Reverse Northern Hybridization Research Articles

Expression Profiling Coupled with In-silico Mapping Identifies Candidate Genes for Reducing Aflatoxin Accumulation in Maize.

Aflatoxin, produced by Aspergillus flavus, is hazardous to health of humans and livestock. The lack of information about large effect QTL for resistance to aflatoxin accumulation is a major obstacle to employ marker-assisted selection for maize improvement. The understanding of resistance mechanisms of the host plant and the associated genes is necessary for improving resistance to A. flavus infection. A suppression subtraction hybridization (SSH) cDNA library was made using the developing kernels of Mp715 (resistant inbred) and B73 (susceptible inbred) and 480 randomly selected cDNA clones were sequenced to identify differentially expressed genes (DEGs) in response to A. flavus infection and map these clones onto the corn genome by in-silico mapping. A total of 267 unigenes were identified and majority of genes were related to metabolism, stress response, and disease resistance. Based on the reverse northern hybridization experiment, 26 DEGs were selected for semi-quantitative RT-PCR analysis in seven inbreds with variable resistance to aflatoxin accumulation at two time points after A. flavus inoculation. Most of these genes were highly expressed in resistant inbreds. Quantitative RT-PCR analysis validated upregulation of PR-4, DEAD-box RNA helicase, and leucine rich repeat family protein in resistant inbreds. Fifty-six unigenes, which were placed on linkage map through in-silico mapping, overlapped the QTL regions for resistance to aflatoxin accumulation identified in a mapping population derived from the cross between B73 and Mp715. Since majority of these mapped genes were related to disease resistance, stress response, and metabolism, these should be ideal candidates to investigate host pathogen interaction and to reduce aflatoxin accumulation in maize.

Identification of Genes Involved in the Biosynthesis of Tripterygium wilfordii Hook.f. Secondary Metabolites by Suppression Subtractive Hybridization

Tripterygium wilfordii Hook.f. (Celastraceae) produces a vast array of natural products, mainly terpenes and sesquiterpene pyridine alkaloids, which have strong biological activities. The limited gene information on the biosynthesis pathways restricts the metabolic engineering of this valuable natural resource. In this study, a suppression subtractive hybridization library of methyl jasmonate (MeJA)-elicited T. wilfordii Hook.f. adventitious root was successfully constructed. To clone the genes involved in the downstream of biosynthetic pathways, a long elicitation time of 12 days was applied. After evaluating the subtraction efficiency through polymerase chain reaction and reverse northern hybridization, 818 clones were randomly sequenced. Basic Local Alignment Search Tool (BLAST) analysis showed that 568 of the 735 successfully expressed sequence tags were annotated. Overrepresentation of gene ontology analysis indicated that the suppression subtractive hybridization library enriched genes that are related to biotic and abiotic stimuli. After BLAST and phylogenetic analyses, 11 genes were found to be putatively involved in the biosynthesis of terpene and sesquiterpene pyridine alkaloid, and nine transcription factors were putatively involved in modulation. The expression modulations of these promising genes were further examined after short MeJA treatment. This study provides valuable information for further research on the production improvement of terpene and sesquiterpene pyridine alkaloids through biotechnology methods.

Construction and analysis of a SSH cDNA library of Eucalyptus grandis × Eucalyptus urophylla 9224 induced by Cylindrocladium quinqueseptatum

Leaf blight caused by Cylindrocladium quinqueseptatum Boedijn & Reitsma is a major disease occurring in Eucalyptus plantlets and saplings worldwide. Current studies on the resistance to this disease in eucalypti have focused primarily on analyzing the relationship between the disease and the metabolic products from the anatomic structure of Eucalyptus leaves and their physiological responses to pathogen invasion. There have been no reports on the mechanisms of molecular responses. Previous research suggests that Eucalyptus grandis × Eucalyptus urophylla 9224 is the highest resistant cultivar among the 11 ones planted in Fujian Province. Therefore, in this study, the molecular responses of E. grandis × E. urophylla 9224 to C. quinqueseptatum were investigated by means of suppression subtractive hybridization (SSH). In total, 928 clones were obtained from the forward SSH cDNA library of E. grandis × E. urophylla 9224, and 25 up-regulated genes were identified by screening the library using the method of reverse northern hybridization. The results show that among these, 23 up-regulated genes with known functions were involved in signal transduction, plant resistance response, photosynthesis, energy transfer, protein metabolism, and plant growth.

