induced transcripts in barley leaves by differential display reverse transcriptase polymerase chain reaction (DDRT-PCR)

Identification of salt stress-induced transcripts in barley leaves by differential display reverse transcriptase polymerase chain reaction (DDRT-PCR)

Somatotropin-mediated gene expression profiling of differentially displayed ESTs during lactation in Indian buffalo (Bubalus bubalis)

The study of bovine mammary gland functional genomics requires appropriate cDNA library collections to access gene expression patterns from different developmental and physiological stages. The present study was undertaken with the objective to identify candidate genes involved in the process of increased milk synthesis following 0, 48 and 96 h of recombinant bovine somatotropin (rbST) treatment to Surti buffalo (Bubalus bubalis) through differential display reverse transcriptase PCR (DDRT-PCR). Of a total 50 sequenced DD bands, 64% of ESTs were differentially expressed (appeared only in post-treatment samples, i.e. 48 h and 96 h) and 36% were up-regulated after rbST treatment. Of the ESTs 32%were found to be located on Bos taurus chromosome 24 (equivalent to buffalo chromosome 22), whereas 16% of ESTs could not be mapped, indicating that they are specific to buffalo. Quantitative real time PCR assay of 15 ESTs revealed transcript level surge in 13 ESTs, and decline in one EST, while one showed up-regulation in expression level at 48 h while down-regulation at 96 h. This study indicates more than 30 novel transcripts, with unknown function, involved in increased milk synthesis and also the involvement of many more genes in the physiology of milk production than once thought.

Differential Display RT-PCR Reveals Genes Associated with Lithium-Induced Neuritogenesis in SK-N-MC Cells

Lithium is shown to be neurotrophic and protective against variety of environmental stresses both in vitro as well as in vivo. In view of the wider clinical applications, it is necessary to examine alterations in levels of expression of genes affected by lithium. Lithium induces neuritogenesis in human neuroblastoma cell line SK-N-MC. Our aim was to elucidate genes involved in lithium-induced neuritogenesis using SK-N-MC cells. The differential display reverse transcriptase polymerase chain reaction (DD-RT-PCR) technique was used to study gene expression profiles in SK-N-MC cells undergoing lithium-induced neuritogenesis. Differential expression of genes in control and lithium (2.5 mM, 24 h)-treated cells was compared by display of cDNAs generated by reverse transcription of mRNA followed by PCR using arbitrary primers. Expression of four genes was altered in lithium-treated cells. Real-time PCR was done to confirm the levels of expression of each of these genes using specific primers. Lithium significantly up-regulated NCAM, a molecule known to stimulate neuritogenesis, occludin, a molecule participating in tight junctions and PKD2, a molecule known to modulate calcium transport. ANP 32c, a gene whose function is not fully known yet, was found to be down-regulated by lithium. This is the first report demonstrating altered levels of expression of these genes in lithium-induced neuritogenesis and contributes four hitherto unreported candidates possibly involved in the process.

Identification of sexually dimorphic gene expression in brain tissue of the fish Leporinus macrocephalus through mRNA differential display and real time PCR analyses

Differentially expressed genes in males and females of vertebrate species generally have been investigated in gonads and, to a lesser extent, in other tissues. Therefore, we attempted to identify sexually dimorphic gene expression in the brains of adult males and females of Leporinus macrocephalus, a gonochoristic fish species that presents a ZZ/ZW sex determination system, throughout a comparative analysis using differential display reverse transcriptase-PCR and real-time PCR. Four cDNA fragments were characterized, representing candidate genes with differential expression between the samples. Two of these fragments presented no significant identity with previously reported gene sequences. The other two fragments, isolated from male specimens, were associated to the gene that codes for the protein APBA2 (amyloid beta (A4) precursor protein-binding, family A, member 2) and to the Rab 37 gene, a member of the Ras oncogene family. The overexpression of these genes has been associated to a greater production of the beta-amyloid protein which, in turns, is the major factor that leads to Alzheimer's disease, and to the development of brain-tumors, respectively. Quantitative RT-PCR analyses revealed a higher Apba2 gene expression in males, thus validating the previous data on differential display. L. macrocephalus may represent an interesting animal model to the understanding of the function of several vertebrate genes, including those involved in neurodegenerative and cancer diseases.

