cDNA-amplified Fragment Length Polymorphism Research Articles

Identification of Cladosporium fulvum infection responsive genes in tomato through cDNA-AFLP

Tomato (Solanum lycopersicum) is one of the highest-value vegetable crops worldwide, but tomato planting can be easily affected by leaf mold. Tomato leaf mold disease is caused by Cladosporium fulvum, and plants carrying the Cf genes have effective resistance to C. fulvum. To identify leaf mold resistant varieties and screen for specific cDNA amplified fragment length polymorphism (cDNA-AFLP) molecular markers, tomato plants harbouring genes Cf-5, Cf-11, Cf-19, and the moneymaker (MM) tomato line (without Cf gene) were used as plant materials. The changes in reactive oxygen species (ROS) levels and antioxidant activities of plant leaves after inoculation of C. fulvum were measured. Ethylene (ETH), abscisic acid (ABA) and salicylic acid (SA) contents were also determined. The results revealed that the ROS levels in Cf-19 tomato were the most sensitive to C. fulvum inoculation, while in MM they were the least. The antioxidant enzymes of Cf-carrying plants exhibited different activities as compared to those of MM. In the cDNA-AFLP analysis data, the expression of ERF109, sedoheptulose-1,7-bisphosphatase (SBPase), L-ascorbic acid oxidase (AAO) and Thioredoxin H2 (TRX2) were different between Cf-gene carriers and MM plants. In summary, in the process of leaf–mold interaction, the outbreak of ROS and development of systemic acquired resistance (SAR) are tightly combined with antioxidant enzymes, including thioredoxin. Tomato plants carrying the Cf-19 gene appear to have stronger C. fulvum resistance.

Caspofungin resistance in clinical Aspergillus flavus isolates

Introduction and Aims: The present study was conducted to determine the candidate genes involved in caspofungin (CAS) resistance in clinical isolates of Aspergillus flavus (A. flavus).Materials and Methods: The antifungal susceptibility assay of the CAS was performed on 14 clinical isolates of A. flavus using the CLSI-M-38-A2 broth micro-dilution protocol. Since CAS had various potencies, the minimum effective concentration (MEC) of anidulafungin (AND) was also evaluated in the present study. The FKS1 gene sequencing was conducted to assess whether mutations occurred in the whole FKS1 gene as well as hot spot regions of the FKS1 gene of the two resistant isolates. A complementary DNA-amplified fragment length polymorphism (CDNA-AFLP) method was performed to investigate differential gene expression between the two resistant and two sensitive clinical isolates in the presence of CAS. Furthermore, quantitative real-time PCR (QRT-PCR) was utilized to determine the relative expression levels of the identified genes.Results: No mutations were observed in the whole FKS1 gene hot spot regions of the FKS1 genes in the resistant isolates. A subset of two genes with known biological functions and four genes with unknown biological functions were identified in the CAS-resistant isolates using the CDNA-AFLP. The QRT-PCR revealed the down-regulation of the P-type ATPase and ubiquinone biosynthesis methyltransferase COQ5 in the CAS-resistant isolates, compared to the susceptible isolates.Conclusion: The findings showed that P-type ATPase and ubiquinone biosynthesis methyltransferase COQ5 might be involved in the CAS-resistance A. flavus clinical isolates. Moreover, a subset of genes was differentially expressed to enhance fungi survival in CAS exposure. Further studies are recommended to highlight the gene overexpression and knock-out experiments in A. flavus or surrogate organisms to confirm that these mentioned genes confer the CAS resistant A. flavus.

Sugarcane mosaic virus mediated changes in cytosine methylation pattern and differentially transcribed fragments in resistance-contrasting sugarcane genotypes.

