Suppression Subtractive Hybridization Library Research Articles

Identification of Differential-Expressed Genes in Banana-Biostimulant Interaction Using Suppression Subtractive Hybridization

The use of environmental-friendly agricultural inputs to increase yield in crops could reduce environmental contamination and alleviate (a)biotic stress. In this study, we applied a foliar liquid biostimulant to banana plants, which was developed from the anaerobic fermentation of microorganisms with organic remains (manure and plant waste) and minerals to use as environmental-friendly product. To determine the effect of this biostimulant in the plants, a Suppression Subtractive Hybridization (SSH) library was developed from banana leaves in greenhouse conditions. Furthermore, salicylic and jasmonic pathways activation was evaluated by gene expression analysis. First, the application of the biostimulant to banana leaves increased the membrane stability index (MSI), which is directly related to a higher CO2 exchange and assimilation rate. Additionally, the total chlorophyll content increased in plants in comparison with the control plants. In addition, the SSH analysis revealed around 300 expressed sequence tags (ESTs). Several induced genes associated with different molecular pathways, including photosynthesis, anthocyanins production, and plant defense, among others, were encountered after bioinformatic analysis. Upregulated gene expression was demonstrated in different time points, after biostimulant application. Furthermore, induced expression of genes related to the jasmonic acid pathway was observed, which may prove beneficial to the plant under stress conditions.

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.

Efficient Anchoring of Erianthus arundinaceus Chromatin Introgressed into Sugarcane by Specific Molecular Markers

Erianthus arundinaceus is a valuable gene reservoir for sugarcane improvement. However, insufficient molecular markers for high-accuracy identification and tracking of the introgression status of E. arundinaceus chromatin impede sugarcane breeding. Fortunately, suppression subtractive hybridization (SSH) technology provides an excellent opportunity for the development of high-throughput E. arundinaceus-specific molecular markers at a reasonable cost. In this study, we constructed a SSH library of E. arundinaceus. In total, 288 clones of E. arundinaceus-specific repetitive sequences were screened out and their distribution patterns on chromosomes were characterized by fluorescence in situ hybridization (FISH). A subtelomeric repetitive sequence Ea086 and a diffusive repetitive sequence Ea009, plus 45S rDNA-bearing E. arundinaceus chromosome repetitive sequence EaITS were developed as E. arundinaceus-specific molecular markers, namely, Ea086-128, Ea009-257, and EaITS-278, covering all the E. arundinaceus chromosomes for high-accuracy identification of putative progeny. Both Ea086-128 and Ea009-257 were successfully applied to identify the authenticity of F1, BC1, BC2, BC3, and BC4 progeny between sugarcane and E. arundinaceus. In addition, EaITS-278 was a 45S rDNA-bearing E. arundinaceus chromosome-specific molecular marker for rapid tracking of the inherited status of this chromosome in a sugarcane background. Three BC3 progeny had apparently lost the 45S rDNA-bearing E. arundinaceus chromosome. We reported herein a highly effective and reliable SSH-based technology for discovery of high-throughput E. arundinaceus-specific sequences bearing high potential as molecular markers. Given its reliability and savings in time and efforts, the method is also suitable for development of species-specific molecular markers for other important wild relatives to accelerate introgression of wild relatives into sugarcane.

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.

Combined physiological responses and differential expression of drought-responsive genes preliminarily explain the drought resistance mechanism of Lotus corniculatus.

Lotus corniculatus L. is a perennial high-quality legume forage species but is vulnerable to drought, and water deficit reduces productivity. To understand the drought response mechanism of L. corniculatus , we investigated physiological responses under drought stress and constructed suppression subtractive hybridisation (SSH) cDNA libraries to isolate drought-inducible genes and quantitatively study the expression levels of candidate drought- responsive genes. Genes encoding calmodulin-like protein, mitogen-activated protein kinase, indole-3-acetic acid-induced protein, ser/thr-protein phosphatase homolog-related proteins, and β -galactosidase-related protein with hydrolysis activity were isolated and considered the main factors that explained the resistance of L. corniculatus to drought. Approximately 632 expressed sequence tags (ESTs) were identified and confirmed in the constructed SSH library. The Gene Ontology (GO) analysis revealed that these genes were involved mainly in transcription processes, protein synthesis, material metabolism, catalytic reactions, sugar metabolism, and photosynthesis. The interaction between the functions of these drought-related genes and the physiological responses preliminarily explains the drought resistance mechanisms of L. corniculatus .