Transcript accumulation of putative drought responsive genes in drought-stressed chickpea seedlings

Differential display reverse transcriptase PCR was used to identify cDNA sequences induced by drought in chickpea seedlings. The sequences of differentially expressed cDNAs: 192, 214, 219 and H1 showed high similarities at the protein level to known drought-inducible genes encoding for alanine aminotransferase, βAKIN1, a proteine kinase from the SnRK1 complex, lipid binding protein and COR protein, respectively. No significant similarity was found with the candidate sequence 277. Semi-quantitative multiplex PCR was used to verify that differentially amplified cDNAs were derived from differentially expressed genes. Relative quantification of the candidate sequences in the controls (well-watered seedlings) of drought-tolerant cv. ICCV2 and drought-susceptible cv. ILC3279 confirmed their induction upon drought in both cultivars. The transcript accumulation of the highly induced sequences, 219 and H1, was more important in the tolerant than the susceptible cultivar. These sequences are likely to be associated with drought tolerance in chickpea seedlings in contrast to sequences 214 and 277 which showed no variation in the mRNA accumulation between cultivars. The effect of ABA treatment on the mRNA accumulation of the isolated sequences 192, 214, 219 and H1 was analyzed. Sequences H1 and 192 were up-regulated by ABA treatment whereas sequences 214 and 219 were not, indicating an ABA-dependent and ABA-independent pathways in signal transduction in response to drought stress in chickpea seedlings. Key words: Differential display, DDRT-PCR, Cicer arietinum, drought stress.

Phosphite induces expression of a putative proteophosphoglycan gene inPhytophthora cinnamomi

The phosphate analogue phosphite is widely used to control diseases of plants caused by oomycete pathogens such as those within the genus Phytophthora. Phosphite inhibits zoospore production and growth of P. cinnamomi. However, very little is known about the underlying mechanism of action. In the present study, we grew P. cinnamomi in Ribero’s liquid medium with 0.1 mM phosphate, with and without 5 μg phosphite/mL, and used differential display reverse transcriptase-PCR (DDRT-PCR) to identify P. cinnamomi genes that are transcriptionally repressed or induced by phosphite. By using this technique, four differentially expressed bands were identified. However, quantitative measurement of the amount of mRNA transcript by RT-PCR revealed that only one gene was actually phosphite inducible. On the basis of the homology of the deduced amino acid sequence, this gene encodes a proteophosphoglycan. The remaining three bands did not show differential expression.

Analysis of Gene Expression in Primary Hepatocellular Carcinoma Using Differentially Displayed Reverse Transcriptase Polymerase Chain Reaction

The investigation of a specific tumor marker for hepatocellular carcinoma (HCC) is needed to examine the carcinogenesis and to select the patients for treatment options. The aim of this study was to find the genes related to HCC. We also examined the expression level of these genes in cancer cell lines and tissue specimens. Three pairs of HCC tissue and non-neoplastic hepatic tissue around the HCC were collected from three patients who underwent resection for HCC. Differential display reverse transcriptase-PCR (DD RT-PCR) using GeneFishingTM PCR was used to detect the differences in the gene expression between in HCC tissue and non-neoplatic tissue. Up- or down-regulated genes in HCC tissue were identified through BLAST searches after cloning and sequencing assays. Real-time RT-PCR assay was employed to detect the expression rate in 11 HCC tissues and human cancer cell lines. Differentially expressed 21 genes were identified, and they were classified as genes involved in protein metabolism, ubiquitin-dependent protein catabolism, carbohydrate metabolism, lipid metabolism, DNA repair, and inflammatory response. We identified differentially expressed genes in HCC, and these genes may play an important role in the study of hepatocarcinogenesis, development of biomarker, and target therapy for HCC.

“Identification and expression of six drought-responsive transcripts through differential display in desi cotton (Gossypium arboreum)”

There is not enough information available on drought-modulated gene(s) in Gossypium arboreum, which can be a valuable gene pool for improving modern cotton cultivars. In the present work differential display reverse transcriptase PCR (DDRT) was used to compare overall differences in gene expression between water stressed and control plants. By screening 93 primer-pair combinations DDRT technique resulted in up-regulation of 30 cDNA transcripts. Through reamplification and quality control assay 10 cDNA transcripts appeared false positive. The remaining 20-cDNA transcripts were extracted from the gel, reamplified, cloned and sequenced. Homology search revealed that 6 transcripts showed significant homology with known genes. Real-time RT-PCR showed that among 6 transcripts 5 showed significant over expression in water stressed leaves as compared to control. This is an important finding since there are only few reports of universal stress protein and transposable elements are available in plants but none in cotton under drought condition.

Molecular Identification and Characterization of a Gene Associated with the Onset of Tapping Panel Dryness (TPD) Syndrome in Rubber Tree (Hevea brasiliensis Muell.) by mRNA Differential Display