Sugarcane mosaic virus (SCMV) is the causal agent of sugarcane mosaic disease (SMD) in Brazil; it is mainly controlled by using resistant cultivars. Studies on the changes in sugarcane transcriptome provided the first insights about the molecular basis underlying the genetic resistance to SMD; nonetheless, epigenetic modifications such as cytosine methylation is also informative, considering its roles in gene expression regulation. In our previous study, differentially transcribed fragments (DTFs) were obtained using cDNA-amplified fragment length polymorphism by comparing mock- and SCMV-inoculated plants from two sugarcane cultivars with contrasting responses to SMD. In this study, the identification of unexplored DTFs was continued while the same leaf samples were used to evaluate SCMV-mediated changes in the cytosine methylation pattern by using methylation-sensitive amplification polymorphism. This analysis revealed minor changes in cytosine methylation in response to SCMV infection, but distinct changes between the cultivars with contrasting responses to SMD, with higher hypomethylation events 24 and 72 h post-inoculation in the resistant cultivar. The differentially methylated fragments (DMFs) aligned with transcripts, putative promoters, and genomic regions, with a preponderant distribution within CpG islands. The transcripts found were associated with plant immunity and other stress responses, epigenetic changes, and transposable elements. The DTFs aligned with transcripts assigned to stress responses, epigenetic changes, photosynthesis, lipid transport, and oxidoreductases, in which the transcriptional start site is located in proximity with CpG islands and tandem repeats. Real-time quantitative polymerase chain reaction results revealed significant upregulation in the resistant cultivar of aspartyl protease and VQ protein, respectively, selected from DMF and DTF alignments, suggesting their roles in genetic resistance to SMD and supporting the influence of cytosine methylation in gene expression. Thus, we identified new candidate genes for further validation and showed that the changes in cytosine methylation may regulate important mechanisms underlying the genetic resistance to SMD.

Cardiac Transcriptomics Reveals That MAPK Pathway Plays an Important Role in Hypoxia Tolerance in Bighead Carp (Hypophthalmichthys nobilis).

Simple SummaryAcute hypoxia treatment was performed in juvenile bighead carp (Hypophthalmicthys nobilis) by decreasing water O2. The results showed that blood lactate and serum glucose increased significantly under hypoxia stress, and some differentially expressed genes were identified among hypoxia tolerant, hypoxia sensitive, and normoxia control groups. Differentially expressed genes between hypoxia tolerant and hypoxia sensitive groups were mainly involved in mitogen-activated protein kinase (MAPK) signaling, insulin signaling, apoptosis, tight junction, and adrenergic signaling in cardiomyocytes pathways, of which MAPK signaling pathway played a key role in cardiac tolerance to hypoxia in bighead carp. These results provide a basis for understanding the physiological and molecular mechanisms underlying hypoxia response in fish and a guide for future genetic breeding programs for hypoxia resistance in bighead carp.As aquatic animals, fishes often encounter various situations of low oxygen, and they have evolved the ability to respond to hypoxia stress. Studies of physiological and molecular responses to hypoxia stress are essential to clarify genetic mechanisms underlying hypoxia tolerance in fish. In this study, we performed acute hypoxia treatment in juvenile bighead carp (Hypophthalmicthys nobilis) by decreasing water O2 from 6.5 mg/L to 0.5 mg/L in three hours. This hypoxia stress resulted in a significant increase in blood lactate and serum glucose. Comparisons of heart transcriptome among hypoxia tolerant (HT), hypoxia sensitive (HS), and normoxia control (NC) groups showed that 820, 273, and 301 differentially expressed genes (DEGs) were identified in HS vs. HT, NC vs. HS, and NC vs. HT (false discovery rate (FDR) < 0.01, Fold Change> 2), respectively. KEGG pathway enrichment showed that DEGs between HS and HT groups were mainly involved in mitogen-activated protein kinase (MAPK) signaling, insulin signaling, apoptosis, tight junction and adrenergic signaling in cardiomyocytes pathways, and DEGs in MAPK signaling pathway played a key role in cardiac tolerance to hypoxia. Combined with the results of our previous cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis of hypoxia stress in this species, such genes as stbp2, ttn, mapk, kcnh, and tnfrsf were identified in both studies, representing the significance of these DEGs in hypoxia tolerance in bighead carp. These results provide insights into the understanding of genetic modulations for fish heart coping with hypoxia stress and generate basic resources for future breeding studies of hypoxia resistance in bighead carp.

