Subtractive Library Research Articles

Wide transcriptional outlook to uncover Penicillium expansum genes underlying fungal incompatible infection

Pathogenesis of P. expansum involved different processes and one of them is the recognition between pathogen-host, which in the case of P. expansum is preferably pome fruit. In this work, the possible mechanisms connected to host recognition are addressed through the generation of a subtractive library carried out during the incompatible P. expansum-orange interaction in the initial stages of infection. The generated library was analyzed by massive sequencing and bioinformatic analysis. Of the identified genes, a total of 24 were selected for subsequent expression analysis by RT-qPCR in two incompatible interaction situations. The characterization of the overexpressed genes revealed the presence of CWDEs, ATPases, aldolases, detoxifying enzymes and virulent determinants that could act as effectors related to fungal virulence independently of the host. However, several identified genes, which could not be associated with the virulence of P. expansum under compatible conditions, were related to enzymes to obtain the nutrients necessary for the growth and development of the pathogen under stress conditions through basal metabolism that contributes to expand the range of adaptation of the pathogen to the environment and different hosts.

INSM1 expression in neuroendocrine tumors in a tertiary care hospital.

Neuroendocrine tumors are heterogenous group of neoplasms that includes benign and malignant tumors that originate from neuroendocrine or nonneuroendocrine organs. Insulinoma-associated protein 1 (INSM1) is a zinc finger transcription factor originally isolated from subtraction library of human insulinoma. The main aim was to study the INSM1 expression in a spectrum of neuroendocrine tumors and a limited spectrum of nonneuroendocrine tumors. A total of 100 cases of which 57 neuroendocrine neoplasms and 43 nonneuroendocrine neoplasms were included in the study. Immunohistochemistry (IHC) was done and expression patterns of INSM1 were analyzed. Pituitary adenoma, medullary carcinoma of thyroid, pheochromocytoma lung, gastrointestinal, and pancreatic neuroendocrine tumors were the neuroendocrine tumors that were included in the study. Papillary carcinoma of thyroid, gastrointestinal adenocarcinoma, lung adenocarcinoma, and squamous cell carcinoma were the nonneuroendocrine tumors that were included in the study. Depending upon the tissue availability, comparison of INSM1 with synaptophysin and chromogranin was also done in few neuroendocrine tumors. All the 57 neuroendocrine tumors showed positive expression for INSM1 and all the nonneuroendocrine tumors were negative for INSM1. This study is statistically significant. Our study suggests that INSM1 is a diagnostic marker for neuroendocrine tumors with high degree of sensitivity and specificity. The study is significant and suggests that INSM1- IHC shows nuclear positivity in a spectrum of neuroendocrine tumors. Being a nuclear marker, interpretation is easy and more reliable than the cytoplasmic markers. INSM1 has a stronger positivity than synaptophysin and chromogranin in the present study especially for small cell carcinoma lung. Hence, INSM1 may be included in the routine panel for neuroendocrine tumors along with synaptophysin and chromogranin.

Redox homeostasis at SAM: a new role of HINT protein.

ScHINT1 was identified at sugarcane SAM using subtractive libraries. Here, by bioinformatic tools, two-hybrid approach, and biochemical assays, we proposed that its role might be associated to control redox homeostasis. Such control is important for plant development and flowering transition, and this is ensured with some protein partners such as PAL and SBT that interact with ScHINT1. The shoot apical meristem transition from vegetative to reproductive is a crucial step for plants. In sugarcane (Saccharum spp.), this process is not well known, and it has an important impact on production due to field reduction. In view of this, ScHINT1 (Sugarcane HISTIDINE TRIAD NUCLEOTIDE-BINDING PROTEIN) was identified previously by subtractive cDNA libraries using Shoot Apical Meristem (SAM) by our group. This protein is a member of the HIT superfamily that was composed of hydrolase with an AMP site ligation. To better understand the role of ScHINT1 in sugarcane flowering, here its function in SAM was characterized using different approaches such as bioinformatics, two-hybrid assays, transgenic plants, and biochemical assays. ScHINT1 was conserved in plants, and it was grouped into four clades (HINT1, HINT2, HINT3, and HINT4). The 3D model proposed that ScHINT1 might be active as it was able to ligate to AMP subtract. Moreover, the two-hybrid approach identified two protein interactions: subtilase and phenylalanine ammonia-lyase. The evolutionary tree highlighted the relationships that each sequence has with specific subfamilies and different proteins. The 3D models constructed reveal structure conservation when compared with other PDB-related crystals, which indicates probable functional activity for the sugarcane models assessed. The interactome analysis showed a connection to different proteins that have antioxidative functions in apical meristems. Lastly, the transgenic plants with 35S::ScHINT1_AS (anti-sense orientation) produced more flowers than wild-type or 35S::ScHINT1_S (sense). Alpha-tocopherol and antioxidant enzymes measurement showed that their levels were higher in 35S::ScHINT_S plants than in 35S::ScHINT1_AS or wild-type plants. These results proposed that ScHINT1 might have an important role with other proteins in orchestrating this complex network for plant development and flowering.