In rubber tree (Hevea brasiliensis), tapping panel dryness (TPD) syndrome is considered as a complex physiological disorder which affects latex biosynthesis. To identify differentially expressed genes between healthy and TPD-affected trees, mRNA differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) analysis was performed. We isolated 10 differentially expressed cDNA fragments of which one cDNA encoding a putative TOM20 like protein was identified. The cDNA (1,024 bp), corresponding to the HbTOM20 gene (H evea b rasiliensis Translocase of the Outer Mitochondrial Membrane), contained an open reading frame to code for 202 amino acid protein with a theoretical pI value of 9.5 and the calculated protein M (W) was 23.5 kDa. The predicted amino acid sequence contained conserved domains of TOM20 like proteins in the N-terminal. The protein HbTOM20 has 32% and 27% similarity to Populus TOM20 and Solanum TOM20, respectively. Both semi-quantitative RT-PCR and Northern blot results revealed that the HbTOM20 expression was significantly down-regulated in TPD-affected trees compared to healthy one. Accumulation of HbTOM20 mRNA transcripts was significantly higher in the bark tissues collected from healthy region than that of partially affected by TPD (partially dried) while barely detectable in completely TPD-affected area. Differential expression pattern was noticed in three rubber clones representing various degrees of TPD tolerance. These results suggest that down-regulation of HbTOM20 in TPD-affected trees may play an important role in alteration of mitochondrial metabolism resulting in impaired latex biosynthesis.

Changes in gene expression following androgen receptor blockade is not equivalent to androgen ablation by castration in the rat ventral prostate

Involution of the rat ventral prostate and concomitant modulation of gene expression post-castration is a well- documented phenomenon. While the rat castration model has been extensively used to study androgen regulation of gene expression in the ventral prostate,it is not clear whether all the gene expression changes post-castration are due to androgen depletion alone. To obtain insights into this, we performed differential display reverse transcriptase polymerase chain reaction (DD-RT-PCR) which resulted in the identification of castration and/or flutamide-regulated genes in the rat ventral prostate. These include clusterin, methionine adenosyl transferase II alpha, and prostate-specific transcripts such as PBPC1BS, S100RVP and A7. While clusterin, PBPC1BS and methionine adenosyl transferase II alpha are regulated by both castration and flutamide, S100 RVP and A7 are regulated by castration alone. Interestingly, we show that flutamide, unlike castration, does not induce apoptosis in the rat ventral prostate epithelium, which could be an underlying cause for the differential effects of castration and flutamide treatment. We propose that castration leads to enrichment and depletion of stromal and epithelial cell types, respectively, resulting in erroneous conclusions on some of the cell type-specific transcripts as being androgen regulated.

Comparison of gene expression patterns between porcine cumulus-oocyte complexes and naked oocytes

Several layers of cumulus cells surround the oocyte in the antral ovarian follicle throughout the follicular development until ovulation. The cumulus cells play an important supportive and regulative role in oocyte development and maturation via intercellular communications between oocytes and cumulus cells. Using the differential display reverse transcriptase polymerase chain reaction (DD-RT-PCR) and semi-quantitative RT-PCR methods, we compared the mRNA expression patterns in porcine oocytes from two sources: cumulus-oocyte complexes (COCs) and naked oocytes (NOs). After a small scale screen, 14 differentially expressed mRNAs were cloned, sequenced, and their expression patterns were verified by semi-quantitative RT-PCR. It was confirmed that four mRNAs including three genes (PELP1, Myo5b and CAST) and a new EST (Expressed Sequence Tag) are expressed preferentially in the oocytes of COCs rather than in NOs. Previous studies of the three genes suggested that they are mainly involved in oestrogen receptor regulation, membrane trafficking, organelle transport, cellular signalling and some other cellular processes. These results suggest that the aberrant of gene expression patterns detected in the oocytes of NOs compared with COCs explains their reduced quality in terms of development and maturation. In conclusion, these differentially expressed mRNAs may be involved in cellular interactions between oocytes and cumulus cells and thus could be considered as essential genes for the competence of oocytes. Keywords: Differential gene expression, DD-RT-PCR, porcine oocytes, cumulus South African Journal of Animal Science Vol. 37 (1) 2007: pp. 57-63

Identification of differentially expressed genes in developing cotton fibers (Gossypium hirsutum L) through differential display

Cotton fibers are differentiated, non-dividing cells that originate from the epidermal layer of developing ovules. To identify genes involved in cotton fiber development, we performed non-radioactive differential display reverse transcriptase PCR (DDRT-PCR) on the purified mRNA. This technique was tested on mRNA isolated from five different developmental stages of cotton fiber including 0, 5, 10, 15 and 20 DPA (days after pollination). The mRNA purified from total RNA was reversibly transcribed using three anchored oligo-dT primers. Polymerase chain reaction (PCR) amplification of each cDNA preparation was carried out in combination with seven arbitrary primers. The amplified products were resolved on 1% agarose gel containing ethidium bromide. DNA was extracted from seventeen differentially expressed bands and cloned in pTZ57R/T vector. The sequencing and BLAST search analysis indicated that 12 of the differentially expressed genes matched the previously characterized genes, while 3 of them matched the uncharacterized sequences of cotton fiber expressed sequence tags (ESTs) reported previously to be associated with cotton fiber and 2 of the clones had homology with putative proteins. The technique can be used to efficiently identify differentially expressed genes and can be expanded to large scale studies by increasing the number of random decamers.