Identification of salinity responsive genes in lavender through cDNA-AFLP

Currently, a global demand exists forlavender as a significant medicinal plant and source of essential oils. Freshwater and arable lands are two major factors that inhibit extensive farming of medicinal plants in Iran. Saline water from seas and salty soil may be new resources for agricultural use, especially for medicinal plants. We sought to extend our knowledge of the Lavandula angustifolia genome and molecular basis of its salinity tolerance by using cDNA amplified fragment length polymorphism (cDNA-AFLP) to investigate the changes in plant transcriptomes in response to NaCl. All identified transcript derived fragments (TDF) were assigned as novel L. angustifolia genes related to signal transduction, regulation of gene expression, alternative splicing, autophagy, and secondary metabolite biosynthesis. qRT-PCR analysis of the TDFs in response to different concentrations of NaCl revealed various levels of mRNA of the identified genes in this plant. Our findings provided primary insights into the molecular response of L. angustifolia to salinity.

Diagnostic accuracy of VIDISCA-NGS in patients with suspected central nervous system infections

ObjectivesConfirming the diagnosis in viral central nervous system (CNS) infections can be difficult with the currently available diagnostic tools. Virus discovery cDNA-amplified fragment length polymorphism next-generation sequencing (VIDISCA-NGS) is a promising viral metagenomic technique that enables the detection of all viruses in a single assay. We performed a retrospective study on the diagnostic accuracy of VIDISCA-NGS in cerebrospinal fluid (CSF) of individuals with suspected CNS infections. MethodsConsecutive adult patients presenting to the Emergency Department or inpatients, who underwent a lumbar puncture for the suspicion of a CNS infection, were included if they were diagnosed with a viral CNS infection, or if a viral CNS infection was initially suspected but eventually a different diagnosis was made. A quantitative PCR panel of the most common causative viruses was performed on CSF of these patients as reference standard and compared with the results of VIDISCA-NGS, the index test. ResultsWe included 38 individuals with viral CNS infections and 35 presenting with suspected CNS infection for whom an alternative aetiology was finally established. Overall sensitivity and specificity were 52% (95% CI 31%–73%) and 100% (95% CI 91%–100%), respectively. One enterovirus, detected by VIDISCA-NGS, was only identified by quantitative PCR upon retesting. Additional viruses identified by VIDISCA-NGS consisted of GB virus C, human papillomavirus, human mastadenovirus C, Merkel cell polyoma virus and anelloviruses. ConclusionIn patients for whom routine diagnostics do not yield a causative pathogen, VIDISCA-NGS can be of additional value as it can detect a broader range of viruses, but it does not perform well enough to replace quantitativePCR.

CDNA-AFLP analysis of salicylic acid- and calcium chloride-induced transcript derived fragments under drought in tomato (Solanum lycopersicum)

Drought seriously threatens tomato production worldwide. Despite much research on exogenous salicylic acid (SA) and Ca2+ improving plant resistance to biotic and abiotic stress, the molecular mechanisms of exogenous SA- and Ca2+-mediated drought resistance response in tomato remain unclear. In this study, we analyzed SA- and Ca2+ -induced differentially expressed transcripts under drought in tomato plants using cDNA-amplified fragment length polymorphism (cDNA-AFLP). In total, 34 transcript derived fragments (TDFs) were differentially expressed. The functions identified through NCBI BLAST alignment mainly involved signal transduction, amino acid metabolism, transcription factors, transfer transport and stress response. The quantitative real-time polymerase chain reaction results of 12 TDFs associated with drought response were consistent with the patterns of changes observed with cDNA-AFLP analysis. These differentially expressed transcripts may be used for functional verification, transgenic research and breeding of drought-resistant tomato varieties.