SSH reveals different functional categories of putative resin synthesizing genes in the industrial insect, Kerria lacca (Kerr)

• SSH library was constructed by using crawler and female cDNA as driver and tester, respectively. • Genes of terpene biosynthesis, fatty acid biosynthesis and transcriptional regulation were present in the SSH library. • Decaprenyl diphosphate synthase and acyl-CoA delta desaturase were cloned from lac insects. • Their expression levels in lac insects were also found to be correlated with resin synthesis. Biosynthesis mechanism of the only animal resin, lac resin remains elusive to the scientific community. An attempt to identify the genes of resin biosynthesis in lac insects was made through SSH (Suppression Subtraction Hybridization) library by subtracting genes of resin non secreting stage (neonate nymphs) from the resin secreting stage (adult female) of the lac insect. SSH library yielded 107 non redundant clones with 61 clones having sequences homologous with known functions (defence, development, metabolism, regulation, transport, cell division, respiration, structural role), 7 clones having hypothetical proteins or uncharacterized proteins and 39 clones did not find any matches. Among the differentially expressed genes, few putative genes of terpene biosynthesis, fatty acid biosynthesis and transcriptional regulation were identified. Out of them, an IDS (Isopentenyl Diphosphate Synthase) type decaprenyl diphosphate synthase and a desaturase gene, acyl-CoA delta desaturase were cloned from lac insects and sequenced. Their expression levels were found to be more in lac resin synthesis stages such as settled nymphs and fertilized female insects in comparison with resin non secreting stage, crawlers (neonate nymphs). It gives a clear indication of their role in lac resin biosynthesis due to the correlation of their expression level with resin biosynthesis in lac insects.

Shrimp Vago5 activates an innate immune defense upon bacterial infection

Acute hepatopancreatic necrosis disease, AHPND, caused by a specific Vibrio parahaemolyticus (VPAHPND) strain, results in a great loss of global shrimp production. This study performed suppression subtractive hybridization (SSH) to identify differentially expressed genes from white shrimp Penaeus vannamei hemocyte upon VPAHPND infection. Among the immune-related genes identified, Vago5, kunitz, secretory leukocyte proteinase inhibitor, and profilin are the most abundant genes classified as the up-regulated genes in the SSH library. The qRT-PCR results show that only Vago5 was highly up-regulated at 3 and 6 h post-VPAHPND challenge, whereas kunitz, secretory leukocyte proteinase inhibitor, and profilin were highly up-regulated at 48 h post-VPAHPND challenge. As an early VPAHPND infection-responsive gene, Vago5 was further functional characterized by RNA interference. Knockdown of Vago5 gene resulted in the significantly rapid increase of shrimp mortality and the number of bacteria in the stomach and hepatopancreas upon VPAHPND infection. Moreover, downstream genes of Toll, IMD, and JAK/STAT pathways and phenoloxidase system were analyzed for the expression in the VPAHPND-infected shrimp hemocyte after dsVago5 treatment. Vago5 gene knockdown resulted in a significant decrease in transcript levels of PEN4, TNF, and PO2 genes as well as PO activity in the hemolymph, suggesting that Vago5 might modulate antibacterial infection through activation of the genes in the NF-κB mediated pathways, JAK/STAT pathway, and phenoloxidase system.

Identification of differentially expressed genes associated with precocious puberty by suppression subtractive hybridization in goat pituitary tissues

The aim of this study was to identify genes related to precocious puberty expressed in the pituitary of goats at different growth stages by suppression subtractive hybridization (SSH). The pituitary glands from Jining Gray (JG) goats (early puberty) and Liaoning Cashmere (LC) goats (late puberty) at 30, 90, and 180 days were used in this study. To identify differentially expressed genes (DEGs) in the pituitary glands, mRNA was extracted from these tissues, and SSH libraries were constructed and divided into the following groups: juvenile group (30-JG vs. 30-LC, API), puberty group (90-JG vs. 180-LC, BPI), and control group (90-JG vs. 90-LC, EPI). A total of 60, 49, and 58 DEGs were annotated by 222 Gene Ontology (GO) terms and 75 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Most of the DEGs were significantly enriched in GO terms related to ‘structural constituent of ribosome’, ‘translation’ and ‘GTP binding’, and numerous DEGs were also significantly enriched in KEGG terms related to the Jak-STAT signaling and oocyte meiosis pathways. Candidate genes associated with precocious puberty and sexual development were screened from the SSH libraries. These genes were analyzed to determine if they were expressed in the pituitary tissues of the goats at different growth stages and to identify genes that may influence the hypothalamic-pituitary-gonadal (HPG) axis. In this study, we found precocious puberty-related genes (such as PRLP0, EIF5A, and YWHAH) that may be interesting from an evolutionary perspective and that could be investigated for use in future goat breeding programs. Our results provide a valuable dataset that will facilitate further research into the reproductive biology of goats.