Characterization of Catharanthus roseus Genes Regulated Differentially by Peanut Witches’ Broom Phytoplasma Infection

AbstractThe differential display (DD) strategy was applied to isolate periwinkle (Catharanthus roseus) cDNAs that were differentially expressed following infection with peanut witches’ broom (PnWB) phytoplasma. Sixty‐four clones were selected from differentially expressed cDNA fragments. Following screening by reverse Northern hybridization, ten transcripts were selected and sequenced. The expression level of each transcript was quantified by real‐time PCR, and seven DD transcripts were identified as truly differentially expressed following PnWB phytoplasma infection. Among these, one that was homologous with phi‐1 gene was up‐regulated, while the others were down‐regulated. Except two genes, other four down‐regulated genes shared homology with the genes encoding psaDa gene, ML domain protein gene, eukaryotic translation initiation factor SUI1 gene and plastidic aldolase NPALDP1 gene, respectively. The identities of homologous genes were further confirmed for three DD transcripts by isolating long cDNA fragments from the cDNA library that was established in this investigation. Verified genes were ML domain protein gene, translation initiation factor SUI1 gene and plastidic aldolase gene and were primarily involved in the innate immune response, the stress response and photosynthesis. The possible role of these genes in the periwinkle that was infected by PnWB phytoplasma is discussed.

Two Internal Origins of Replication in Streptomyces Linear Plasmid pFRL1

Previous reports showed that Streptomyces linear plasmids usually contain one internal replication locus. Here, we identified two new replication loci on pFRL1, one (rep1A-ncs1) next to a telomere and another (rep2A-ncs2) approximately 10 kb from it. The rep1A-ncs1 locus was able to direct replication independently in both linear and circular modes, whereas rep2A-ncs2 required an additional locus, rlrA-rorA, in order to direct propagation in linear mode. Rep1A protein bound to ncs1 in vitro. By quantitative reverse transcription-PCR and Northern hybridization, we showed that transcription of rep1A and rep2A varied during development and that each dominated at different time points. pFRL1-derived linear plasmids were inherited through spores more stably than circular plasmids and were more stable with pSLA2 telomeres than with pFRL1 telomeres in Streptomyces lividans.

Identification of cDNAs for jasmonic acid-responsive genes in Polygonum minus roots by suppression subtractive hybridization

Elicitation, the plant-based biotechnology approach that utilizes the ability of plant roots to absorb and secrete a vast variety of bioactive compounds, was studied on Polygonum minus using jasmonic acid (JA) as an elicitor. To understand the overall molecular responses of P. minus roots to JA induction, a subtracted cDNA library was constructed using the suppression subtractive hybridization (SSH) method. From a total of 1,344 randomly selected colonies, 190 clones were shown to be differentially expressed using Reverse Northern hybridization. BLAST analysis revealed that clones were similar to genes associated with the biosynthesis of aromatic compounds through the oxylipin pathway, such as alcohol dehydrogenase and lipoxygenase. Putative clones involved in the shikimate pathway, including S-adenosyl-l-methionine synthetase and S-adenosyl-l-homocysteine hydrolase, were identified with predicted roles in phenylpropanoids’ biosynthesis. Genes responding to abiotic stress unique to JA elicitation, such as ELI3-1, glutathione S-transferase and peroxidase 1, were also identified. The kelch-repeat containing F-box family protein, a possible transcription factor in response to JA elicitation was also found. The results of the RT-PCR showed that the eight selected clones were strongly up-regulated, except for lipoxygenase, which showed a slightly higher expression of the transcript levels in response to the JA elicitation.