BcERF070, a novel ERF (ethylene-response factor) transcription factor from non-heading Chinese cabbage, affects the accumulation of ascorbic acid by regulating ascorbic acid-related genes

L-Ascorbic acid (AsA) is an important nutrient in non-heading Chinese cabbage (Brassica rapa ssp. chinensis). To understand the regulatory mechanism of AsA accumulation, we crossed “Wutacai” and “Erqing” to obtain F2 plants and then constructed two pools (AsA-High and AsA-Low) of F2-generation plants to further analyze the differentially expressed genes (DEGs) by cDNA-amplified fragment length polymorphism (cDNA-AFLP) technology. Among the DEGs, BcERF070 showed a significantly higher expression level in “Wutacai” and F2 plants with high AsA than in “Erqing” and F2 plants with low AsA, suggesting that BcERF070 is involved in the accumulation of AsA. To further understand the function of BcERF070 in AsA accumulation, we obtained BcERF070-silenced plants and BcERF070-overexpressed Arabidopsis thaliana plants. It was found that the downregulation of BcERF070 resulted in a decrease in AsA content, and the upregulation of BcERF070 resulted in an increase in AsA content. To evaluate the mechanism of BcERF070 involvement in the accumulation of AsA, the leaves of BcERF070-silenced and control plants were subjected to transcriptome sequencing. Twelve DEGs including BrAPXs were found to be associated with ascorbic acid. The qRT-PCR analysis on BcERF070-silenced plants and BcERF070-overexpressed plants further demonstrated the reliability of the transcriptome sequencing. Finally, based on analysis of the cis-acting elements of the promoter of 12 AsA-related genes, we speculated that BcERF070 may act on the DRE (dehydration-responsive element) of seven target gene promoters to regulate AsA content. In conclusion, a novel transcription factor, BcERF070, was revealed in this study, which affected the accumulation of AsA by regulating genes involved in the ascorbate biosynthesis and metabolic pathway. This information will help in understanding the regulatory mechanism of BcERF070 involvement in the accumulation of AsA in plants.

ShORR-1, a Novel Tomato Gene, Confers Enhanced Host Resistance to Oidium neolycopersici.

A previous complementary cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis examined responses to the powdery mildew pathogen Oidium neolycopersici (On) of the resistant cultivar Solanum habrochiates G1.1560, carrying the Ol-1 resistance gene, and susceptible cultivar S. lycopersicum Moneymaker (MM). Among other findings, a differentially expressed transcript-derived fragment (DE-TDF) (M14E72-213) was upregulated in near isogenic line (NIL)-Ol-1, but absent in MM. This DE-TDF showed high homology to a gene of unknown function, which we named ShORR-1 (Solanum habrochaites Oidium Resistance Required-1). However, MM homolog of ShORR-1 (named ShORR-1-M) was still found with 95.26% nucleic acid sequence similarity to ShORR-1 from G1.1560 (named ShORR-1-G); this was because the cut sites of restriction enzymes in the previous complementary cDNA-AFLP analysis was absent in ShORR-1-M and differs at 13 amino acids from ShORR-1-G. Transient expression in onion epidermal cells showed that ShORR-1 is a membrane-localized protein. Virus-induced gene silencing (VIGS) of ShORR-1-G in G1.1560 plants increased susceptibility to On. Furthermore, overexpressing of ShORR-1-G conferred MM with resistance to On, involving extensive hydrogen peroxide accumulation and formation of abnormal haustoria. Knockdown of ShORR-1-M in MM did not affect its susceptibility to On, while overexpressing of ShORR-1-M enhanced MM’s susceptibility to On. We also found that changes in transcript levels of six well-known hormone signaling and defense-related genes are involved in ShORR-1-G-mediated resistance to On. The results indicate that ShORR-1-M and ShORR-1-G have antagonistic effects in tomato responses to On, and that ShORR-1 is essential for Ol-1-mediated resistance in tomato.

Exploiting Differential Gene Expression to Discover Ionic and Osmotic-Associated Transcripts in the Halophyte Grass Aeluropus littoralis