Jasmonic acid and ethylene signaling pathways participate in the defense response of Chinese cabbage to Pectobacterium carotovorum infection

Chinese cabbage (Brassica rapa subsp. pekinensis) suffers from soft rot disease caused by Pectobacterium carotovorum (Pc). To uncover the mechanisms underlying the defense response of Chinese cabbage to Pc, we constructed a suppression subtractive hybridization (SSH) library from Pc-infected cabbage and obtained 1 919 non-redundant expressed sequence tags (ESTs), which were used for cDNA microarray. We detected 800 differentially expressed genes (DEGs) in cabbage at different time points post-Pc inoculation, which were further confirmed by quantitative real-time PCR. One quarter of these DEGs were involved in the biotic stress pathways visualized by MapMan. Among them, 8, 8, 1, 3, and 2 DEGs were related to jasmonic acid (JA), ethylene (ET), JA+ET, auxin, and abscisic acid (ABA) signaling pathways, respectively, while no DEG was detected for salicylic acid (SA) signaling. Assessment of phytohormone production in the Pc-infected leaves showed that JA and ET production was increased, while SA production was decreased. Treatment with JA, methyl jasmonate (MeJA), the ET precursor 1-aminocyclopropane-1-carboxylate (ACC), or combinations thereof, reduced the disease severity, and the JA and JA+ACC treatments were superior and performed equally well. Our findings suggest that JA and ET may act synergistically against Pc infection in Chinese cabbage, and JA-mediated signaling might be the most significant.

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.

Novel ASR isolated from drought stress responsive SSH library in pearl millet confers multiple abiotic stress tolerance in PgASR3 transgenic Arabidopsis

A genomic resource of drought stress responsive genes/ESTs was generated using Suppression Subtractive Hybridization (SSH) approach in a drought stress tolerant Pennisetum glaucum genotype 841B. Fifty five days old plants were subjected to drought stress after withholding water for different time intervals (10 days, 15 days, 20 days and 25 days). A forward subtractive cDNA library was prepared from isolated RNA of leaf tissue. Differential gene expression under drought stress was validated for selected nine contigs by RT-qPCR. A transcript homologous to Setaria italica ASR3 upregulated under drought stress was isolated from genotype 841B and characterized. Heterologous expression of PgASR3 was validated in Arabidopsis and confirmed under multiple abiotic stress conditions. A total of four independent transgenic lines overexpressing gene PgASR3 were analyzed by Southern blot at T1 stage. For drought stress tolerance, three independent lines (T2 stage) were analyzed by biochemical and physiological assays at seedling stage. The growth rate (shoot and root length) of transgenic seedlings improved as compared to WT seedling under differenct abiotic stress conditions. The three transgenic lines were also validated for drought stress tolerance and RT-qPCR analysis, at maturity stage. Under drought stress conditions, the mature transgenic lines showed higher levels of RWC, chlorophyll and proline but lower levels of MDA as compared to WT plants. PgASR3 gene isolated and validated in this study can be utilized for developing abiotic stress tolerant crops.

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.

Regulation of vitellogenin gene expression under the negative modulator, gonad-inhibiting hormone in Penaeus monodon

Vitellogenesis is a principal process during ovarian maturation in crustaceans. This process is negatively regulated by gonad-inhibiting hormone (GIH), a neuronal peptide hormone from eyestalks. However, the detailed mechanism through which GIH regulates Vg expression is still ambiguous. In this study, suppression subtractive hybridization (SSH) under specific GIH-knockdown condition was utilized to determine the expression of genes in the ovary that may act downstream of GIH to control vitellogenin synthesis in Penaeus monodon. The total of 102 and 82 positive clones of up-regulated and down-regulated genes in GIH- knockdown shrimp were identified from the forward and reverse SSH libraries, respectively. Determination of the expression profiles of these reproduction-related genes during ovarian development revealed that the expression of calreticulin (CALR) was significantly reduced in vitellogenic ovary suggesting its role in vitellogenesis. Suppression of CALR by specific dsRNA showed elevated vitellogenin (Vg) transcript level in the ovary at day 7 post-dsRNA injection. Since CALR can bind to steroid hormone receptors and prevents the binding of the receptor to its responsive element to regulate gene expression, it is possible that CALR is an inhibitory mediator of vitellogenin synthesis via steroidal pathway. Our results posted a possible novel pathway of GIH signaling that might interfere the steroid signaling cascade to mediate Vg synthesis in the shrimp.

Transcript profiling of chickpea pod wall revealed the expression of floral homeotic gene AGAMOUS-like X2 (CaAGLX2).