Construction and analysis of suppression subtractive library of <I>Festuca arundinacea</I> to low temperature stress

A forward subtractive cDNA library was constructed from tall fescue "Houndog 5" cultivar exposed to low temperature stress via suppression subtractive hybridization (SSH). Then cold-resistance of tall fescue (induced by low temperaturing) was determined through genes expression analysis. A total of 36 ESTs from SSH-cDNA library were selected and sequenced after reverse northern hybridization screening. The sequencing results were analyzed by means of retrieval and alignment in unigene GeneBank database. Based on the analysis, 34 genes exhibited different expressions, of which the functions of 33 genes were known and identified. These functionally known genes were detected in signal transduction, photosynthesis, sugar metabolism, material transportation and stress resistance.

RNA Extraction from Arabidopsis for Northern Blots and Reverse Transcriptase-PCR

INTRODUCTIONAnalysis of the cellular distribution of gene products (RNA and protein) can provide important clues to the function of genes in vivo. RNA expression can be monitored either after extraction from plants or tissues or in situ. The former procedure is easier to perform and more quantitative, whereas the latter provides much more detailed resolution of spatial and temporal expression patterns but is often more technically challenging. This protocol describes a method for RNA extraction from Arabidopsis thaliana. Frozen plant tissue is homogenized using a hand drill fitted with a plastic micropestle instead of using a conventional mortar and pestle, which can be inefficient and physically demanding. This homogenization method allows samples to be processed quickly (up to 12 samples/hour). Typical yields are 20-40 μg of total RNA from 100 mg of Arabidopsis seedlings. The RNA extracted using this method is suitable for reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern hybridization.

Differential Gene Expression in Response to Drought Stress in Maize Seedling

To provide the useful information for the choice of molecular marker used in marker-assisted selection of drought tolerance, it is necessary to find out more candidate genes and fulfill the information gaps in gene expression regulation under drought stress. In this study, we isolated four differentially expressed cDNA fragments from leaves of a drought-tolerant inbred line by suppression subtractive hybridization and reverse Northern hybridization, and validated their differential expression patterns among six inbred lines with different drought tolerance in response to drought stress by quantitative real-time PCR. Sequence similarity analysis indicated that two of four differentially expressed cDNA showed homology to gene DegP encoding trypsin-like serine protease, and gene PGAM-i encoding cofactor-independent phosphoglyceromutase, respectively. Expressions of the genes corresponding to four cDNA fragments was decreased at 6 h after drought stress treatment in most of the six inbred lines, and then returned to the control level with further stress in three of the tolerant inbred lines. The expression of the gene PGAM-i and the genes corresponding to fragments E4 and F4 were increased to a high level in tolerant inbred line 81565. In the two drought-sensitive inbred lines (Dan340 and ES40), the expression of these genes was still down-regulated. The probable mechanisms of these genes in response to drought stress were discussed. These results indicated that the drought-tolerant inbred lines up-regulated the expression of the drought-tolerant candidate genes, in contrast, drought-sensitive inbred lines down-regulated the expression of the genes.

Profiling of differentially expressed genes in human cervical carcinoma

Using the DDRT‐PCR, a series of differentially expressed genes in human primary cervical cancer was isolated. Among the 250 PCR amplimers, 88 gene fragments were confirmed by reverse Northern hybridization. Homology searches indicated that 26 out of 88 were previously known genes including calmodulin, human BBC1, histone H3.3, a series of ribosomal proteins (RPL19, RPS19, and RPS12), translation initiation factor (elF‐4AI), lactoferrin, integrin a6, cell‐surface antigens (CD9 and CD59), transcription factor (mbp‐1), and mitochondrial proteins. Several unknown clones showed sequence homology with known genes. Furthermore, six of the unknown genes showed identical sequence with expressed sequence tags (EST) of unknown function. Differential expression patterns of identified genes were further examined and confirmed with multiple pairs of cervical cancer samples using Northern hybridization. Our profiling of differentially expressed genes may provide useful information about the underlying genetic alterations in human cervical carcinoma and diagnostic markers for this disease. The precise roles of these genes in cancer development remain to be elucidated

Partial Sequence and Survey Analysis Identify a Multipartite, Negative-Sense RNA Virus Associated with Fig Mosaic.