BackgroundSalinity as a most significant environmental challenges affects the growth and productivity of plants worldwide. In this study, the ionic and iso-osmotic effects of salt stress were investigated in Aeluropus littoralis L., a halophyte grass species from Poaceae family, by cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique. To dissect the two different effects (ionic and osmotic) exerted by salt stress, various ionic agents including 200 and 400 mM sodium chloride (NaCl), 200 and 400 mM potassium chloride (KCl) as well as 280 and 406 gl− 1 (− 0.9 and − 1.4 MPa) polyethylene glycol 6000 (PEG) as their iso-osmotic concentrations were applied.ResultsApplication of KCl and PEG significantly reduced the fresh weight (FW) of A. littoralis seedlings compared to control while NaCl treatment markedly enhanced the FW. At the transcriptome level, different observations of changes in gene expression have been made in response of A. littoralis to ionic and osmotic stresses. Out of 69 transcript derived fragments (TDFs), 42 TDFs belong to 9 different groups of genes involved in metabolism (11.6%), transcription (10.2%), ribosomal protein (8.7%), protein binding (8.7%) transporter (5.8%), translation (5.8%), signal transduction (4.3%), nucleosome assembly protein (2.9%) and catabolism (2.9%). The 44 and 28 percent of transcripts were expressed under ionic stress (NaCl-specific and KCl-specific) and osmotic stress (common with NaCl, KCl and PEG), respectively which indicating a greater response of plants to ionic stress than osmotic stress. Expression pattern of eight candidate TDFs including; SYP81, CAND1, KATN, ISB1, SAMDC, GLY1, HAK18 and ZF30 was evaluated by RT-qPCR at high salinity levels and recovery condition.ConclusionDifferential regulation of these TDFs was observed in root and shoot which confirm their role in salt stress tolerance and provide initial insights into the transcriptome of A. littoralis. Expression pattern of ionic and osmotic-related TDFs at A. littoralis can be taken as an indication of their functional relevance at different salt and drought stresses.

Association between seed yield-related traits and cDNA-AFLP markers in cumin (Cuminum cyminum) under drought and irrigation regimes

Cumin (Cuminum cyminum L.) is an aromatic medicinal herb with beneficial essential oil being used in antibacterial activities and industry of creams, lotions, and perfumes. Use of functional markers expedites breeding cumin for drought tolerance. The objective of this study was to identify association of plant height and several seed yield- related traits with complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) markers in 15 cumin (Cuminum cyminum) accessions under normal irrigation (NI) and drought stress (DS) conditions. Cumin accessions were grown in the field and subjected to NI practice and post-heading DS in two years in Kerman, Iran. The mean seed yield ranged between 448.72 and 994.18 kg/ha in NI and was from 211.05 to 452.05.06 kg/ha in DS. Reduction in plant height and seed yield-related traits ranged 27–28% in the first and 29–50% in the second year in DS compared with NI. Twelve EcoR I-Mse I primer combinations were used to amplify AFLP markers in cDNA fragments isolated from RNA samples. The identified markers were subjected to analysis of marker-trait associations (MTAs) using the generalized linear model (GLM) and mixed linear model (MLM). A number of 239 and 208 reliable transcript-derived fragments (TDFs) were amplified in cumin genotypes tested in NI and DS, respectively. Of the identified TDFs, highly significant markers were used for association analysis, only. Higher number of MTAs was identified for traits in NI compared with DS. Of the identified linked markers, 5 in NI and 3 in DS were significantly associated with more than one trait under two water status conditions. None of markers were identified as common MTAs between the two moisture conditions. Analysis of population structure demonstrated cumin accessions could be divided into 3 and 4 sub-populations. Overall, high variation was identified in cumin with respect to biological yield, seed yield and seed related traits and the identified linked markers can be incorporated into marker-assisted selection programs for the improvement of seed yield in drought condition.

Colinearity of putative flowering gene in both sugarcane and sorghum

Sugarcane (Saccharum spp.) and sorghum (Sorghum spp.) have become increasingly important crops for biofuels production. Sugarcane has an autopolyploid complex genome, whereas sorghum has a diploid simple genome. Flowering is one of the sugar-related agronomic traits in both species. Here, we obtained cDNA of inflorescences at 0–15 cm from S. spontaneum using cDNA-amplified restriction fragment length polymorphisms to develop flower transcriptome profiling with 26 primer combinations. A total of 183 transcript-derived fragments (TDFs) were screened and 96 TDFs were sequenced. Out of 96 TDFs, 26 were selected as putative flowering genes to study collinearity with the sorghum genome. For gene collinearity, a genetic linkage map with 169 SSR co-dominant markers and 12 TDF marker loci were mapped to 14 linkage groups collectively spanning 1077.8 cM and corresponding to the 10 sorghum chromosomes. Interestingly, 9 TDF marker loci could be mapped to 5 linkage groups. In this study, we successfully identified the homologous location of sugarcane flowering TDFs in the sorghum genome and found that 4DS_1X and 2DS_3E TDFs may serve as candidate specific markers linked to flowering in both sorghum and sugarcane.