Differential expression of iron transporters in Amaranthus cruentus roots when are subjected to salt stress: The influence of root endophytes

Salt-stress is one the main abiotic factors that negatively affect plant growth leading to crop losses throughout the world. Amaranth (Amaranthus L.) is a plant with outstanding agronomic properties and can grow under several environmental stresses including salinity. But, despite its agronomic qualities, molecular changes in plants subjected to salt stress are not well studied. Plant roots are the primary sensors responsible for sensing the rhizosphere conditions to adjust the adequate nutrient uptake for plant growth, and they also house endophytic microbes that play important roles in plant growth promotion. In this work, suppression subtractive hybridization libraries from amaranth roots growing under control conditions and in plants exposed to 150 mM NaCl were constructed. Identified differential expressed sequences tags (EST) were related with Fe, Mo, and amino acids transport, interestingly those EST showed high homology with bacterial sequences. Bacterial endophytes were isolated and identified by 16S ARNr gene sequencing. Under control conditions culturable diazotrophic endophytes were mainly composed by Pseudomonas and Variovorax, while under salt stress were Pseudomonas, Kosakonia, and Enterobacter. Most of the isolated bacteria were able to produce siderophores, solubilize phosphates, and showed the ability to grow under high NaCl conditions. These results open new venues on the relevance of amaranth endophytes on salt-stress tolerance.

ARNSHL gene identification: past, present and future

Autosomal recessive non-syndromic hearing loss (ARNSHL) is the most common hereditary deafness. It is genetically highly heterogeneous and about 89 gene loci and 76 gene's mutations have been implicated in the etiology of ARNSHL. Molecular basis of ARNSHL remains unresolved in 60% of cases and gene mutations are unknown for 23 of 89 reported loci. Techniques used to identify reported ARNSHL gene mutations can be divided into position-dependent and position-independent approaches. The localization of the loci has been facilitated by homozygosity mapping or linkage studies using STR or SNP genotyping in large consanguineous families. First few genes identified for hearing loss exhibited such wide diversity of function and expression patterns that candidate gene approach was not a viable option. The mapping of the disorder to a chromosomal location has been followed by Sanger sequencing of all genes in the target region or confining of the massively parallel sequencing data analyses to the linkage region. Sometimes genes located in the linkage interval were prioritized because there was a reported orthologs with mutations causing hearing loss in mouse or when mutations in the gene caused a related disorder. Position-independent approaches involving use of mouse subtractive cochlear libraries, forward genetic screening, and position-independent analyses of massively parallel sequencing data have helped identify 17 of 68 reported ARNSHL gene mutations. A thorough study of the strategies used in the identification of reported ARNSHL genes and of their relative success can help increase the success rate of future studies.

MaNCP1, a C2H2 Zinc Finger Protein, Governs the Conidiation Pattern Shift through Regulating the Reductive Pathway for Nitric Oxide Synthesis in the Filamentous Fungus Metarhizium acridum.