RNA and nucleotide sequence-based analyses were used to identify viruses in fig mosaic (FM)-affected fig (Ficus carica) trees. Nucleotide sequence analyses of 267 cloned cDNAs identified sequences corresponding to four viruses representing four distinct taxa from fig trees in California. Virus sequences corresponding to members of the family Closteroviridae were most common (55 sequences). We also found two sequences for an Umbravirus, one sequence corresponding to a Luteovirus-associated RNA, and two sequences that showed homology to European mountain ash ringspot-associated virus (EMARAV). Reverse transcription-polymerase chain reaction (RT-PCR) and northern hybridization analyses were used to confirm the presence of specific virus RNAs in fig trees. A survey of 184 fig trees from a germplasm collection, a commercial orchard, backyards, and feral fig trees showed that one virus was most common (detected in 96% of tested samples), while none of the other virus sequences were detected in more than 36% of the fig trees. Based on its association with FM-affected trees, nucleotide sequence-based phylogenetic association, and previous reported properties, we suggest the name of this virus as Fig mosaic-associated virus (FMaV).

Differential Gene Expression in Uppermost Internode between Wheat Hybrid and Its Parents

Heterosis is defined as the advantage of hybrid performance over its parents in growth and productivity. Previous studies showed that differential gene expression in hybrids and their parents is responsible for the heterosis. Plant hormones, espe-cially GAs are found to be related to heterosis in development of shoot and plant height. However, information on systematic identification and characterization of the differentially expressed genes in shoots of hybrid and their parents are not found, and molecular evidence for the contribution of plant hormone to plant heterosis are also absent. The purpose of this paper was to in-vestigate the differential gene express in stems of hybrid and their parents, and further, to investigate the possible role of genes participating hormone signaling in the formation of heterosis of plant stem. Using a wheat cross combination that exhibited vig-orous heterosis in plant height, the differential gene expression in upper-most internode of wheat hybrids and their parents was analyzed by cDNA-AFLP. Ninety-eight differentially expressing cDNA fragments were obtained, which were sequenced and analyzed by Blast-x. Reverse Northern hybridization was performed in order to test the expression pattern of isolated ESTs in hybrid and their parents. The differentially expressed genes included a cdc2 gene and a PAS1 gene, which were induced by GAs and CTKs respectively and participated in plant cell division. The cdc2 gene was up-regulated in hybrid while the expression of PAS1 in hybrid was near the higher parent. The differentially expressed genes also included kinase participating in signal trans-duction, genes involved in metabolism and genes related to transcription regulations. It can be inferred that numerous genes were differentially expressed in the upper-most internode of hybrids and their parents, including genes participating in cell division and cell expansion, kinase related to signal transduction and so on. Reverse Northern blot analysis indicated that the expression pattern in our cDNA-AFLP analysis is credible. Since it has been much reported that genes participating in cell division and cell expan-sion are involved in plant stem growth and elongation, and genes that participate in gibberellins signals have been proved to be up-regulated in wheat hybrid and contributed to heterosis in wheat plant height, it can be modestly speculated that the differential expression of genes involved in phytohormone signals may be related to heterosis in wheat intenode elongation and plant height.

Identification of new up-regulated genes under drought stress in soybean nodules

Legumes/rhizobium biological N 2 fixation (BNF) is dramatically affected under abiotic stress such as drought, salt, cold and heavy metal stresses. Nodule response to drought stress at the molecular level was analysed using soybean ( Glycine max) and Bradyrhizobium japonicum as a model, since this symbiotic partnership is extremely sensitive to this stress. To gain insight into molecular mechanisms involved in drought-induced BNF inhibition, we have constructed a SSH (Suppression Subtractive Hybridisation) cDNA library from nodular tissue of plants irrigated at field capacity or plants water deprived for 5 days. Sequence analysis of the first set of 128 non redundant ESTs using protein databases and the Blastx program indicated that 70% of ESTs could be classified into putative known functions. Using reverse northern hybridization, 56 ESTs were validated as up-regulated genes in response to drought. Interestingly, only a few of them had been previously described as involved in plant response to drought, therefore most of the ESTs could be considered as new markers of drought stress. Here we discuss the potential role of some of these up-regulated genes in response to drought. Our analysis focused on two genes, encoding respectively a ferritin and a metallothionein, which are known to be involved in homeostasis and detoxification of metals and in response to oxidative stress. Their spatiotemporal expression patterns showed a high accumulation of transcripts restricted to infected cells of nodules in response to drought.