Genetic and expression differences between putative ecotypes of Sphagnum denticulatum Brid. (Sphagnaceae: Bryophyta) subjected to drought stress and rehydration

Adverse changes in vegetation occur as the effect of the progressive drying of peatlands, leading to the disappearance of peat mosses (Sphagnum), a key component of these ecosystems. We report gene expression divergence for two putative ecotypes of S. denticulatum collected from contrasted habitats under the threat of drought. We used cDNA-amplified fragment length polymorphism (cDNA-AFLP) as the first screening method. We then used real-time, quantitative PCR to identify genes whose transcription changes in response to drought and rehydration treatments. Using 133 of 900 pretested cDNA-AFLP primer combinations, we have identified 949 drought-induced transcript-derived fragments, 6 (0.6%) of which are significantly differentially expressed genes (DEGs) in the putative ecotypes. We cloned and sequenced 378 fragments using designed fragment-specific primers. Transcription patterns were quantified for 21 target genes using qRT-PCR. When upregulated under drought, nine genes showed fold changes in a range of 4.64–150.03. The DEGs represented functional categories including protein/nucleotide binding, catalytic activity, transporter activity, and enzyme regulation activity. These genes are known to be involved in various metabolic pathways and physiological processes which emphasize the complexity of plant response to stress. Most of the significant differences in transcription patterns were evident in the aquatic plants, suggesting induced responses to drought in these plants. The expression was significantly less pronounced in the terrestrial plants, indicating that they are preconditioned to withstand drought stress. We detected positive selection on individual codons for specific branches using the ratio of substitution rates at nonsynonymous and synonymous sites (dN/dS). For the first time, this study provides insight into the genetic basis of the putative adaptive evolution in Sphagnum and allows for the discovery of genes involved in drought stress response.

CDNA-AFLP analysis of transcript derived fragments during seed development in castor bean (Ricinus communis L.)

Ricinoleic acid, one of the biggest components in castor oil, is an unusual fatty acid with numerous industrial applications. Despite much research on the oil content of Ricinus communis L., the genes encoding the oil biosynthesis function in the seed developmental stages are still poorly known. Here, we analyzed differentially expressed transcripts during seed development using cDNA-amplified fragment length polymorphism. Almost all ingredients significantly decreased along with seed maturation, except for ricinoleic acid, which increased greatly from 6.7% to 86.8%. A total of 534 distinguished fragments were differentially expressed in six development stages. Among these, 67 fragments showed high homology with known-function genes, and 26 sequences were successfully annotated with fatty acid synthesis or storage proteins in castor and other species. The results showed that fatty acid synthesis and accumulation in castor seeds displayed various temporal patterns. Different cDNA-fragment patterns were mainly found in later development stages, coinciding with the onset of oil synthesis.

In vivo identification of novel TGIF2LX target genes in colorectal adenocarcinoma using the cDNA-AFLP method

Background and study aimsHomeobox-containing genes are composed of a group of regulatory genes encoding transcription factors involved in the control of developmental processes. The homeodomain proteins could activate or repress the expression of downstream target genes. This study was conducted to in vivo identify the potential target gene(s) of TGIF2LX in colorectal adenocarcinoma. MethodsA human colorectal adenocarcinoma cell line, SW48, was transfected with the recombinant pEGFPN1-TGIF2LX. The cells were injected subcutaneously into the flank of the three groups of 6-week-old female athymic C56BL/6 nude mice (n = 6 per group). The transcript profiles in the developed tumours were investigated using the cDNA amplified fragment length polymorphism (cDNA-AFLP) technique. ResultsThe real-time RT-PCR and DNA sequencing data for the identified genes indicated that the N-terminal domain-interacting receptor 1 (Nir1) gene was suppressed whereas Nir2 and fragile histidine triad (FHIT) genes were upregulated followed by the overexpression of TGIF2LX gene. ConclusionDownregulation of Nir1 and upregulation of Nir2 and FHIT genes due to the overexpression of TGIF2LX suggests that the gene plays an important role as a suppressor in colorectal adenocarcinoma.