ABSTRACTAsexual sporulation is the most common reproduction mode of fungi. Most filamentous fungi have two conidiation patterns, normal conidiation and microcycle conidiation, which may be regulated by nutritional conditions. Nitrogen source can affect the fungal conidiation pattern, but the regulatory mechanism is not fully understood. In this study, we report a C2H2 zinc finger protein, MaNCP1, which has typical transcription factor characteristics and is screened from the subtractive library regulated by nitrate in the entomopathogenic fungus Metarhizium acridum. MaNCP1 and its N-terminal play critical roles in the conidiation pattern shift. Further study shows that MaNCP1 interacts with MaNmrA, which also contributes to the conidiation pattern shift and is involved in the reductive pathway of nitric oxide (NO) synthesis. Intriguingly, the conidiation pattern of the MaNCP1-disruption strain (ΔMaNCP1) can be restored to microcycle conidiation when grown on the microcycle conidiation medium, SYA, supplemented with NO donor or overexpressing MaNmrA in ΔMaNCP1. Here, we reveal that MaNCP1 governs the conidiation pattern shift through regulating the reductive synthesis of NO by physically targeting MaNmrA in M. acridum. This work provides new mechanistic insights into how changes in nitrogen utilization are linked to the regulation of fungal morphological changes.IMPORTANCE Fungal conidia play important roles in the response to environmental stimuli and evasion of the host immune system. The nitrogen source is one of the main factors affecting shifts in fungal conidiation patterns, but the regulatory mechanism involved is not fully understood. In this work, we report that the C2H2 zinc finger protein, MaNCP1, governs the conidiation pattern shift in M. acridum by targeting the MaNmrA gene, thereby altering the regulation of the reductive pathway for NO synthesis. This work provides further insights into how the nutritional environment can regulate the morphogenesis of filamentous fungi.

Differentially Expressed Long-Term Salinity Responsive Sequences in Halophyte Suaeda maritima (L.) Dumort

Salinity is a major abiotic stress that affects plant growth and productivity. To cope with salt stress, plants express large number of salt responsive genes and proteins that are involved in a wide range of cellular functions. In the present study, halophytic plant Suaeda maritima (L.) Dumort. were hydroponically exposed to NaCl for understanding the molecular mechanisms behind salinity tolerance using PCR-based Suppressive Subtractive Hybridization (SSH). Two cDNA subtraction libraries were constructed between Suaeda maritima X Sesuvium portulacastrum and Suaeda maritima X Salicornia brachiata to identify differentially expressed genes from leaves exposed to 200mM NaCl treatment for 14 days. A total of 224 clones from both libraries were assembled into 109 unique-ESTs grouped into different functional categories. Based on GO functional annotation, the expressed sequences like Oxygen-evolving enhancer protein1, AARF domain-containing kinase protein, V-type proton ATPase subunitd2, RMD5 homologA, and ABC transporter G35 that are involved in photosynthesis, cellular transport, cell rescue and defense, polyubiquitination and secondary metabolism played a significant role implying a complex response to salt in S. maritima. This is the first report that SSH could facilitate screening across species and family specific identification of salt responsive genes provides insight into biological mechanisms underlying salinity response.

Deep sequencing of suppression subtractive library identifies differentially expressed transcripts of Saccharum spontaneum exposed to salinity stress

Saccharum spontaneum, a wild relative of sugarcane, is highly tolerant to drought and salinity. The exploitation of germplasm resources for salinity tolerance is a major thrust area in India. In this study, we utilized suppression subtractive hybridization (SSH) followed by sequencing for the identification of upregulated transcripts during salinity stress in S. spontaneum clones coming from different geographical regions of India. Our sequencing of the SSH library revealed that 95% of the transformants contained inserts of size 200-1500 bp. We have identified 314 differentially expressed transcripts in the salinity-treated samples after subtraction, which were subsequently validated by quantitative real-time polymerase chain reaction. Functional annotation and pathway analysis revealed that the upregulated transcripts were a result of protein modifications, stress, and hormone signaling along with cell wall development and lignification. The prominently upregulated transcripts included UDP glucose dehydrogenase, cellulose synthase, ribulose, cellulose synthase COBRA, leucine-rich protein, NAC domain protein, pectin esterase, ABA-responsive element binding factor 1, and heat stress protein. Our results is a step forward the understanding of the molecular response of S. spontaneum under salinity stress, which will lead to the identification of genes and transcription factors as novel targets for salinity tolerance in sugarcane.

Real-time performance evaluation of BGSLibrary algorithms for intelligent surveillance

<p>Background subtraction is the first and basic stage in video analysis and smart surveillance to extract moving objects. In fact, the background subtraction library (BGSLibrary) was created by Andrews Sobral in 2012, which currently combines 43 background subtraction algorithms from the most popular and widely used in the field of video analysis. Each algorithm has its own characteristics, strengths and weaknesses in extracting moving objects. The evaluation allows the identification of these characteristics and helps researchers to design the best methods. Unfortunately, the literature lacks a comprehensive evaluation of the algorithms included in the library. Accordingly, the present work will evaluate these algorithms in the BGSLibrary through the segmentation performance, execution time and processor, so as to, achieve a perfect, comprehensive, real-time evaluation of the system. Indeed, a background modeling challenge (BMC) dataset was selected using the synthetic video with the presence of noise. Results are presented in tables, columns and foreground masks.</p>

Probing the Energetic Metabolism of Resting Cysts under Different Conditions from Molecular and Physiological Perspectives in the Harmful Algal Blooms-Forming Dinoflagellate Scrippsiella trochoidea.