Isolation of EF1γ from calli regenerating SSH library in maize (Zea mays)

18599Hong, a good Maize (Zea mays) inbred line as well as good transformation acceptor with high regeneration capacity, was used for isolating embryonic callus regeneration genes. Subtractive library was constructed by Suppression subtractive hybridization and screened by Reverse Northern Hybridization. The clones of No. 27 was randomly picked to sequence. NCBI blastx results showed the similarity to elongation factor 1gamma in rice.

Changes in Photosynthetic Rates and Gene Expression of Leaves during a Source–Sink Perturbation in Sugarcane

In crops other than sugarcane there is good evidence that the size and activity of carbon sinks influence source activity via sugar-related regulation of the enzymes of photosynthesis, an effect that is partly mediated through coarse regulation of gene expression. In the current study, leaf shading treatments were used to perturb the source-sink balance in 12-month-old Saccharum spp. hybrid 'N19' (N19) by restricting source activity to a single mature leaf. Changes in leaf photosynthetic gas exchange variables and leaf and culm sugar concentrations were subsequently measured over a 14 d period. In addition, the changes in leaf gene response to the source-sink perturbation were measured by reverse northern hybridization analysis of an array of 128 expressed sequence tags (ESTs) related to photosynthetic and carbohydrate metabolism. Sucrose concentrations in immature culm tissue declined significantly over the duration of the shading treatment, while a 57 and 88% increase in the assimilation rate (A) and electron transport rate (ETR), respectively, was observed in the source leaf. Several genes (27) in the leaf displayed a >2-fold change in expression level, including the upregulation of several genes associated with C(4) photosynthesis, mitochondrial metabolism and sugar transport. Changes in gene expression levels of several genes, including Rubisco (EC 4.1.1.39) and hexokinase (HXK; EC 2.7.1.1), correlated with changes in photosynthesis and tissue sugar concentrations that occurred subsequent to the source-sink perturbation. These results are consistent with the notion that sink demand may limit source activity through a kinase-mediated sugar signalling mechanism that correlates to a decrease in source hexose concentrations, which, in turn, correlate with increased expression of genes involved in photosynthesis and metabolite transport. The signal feedback system reporting sink sufficiency and regulating source activity may be a potentially valuable target for future genetic manipulation to increase sugarcane sucrose yield.

The genes associated with termination of the critical period of neuroplasticity in visual cortex

To screening genes associated with termination of the critical period of neuroplasticity in visual cortex and investigate the molecular mechanism. To construct a cDNA library using visual cortex of dark rearing rats and normal light rearing 1 week after dark rearing 60 days rats with suppression subtractive hybridization technique. PCR, reverse Northern hybridization, sequencing and homology search were used to analysis the differential expression genes fragments. The cDNA library of termination the critical period was set up successfully. After screening, sequencing and homology search 14 sequences were obtained. Among of the genes 13 were known genes and one was novel gene fragment. They were up-regulated genes in visual cortex when the critical period termination. Through construction the cDNA library, we screened a crop of candidate genes related to termination the critical period. The results provided important foundation to further illuminate the molecular mechanism of termination the critical period of neuroplasticity in visual cortex.

Identification of genes displaying differential expression in the nuc-2 mutant strain of the mold Neurospora crassa grown under phosphate starvation

Subtractive hybridization was used to isolate transcripts up-regulated in the nuc-2 mutant strain of Neurospora crassa grown under phosphate starvation. Following differential screening, 66 cDNA clones of the total enriched were screened in a second round by reverse Northern hybridization. The 17 cDNA candidates displaying visual positive differential expression were sequenced, and functional grouping identified putative proteins possibly involved in diverse cellular processes as, for example, protein synthesis, signal transduction mechanisms, and transport facilitation. Four of them, confirmed by both virtual and Northern blot analyses, revealed genes involved in the initiation of mRNA translation that are significantly up-regulated in the nuc-2 mutant strain, which may be relevant to a further understanding of the molecular events involved in the phosphorus sensing in N. crassa.