Transcriptome analysis reveals novel insights into the response to Pb exposure in Phanerochaete chrysosporium

Metals released into the environment continue to be of concern for human health. Using white-rot fungi as biosorbents for heavy metals removal is an attractive alternative owing to its good performance and low cost. However, the molecular mechanism underlying heavy metal tolerance in white-rot fungi has not yet been fully elucidated. This study identified and analyzed the lead (Pb)-induced transcriptional changes in Phanerochaete chrysosporium, a well-known heavy metal hyperaccumulating white-rot fungus. The results confirmed its outstanding ability in Pb tolerance and effective defense system. By comparative analysis of gene expression profiles obtained from cDNA-amplified fragment length polymorphism (cDNA-AFLP), we isolated 43 transcript-derived fragments (TDFs) differentially regulated by Pb exposure in P. chrysosporium, and 23 TDFs presented significant similarities to genes encoding known or putative proteins which belong to different functional categories involving ion binding, energy and carbohydrate metabolism, and signal transduction. The detailed characterization of these Pb-responsive genes was presented and the expression patterns of some interesting genes were validated by quantitative RT-PCR. This work provides the first evidence of Pb-responsive genes along with their putatively functional annotations in P. chrysosporium, which may help to understand the mechanism underlying heavy metal accumulation and tolerance in P. chrysosporium.

Effects of ethephon on DNA methylation and gene expressions associated with shortened internodes in maize

ABSTRACTIn this study, the molecular mechanism underlying ethephon-induced shortened internodes in maize was investigated using Zhengdan958 maize variety. The ethephon treatment was performed by spraying 225 mL/ha Ethephon 40% SL onto the foliage at the eight-expanded-leaves stage. The differentially expressed genes in the young internodes under ethephon treatment were identified through methylation-sensitive amplified fragment length polymorphism (MSAP), start codon targeted polymorphism (SCoT) and cDNA-amplified fragment length polymorphism (AFLP) analyses. MSAP results showed the methylation sites were widely distributed in both the ethephon-treated (at 27.8%) and control plants (30.1%). This suggested that ethephon treatment modified the methylation patterns; with 3.0% of the sites being hyper-methylated and 7.3% demethylated compared with the control. Based on SCoT analysis, 148 transcript derived fragments (TDFs) were obtained in the ethephon-treated plants. Among them, 38 were up-regulated (25.7%) and 47 down-regulated (31.8%). cDNA-AFLP analysis using 70 primer pairs identified 1635 TDFs in the ethephon-treated and the control plants. Of these, 600 and 564 TDFs were up- and down-regulated by the ethephon treatment, respectively. BLASTX analysis on 50 (randomly selected) differentially expressed TDFs divided them into several categories based on their putative biological functions: signal transduction (6%), resistance-related (14%), energy and metabolism (22%), transcription (4%), cell apoptosis (2%), unknown functional proteins (42%) and unknown genes (10%). Our results revealed that ethephon treatment could induce DNA methylation variation principally by increasing the demethylation tendency. This is suggested to play roles in stress-defence genes expression regulation and the differentially expressed genes could be associated with shortened internodes in maize.

Isolation and expression pattern analysis of CmRNF5 and CmNPH3L potentially involved in graft compatibility in cucumber/pumpkin graft combinations