Energetic metabolism is essential in maintaining the viability of all organisms. Resting cysts play important roles in the ecology of dinoflagellates, particularly for harmful algal blooms (HABs)-causative species. However, the energetic metabolism underlying the germination potency maintenance of resting cysts of dinoflagellate have been extremely scarce in studies from physiological and, particularly, molecular perspectives. Therefore, we used the cosmopolitan Scrippsiella trochoidea as a representative of HABs-forming and cyst-producing dinoflagellates in this work to obtain novel insights into the molecular mechanisms, regulating the energetic metabolism in dinoflagellate resting cysts, under different physical condition. As the starting step, we established a cDNA subtractive library via suppression subtractive hybridization (SSH) technology, from which we screened an incomplete sequence for the β subunit of ATP synthase gene (β-F1-ATPase), a key indicator for the status of cell’s energetic metabolism. The full-length cDNA of β-F1-ATPase gene from S.trochoidea (Stβ-F1-ATPase) was then obtained via rapid amplification of cDNA ends (RACE) (Accession: MZ343333). Our real-time qPCR detections, in vegetative cells and resting cysts treated with different physical conditions, revealed that (1) the expression of Stβ-F1-ATPase in resting cysts was generally much lower than that in vegetative cells, and (2) the Stβ-F1-ATPase expressions in the resting cysts under darkness, lowered temperature, and anoxia, and during an extended duration of dormancy, were significantly lower than that in cysts under the condition normally used for culture-maintaining (a 12 h light:12 h dark cycle, 21 °C, aerobic, and newly harvested). Our detections of the viability (via Neutral Red staining) and cellular ATP content of resting cysts, at the conditions corresponding to the abovementioned treatments, showed that both the viability and ATP content decreased rapidly within 12 h and then maintained at low levels within the 4-day experimentation under all the three conditions applied (4 °C, darkness, and anoxia), which are well in accordance with the measurements of the transcription of Stβ-F1-ATPase. These results demonstrated that the energy consumption of resting cysts reaches a low, but somehow stable, level within a short time period and is lower at low temperature, darkness, and anoxia than that at ambient temperature. Our work provides an important basis for explaining that resting cysts survive long-term darkness and low temperature in marine sediments from molecular and physiological levels.

HaYABBY Gene Is Associated with the Floral Development of Ligulate-Like Tubular Petal Mutant Plants of Sunflower

As an ornamental organ, petal plays an important role in attracting insect pollination, it’s development attracts the attention of many researchers. In this study, a sunflower mutant line with ligulate-like tubular petal (lpm) was screened out, and its disc tubular florets were characterized with elongated ligulate-like petals, longer filaments size, fewer anthers and lower seed setting rate, compared with wide type (WT) plants. From the suppression subtractive hybridization library constructed using inflorescence of lpm and WT plants, the HaYABBY was identified with expressing differentially. The cDNA and structural sequences of HaYABBY gene were 997 and 4339 bp, respectively, encoding a peptide with 219 amino acids. The analysis of HaYABBY expression showed that it presented higher transcription level in flower than root, stem and leaf of sunflower. The expression level of YABBY in disc florets of lpm was significantly higher than those of WT, especially at early floral developmental stage. The results suggested that HaYABBY could impact the floral development at early stage, particularly on petal elongation and floral symmetry morphogenesis in sunflower. Furthermore, the results implied that HaYABBY would be involved in the regulation of reproductive system development and affect their fertility. Our findings will provide some insight to understand the regulating network of floral development in sunflower, and help to cultivate new sunflower varieties with high ornamental value.

Monoclonal antibody specific to the Dia blood group antigen generated by phage display technology.