Screening of Genes Associated with Termination of the Critical Period of Visual Cortex Plasticity in Rats

Purpose: To investigate the molecular mechanism involved in the termination of the critical period of visual cortex plasticity in the rat. Methods: The rats were divided into two groups, one group was dark-reared for 67 days after birth, while the other group was dark-reared for 60 days and then put under a normal light/dark cycle for 7 days. A subtracted cDNA library was constructed from the visual cortex, and differentially expressed genes were screened by nested PCR, reverse Northern hybridization, sequencing, and homology analysis. Results: Fourteen genes were found to be up-regulated in the visual cortex. These included 13 known genes and a novel fragment (Genbank submission EB174193). Of the known genes, three genes encoding β -tubulin, myelin basic protein, and cyclophilin were previously reported to be associated with visual cortex plasticity. Conclusion: A set of candidate genes related to the termination of the critical period was identified using the subtracted cDNA library. This work provides an important basis for understanding the molecular mechanisms involved in the termination of plasticity in the visual cortex.

OCIA domain containing 2 is highly expressed in adenocarcinoma mixed subtype with bronchioloalveolar carcinoma component and is associated with better prognosis

Although lung adenocarcinoma is a major cause of cancer death worldwide, details of its molecular carcinogenesis and stepwise progression are still unclear. To characterize the sequential progression from bronchioloalveolar adenocarcinoma of the lung (BAC, in situ carcinoma) to adenocarcinoma mixed subtype with BAC component polymerase chain reaction-based cDNA suppression subtractive hybridization (SSH) was carried out using two representative cases of BAC (non-invasive tumors) and adenocarcinoma mixed subtype with BAC (invasive tumors). Through differential screening, virtual reverse northern hybridization and quantitative real-time reverse-transcription-polymerase chain reaction (qRT-PCR) we selected five genes (TncRNA, OCIAD2, ANXA2, TMED4 and LGALS4) that were expressed at significantly higher levels in invasive adenocarcinoma mixed subtype with BAC than in BAC. After in situ hybridization and qRT-PCR analyses, we confirmed that only the OCIAD2 gene showed significantly higher expression in the tumor cells of invasive adenocarcinoma mixed subtype with BAC than in BAC (P = 0.026). We then carried out in situ hybridization of OCIAD2 in 56 adenocarcinoma mixed subtype with BAC component and assessed the correlation between OCIAD2 expression and clinicopathological features. In contrast to our expectation, the patients with OCIAD2 expression showed a better clinical outcome than those without OCIAD2 expression, and OCIAD2 expression showed an inverse correlation with lymphatic invasion, blood vessel invasion and lymph node metastasis. These results suggest that OCIAD2 begins to express at the progression from in situ to invasive carcinoma, and is associated with the favorable prognosis of adenocarcinoma mixed subtype with BAC component.

Identification of genes induced during Medicago sativa nodule development by using the cDNA-AFLP technique

A variety of genes involved in nodule development and function were induced during symbiotic root nodule development in alfalfa. We identified those genes by comparing the cDNA-amplified fragment length polymorphism (cDNA-AFLP) patterns of infected nodules and uninfected roots. Seventy-one transcript-derived fragments (TDFs) were isolated from about 3000 TDFs, which are enhanced or specifically expressed in nodule. The differential expression patterns of 61 genes were confirmed by reverse Northern hybridization or Northern hybridization analyses. Among these, 31 exhibited significant similarities to characterized database entries. These results suggest that the genes corresponding to the 31 TDFs may play important roles in signal transduction, gene expression and regulation, substance transportation or auto-regulation of nodulation during nodule development. The Northem hybridization analysis also indicates that the gene corresponding to TDF RX89 was not expressed in the nodule elicited by bacA mutant, presumably it is specially induced at the stage of nodule development when bacteriods begin to be differentiated.