Abstract Graft compatibility between the rootstock and scion is a prerequisite for grafting and is presumably regulated by multiple genes involved in a complicated regulatory network. To better understand the mechanism of graft compatibility, it is necessary to analyze the function of differentially expressed genes in graft combinations with different degrees of graft compatibility. The objective of this study was to identify transcriptionally regulated genes and their possible functions in graft compatibility. Using cucumber (Cucumis sativus L.) as scions, pumpkin (Cucurbita L.) with different degrees of graft compatibility as rootstocks, and both self-grafting and non-grafting as controls, 18 different transcript-derived fragments (TDFs) in the leaves of grafted cucumber plants were cloned and sequenced using complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) technique. qRT-PCR showed that 6 TDFs exhibited significant differences between compatible and incompatible grafted cucumber leaves, and two of these TDFs also displayed differences between the two compatible and incompatible rootstocks at 25 days after grafting (DAG). Subsequently, a RING finger protein gene named CmRNF5 and a BTB domain-containing protein gene named CmNPH3L were cloned from pumpkin. Amino acid sequence alignments and phylogenetic analysis showed that these two genes were highly orthologous with those in other plants. The expression patterns of the two genes were investigated in rootstocks with different degrees of compatibility over time (5, 10, 15, 20, and 25 DAG). The results indicated that CmRNF5 and CmNPH3L transcripts were differentially expressed between compatible and incompatible unions throughout graft development. The expression differences of the two genes observed in the scion and different compatibility rootstocks suggested that these two genes could be related to graft compatibility/incompatibility responses. To gain more insight into the regulation of graft development, analysis of the expression patterns of the two genes under different environmental stimuli was performed via qRT-PCR, and the results showed that both genes are stress-induced genes. Based on the bioinformatics analysis of CmRNF5 and CmNPH3L, we hypothesized that CmRNF5 and CmNPH3L may trigger a compatibility/incompatibility response between the two graft partners that constitutes a compatible/incompatible combination via the ABA and auxin signal transduction pathways, respectively.

Extrauterine placenta-perfusing fetal incubation of midgestational rabbits: a pilot study

We evaluated the effect of TiO2 nanoparticles (NPs) on cold tolerance (CT) development in two chickpea (Cicer arietinum L.) genotypes (Sel96Th11439, cold tolerant, and ILC533, cold susceptible) by using cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique during the first and sixth days of cold stress (CS) at 4 °C. Selective amplification by primer combinations generated 4200 transcript-derived fragments (TDFs) while 100 of them (2.62%) were differentially expressed. During CS, 60 differentially expressed TDFs of TiO2 NPs-treated plants were cloned and 10 of them produced successfully readable sequences. These data represented different groups of genes involved in metabolism pathways, cellular defense, cell connections and signaling, transcriptional regulation and chromatin architecture. Two out of 10 TDFs were unknown genes with uncharacterized functions or sequences without homology to known ones. The network-based analysis showed a gene-gene relationship in response to CS. Quantitative reverse-transcriptase polymerase chain reaction (qPCR) confirmed differential expression of identified genes (six out of 10 TDFs) with potential functions in CT and showed similar patterns with cDNA-AFLP results. An increase in transcription level of these TDFs, particularly on the first day of CS, was crucial for developing CT through decreasing electrolyte leakage index (ELI) content in tolerant plants compared to susceptible ones, as well as in TiO2 NPs-treated plants compared to control ones. It could also indicate probable role of TiO2 NPs against CS-induced oxidative stress. Therefore, a new application of TiO2 NPs in CT development is suggested for preventing or controlling the damages in field conditions and increasing crop productivity.

Differential expression analysis of genes related to graft union healing in Pyrus ussuriensis Maxim by cDNA-AFLP

The purpose of the present study was to understand the molecular mechanisms during the graft union healing process. In the present study, cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis was used to examine gene expression in Pyrus ussuriensis Maxim micro-grafting plantlets at four time points (at 0, 4, 7, and 11days) during grafting tissue healing process. Twenty-six transcript-derived fragments (TDFs) involved in the graft healing mechanism were successfully obtained, which comprised 14 genes of known function, 9 genes of unknown function, and 3 novel genes. These 14 genes of known functions were involved in the functions of kinase, carbohydrate metabolism, protein metabolism, nuclear metabolism, cell activity and development, energy, and others. The expression analysis of 3 TDFs by qPCR showed that the results analyzed by cDNA-AFLP were basically reliable. These results suggest that P. ussuriensis undergoes complex metabolic processes during the graft union healing process. Detailed characterization of the genes involved in the specific process can help us uncover the molecular mechanisms for the healing process in plants.