Alloanti-Dia can be implicated in mild to severe blood transfusion reactions. Given the concomitance of a high prevalence of the Dia antigen and antibody circulating in some populations, an anti-Dia typing reagent is required in order to enable safe blood transfusions. Limitations of hybridoma technology to produce such a reagent led to the use of phage display technology to generate an anti-Dia monoclonal antibody. A library of phages displaying murine single-chain variable fragment antibody (scFv-phages) was consecutively adsorbed with different panels of Di(a-b+) red cells to eliminate scFc-phages that potentially bind irrelevant blood group antigens. Thereafter, the subtractive library was specifically selected for the scFv-phages that bound Dia antigen by sequentially biopanning the library with Di(a+b+) cell ghosts and Di(a+b-) intact red cells. A specific interaction between the selected scFv-phages and Dia epitope was validated with the Dia peptide by a competitive haemagglutination inhibition assay and confirmed with the red cells by flow cytometry. An scFv-phage clone specifically bound the Dia epitope, as shown by its binding competition with the human anti-Dia to the Dia peptide in a haemagglutination inhibition test. Moreover, it was highly reactive to Di(a+b+) red cells but not to Di(a-b+) red cells, as determined by flow cytometry. In this study, a Dia-specific scFv-phage antibody was successfully produced. The selection protocol might be a prototypic platform for producing monoclonal antibodies to relevant blood group antigens. The scFv-phage produced in this way warrants further development for use as a reagent for Dia red cell typing.

LreEF1A4, a Translation Elongation Factor from Lilium regale, Is Pivotal for Cucumber Mosaic Virus and Tobacco Rattle Virus Infections and Tolerance to Salt and Drought.

Eukaryotic translation elongation factors are implicated in protein synthesis across different living organisms, but their biological functions in the pathogenesis of cucumber mosaic virus (CMV) and tobacco rattle virus (TRV) infections are poorly understood. Here, we isolated and characterized a cDNA clone, LreEF1A4, encoding the alpha subunit of elongation factor 1, from a CMV-elicited suppression subtractive hybridization library of Lilium regale. The infection tests using CMV remarkably increased transcript abundance of LreEF1A4; however, it also led to inconsistent expression profiles of three other LreEF1A homologs (LreEF1A1–3). Protein modelling analysis revealed that the amino acid substitutions among four LreEF1As may not affect their enzymatic functions. LreEF1A4 was ectopically overexpressed in petunia (Petunia hybrida), and transgenic plants exhibited delayed leaf and flower senescence, concomitant with increased transcription of photosynthesis-related genes and reduced expression of senescence-associated genes, respectively. A compromised resistance to CMV and TRV infections was found in transgenic petunia plants overexpressing LreEF1A4, whereas its overexpression resulted in an enhanced tolerance to salt and drought stresses. Taken together, our data demonstrate that LreEF1A4 functions as a positive regulator in viral multiplication and plant adaption to high salinity and dehydration.

Identification of salt-responsive genes from C4 halophyte Suaeda nudiflora through suppression subtractive hybridization and expression analysis under individual and combined treatment of salt and elevated carbon dioxide conditions

Salinization of soil is a prime abiotic stress that limits agriculture productivity worldwide. To Study the mechanisms that halophytes take up to survive under high salt condition is important in engineering salinity stress tolerance in sensitive species. Suaeda nudiflora is a halophyte plant that grows in the saline environment and extreme high tidal belt. The species have high capability to produce high protein biomass in salty soils due to C4 photosynthesis. The physiological and biochemical changes in S. nudiflora under salinity stress were studied by measuring chlorophyll content, electrolytic leakage, level of lipid peroxidation and total soluble sugars. Increased lipid peroxidation and electrolytic leakage was observed in salt stressed S. nudiflora compared to control plants. A suppression subtractive hybridization strategy was employed to identify differentially expressed genes under salt treatment in S. nudiflora. A total of 333 positive clones were identified and screened. Of these, 250 expressed sequence tags were identified. cDNA subtraction library resulted in 33 contigs and 138 singletons. The functional annotation and metabolic pathways identification were performed using the Blast2GO program. In addition, we analyzed the expression patterns of 18 genes associated with salt stress-responsive pathways by semi-quantitative PCR under salt and elevated carbon dioxide (CO2) conditions. Several of the analyzed genes showed an increase in expression levels under different time points of salt treatment and at different concentrations of salt. When the same genes were studied for its expression under elevated CO2 concentrations, most of the known salt responsive genes showed higher expression under the combined treatment of elevated CO2 concentrations (500ppm) and NaCl treatment (200mM) compare to ambient condition. This implies that salt responsive genes are enhanced at elevated CO2 concentrations.

Insulinoma-Associated-1: From Neuroendocrine Tumor Marker to Cancer Therapeutics.

Insulinoma-associated-1 (IA-1 or INSM1) encodes a zinc-finger transcription factor, which was isolated from a human insulinoma subtraction library, with specific expression patterns, predominantly in developing neuroendocrine tissues and tumors. INSM1 is key in early pancreatic endocrine, sympatho-adrenal lineage, and pan-neurogenic precursor development. Insm1 gene ablation results in impairment of pancreatic β cells, catecholamine biosynthesis, and basal progenitor development during mammalian neocortex maturation. Recently, INSM1 has emerged as a superior, sensitive, and specific biomarker for neuroendocrine tumors. INSM1 regulates downstream target genes and exhibits extranuclear activities associated with multiple signaling pathways, including Sonic Hedgehog, PI3K/AKT, MEK/ERK1/2, ADK, p53, Wnt, histone acetylation, LSD1, cyclin D1, Ascl1, and N-myc. Novel strategies targeting INSM1-associated signaling pathways facilitate the suppression of neuroendocrine tumor growth. In addition, INSM1 promoter-driven reporter assay and/or suicide gene therapy are promising effective therapeutic approaches for targeted specific neuroendocrine tumor therapy. In this review, the current knowledge of the biological role of INSM1 as a neuroendocrine tumor biomarker is summarized, and novel strategies targeting multiple signaling pathways in the context of INSM1 expression in neuroendocrine tumors are further explored. IMPLICATIONS: Neuroendocrine transcription factor (INSM1) may serve as a neuroendocrine biomarker for the development of novel cancer therapeutics against neuroendocrine tumors.

Ectopic Expression of Cold Responsive LlaCIPK Gene Enhances Cold Stress Tolerance in Nicotiana tabacum.

Low-temperature stress severely affects the growth, development, and geographical distribution of various crop plants, resulting in significant economic loss to producers. In a quest to identify cold-regulated genes, we constructed a cDNA suppression subtractive library from a high altitude adapted ecotype of Lepidium. We cloned a cold-induced gene LlaCIPK from the subtracted cDNA library which gave homology to Arabidopsis CIPK15 gene. The predicted 3D structure of LlaCIPK protein also showed homology with Arabidopsis CIPK protein. Quantitative real-time PCR analysis in Lepidium seedlings exposed to 6 h of cold stress shows a 3-fold increase in the expression of LlaCIPK transcript. The expression of LlaCIPK was also differentially regulated by ethylene, CaCl2, ABA, and SA treatments. Ethylene and CaCl2 treatments up regulated LlaCIPK expression, whereas ABA and SA treatments down regulated the LlaCIPK expression. Transgenic plants overexpressing LlaCIPK gene under constitutive promoter show an increased level of proline and cell membrane stability. Taken together, our results suggest that the LlaCIPK contributes to the cold-response pathway in Lepidium plants.

MiR-451a suppressing BAP31 can inhibit proliferation and increase apoptosis through inducing ER stress in colorectal cancer

The global morbidity and mortality of colorectal cancer (CRC) are ranked the third among gastrointestinal tumors in the world. MiR-451a is associated with several types of cancer, including CRC. However, the roles and mechanisms of miR-451a in CRC have not been elucidated. BAP31 is a predicted target gene of miR-451a in our suppression subtractive hybridization library. Its relationship with miR-451a and function in CRC are unclear. We hypothesized that miR-451a could induce apoptosis through suppressing BAP31 in CRC. Immunohistochemistry and real-time PCR were used to measure BAP31 expressions in CRC tissues and pericarcinous tissues from 57 CRC patients and CRC cell lines. Dual-luciferase reporter assay was used to detect the binding of miR-451a to BAP31. The expression of BAP31 protein in CRC tissues was significantly higher than that in pericarcinous tissues, which was correlated with distant metastasis and advanced clinical stages of CRC patients. The expression of BAP31 was higher in HCT116, HT29, SW620, and DLD cells than that in the normal colonic epithelial cell line NCM460. The expression of BAP31 was absolutely down-regulated when over-expressing miR-451a in HCT116 and SW620 cells compared with control cells. Mir-451a inhibited the expression of BAP31 by binding to its 5’-UTR. Over-expressing miR-451a or silencing BAP31 suppressed the proliferation and apoptosis of CRC cells by increasing the expressions of endoplasmic reticulum stress (ERS)-associated proteins, including GRP78/BIP, BAX, and PERK/elF2α/ATF4/CHOP, which resulted in increased ERS, cytoplasmic calcium ion flowing, and apoptosis of CRC cells. These changes resulting from over-expressing miR-451a were reversed by over-expressing BAP31 with mutated miR-451a-binding sites. Over-expressing miR-451a or silencing BAP31 inhibited tumor growth by inducing ERS. The present study demonstrated that miR-451a can inhibit proliferation and increase apoptosis through inducing ERS by binding to the 5’-UTR of BAP31 in CRC.

A CkDREB1 gene isolated from Caragana korshinskii Kom. enhances Arabidopsis drought and cold tolerance

Caragana korshinskii Kom., an arbuscular legume with important economic and ecological value in feed, processing industry, and environmental protection, also has great tolerance potential to abiotic stress conditions. An AP2 domain-containing gene was isolated from the suppression subtractive hybridization library of C. korshinskii under drought stress. In addition, the isolated gene was also found to be responsive to cold and ABA treatment. Phylogenetic analysis indicates that the deduced protein belongs to the DREB A-1 subfamily and is designated as CkDREB1. Overexpression of CkDREB1 in Arabidopsis thaliana (L.) Heynh increased drought and cold tolerance compared with the wild type. The drought responsive genes RD29A, RD29B, KIN1, and KIN2, as well as cold-responsive marker genes COR15A and COR47, were also highly induced in the overexpression lines under drought and cold conditions. These results should shed light on our understanding on the mechanisms of abiotic resistance of C. korshinskii.

PK-profiling method for identifying the expression of resistance-associated genes in partially resistant oats to crown rust

BackgroundProtein kinases play a key role in plant cell homeostasis and the activation of defense mechanisms. Partial resistance to fungi in plants is interesting because of its durability. However, the variable number of minor loci associated with this type of resistance hampers the reliable identification of the full range of genes involved. The present work reports the technique of protein kinase (PK)-profiling for the identification of the PK genes induced in the partially resistant oats line MN841801–1 following exposure to the fungus Puccinia coronata. This is the first time this technique has been used with cDNA (complementary DNA) from a suppression subtractive hybridization library obtained after the hybridization of cDNAs from inoculated and mock-inoculated plants.ResultsSix degenerate primers based on the conserved domains of protein kinases were used in a PK-profiling assay including cDNA from mock-inoculated leaves and subtracted cDNA. Of the 75.7% of sequences cloned and sequenced that showed significant similarity to resistance genes, 76% were found to code for PKs. Translation and ClustalW2 alignment of each sequence cloned with the complete sequences of the most similar B. distachyon PKs allowed those of the partially resistant oat line to be deduced and characterized. Further, a phylogenetic study carried out after alignment of these B. distachyon PK sequences with the most similar protein sequences of related species also allowed to deduce different functions for the PK cloned. RT-qPCR (Reverse Transcription-quantitative PCR) was analyzed on nine representative sequences to validate the reliability of the employed PK-profiling method as a tool for identifying the expression of resistance-associated genes.ConclusionsPK-profiling would appear to be a useful tool for the identification of the PKs expressed in oats after challenge by P. coronata, and perhaps other pathogens. Most of the PKs studied are related to receptor-like protein kinases expressed shortly after infection. This is in agreement with previous studies indicating a close relationship between partial resistance and the first layer of defense against pathogen used by plants.

Overexpression of TaMADS from wheat promotes flowering by upregulating expression of floral promoters and provides protection against thermal stress

MADS-box genes belong to a large family of transcription factors. They are known to play key role in flower development and organ differentiation in plants. In this study an attempt was made to characterize a type II (MIKC) TaMADS identified through subtractive hybridization heat stress library of Triticum aestivum (CPAN1676). Overexpression of TaMADS resulted in early flowering under both long day and short day conditions as well as after cold treatment. This early flowering has been attributed to the upregulation of FCA and LD, both of which are floral promoters within the autonomous pathway. Expression analysis suggests the possible role of TaMADS in both heat stress and recovery. Moreover, heat stress induces a temporal and tissue specific regulation. The overexpression of TaMADS conferred increased tolerance in terms of growth and yield of transgenics under heat stress conditions. The expression studies revealed the additional role of TaMADS in various abiotic stress conditions. Conclusively, the data suggests that TaMADS is heat inducible and functions as a floral promoter.