The differentially expressed genes in the chickpea pod wall have been identified for the first time using a forward suppression subtractive hybridization (SSH) library. In all, 226 clones of SSH library were sequenced and analyzed. A total of 179 high-quality expressed sequence tags (ESTs) were generated and based on the CAP3 assembly of these ESTs, 126 genes (97 singletons and 29 contigs) were computationally annotated. The mapping of 88.26% ESTs by gene ontology (GO) annotation distributed them into 751 GO terms of three categories, cellular location, molecular function, and biological process. The KEGG pathway analysis revealed 45 ESTs are involved in 49 different biological pathways. Also, 67 ESTs encodes four different classes of enzymes such as oxidoreductases (29), transferase (20), hydrolases (16) and isomerase (2). Six genes were selected and subjected to qPCR analysis, of these, two genes (FHG Floral homeotic AGAMOUS-like isoform X2, MADS1 MADS-box transcription factor) showed significant up-regulation in the pod wall compared to leaves. Surprisingly, one of the MADS1 box gene, FHG (CaAGLX2), responsible for flower development expressed in the pod wall. Therefore, understanding its specific role in the pod wall could be interesting. Thus, the transcript dynamics of the chickpea pod wall revealed differentially expressed genes in the pod wall, which may be participating in the metabolic build-up of both pod wall and seeds.

Towards sex identification of Asian Palmyra palm (Borassus flabellifer L.) by DNA fingerprinting, suppression subtractive hybridization and de novo transcriptome sequencing.

BackgroundAsian Palmyra palm, the source of palm-sugar, is dioecious with a long juvenile period requiring at least 12 years to reach its maturity. To date, there is no reliable molecular marker for identifying sexes before the first bloom, limiting crop designs and utilization. We aimed to identify sex-linked markers for this palm using PCR-based DNA fingerprinting, suppression subtractive hybridization (SSH) and transcriptome sequencing.MethodsDNA fingerprints were generated between males and females based on RAPD, AFLP, SCoT, modified SCoT, ILP, and SSR techniques. Large-scale cloning and screening of SSH libraries and de novo transcriptome sequencing of male and female cDNA from inflorescences were performed to identify sex-specific genes for developing sex-linked markers.ResultsThrough extensive screening and re-testing of the DNA fingerprints (up to 1,204 primer pairs) and transcripts from SSH (>10,000 clones) and transcriptome data, however, no sex-linked marker was identified. Although de novo transcriptome sequencing of male and female inflorescences provided ∼32 million reads and 187,083 assembled transcripts, PCR analysis of selected sex-highly represented transcripts did not yield any sex-linked marker. This result may suggest the complexity and small sex-determining region of the Asian Palmyra palm. To this end, we provide the first global transcripts of male and female inflorescences of Asian Palmyra palm. Interestingly, sequence annotation revealed a large proportion of transcripts related to sucrose metabolism, which corresponds to the sucrose-rich sap produced in the inflorescences, and these transcripts will be useful for further understanding of sucrose production in sugar crop plants. Provided lists of sex-specific and differential-expressed transcripts would be beneficial to the further study of sexual development and sex-linked markers in palms and related species.

Identification and characterization of catalase genes in Eleusine coracanaunder abiotic stresses

Reactive oxygen species (ROS) are byproducts of metabolic processes such as respiration and photosynthesis in plants. Production of ROS leads to rapid cell damage, and plants developed a complex system of enzymatic and non-enzymatic antioxidants to scavenge these ROS. Catalase is an important enzyme, which plays a key role in elimination of toxic effects of hydrogen peroxide and plays a major role as an antioxidant. When characterizing heat responsive genes in finger millet (Eleusine coracana L.) using a suppression subtractive hybridization (SSH) library, we isolated two catalase genes and named them as EcCATA1 and EcCATB1. The lengths of the EcCATA1 and EcCATB1 open reading frames were 1 482 and 1 426 bp, respectively. We characterized these genes under different abiotic stresses and in different tissues. The tissue wise expression revealed that EcCATA1 expression was higher in leaves whereas EcCATB1 expression was higher in roots than in other organs. Under stress conditions, the expression of EcCATA1 was highest under salt stress followed by mannitol treatment. In the case of EcCATB1, the highest expression was observed under mannitol treatment followed by cold and dehydration. We also studied expression of both the genes under heat stress in different finger millet genotypes and observed that expressions of these genes can be correlated with heat tolerance. For both the genes, a detailed computational investigation was also performed for understanding their structural properties and physicochemical characteristics. Overall, this is the first study to identify and characterize catalase genes from climate resilient finger millet crop.

Regulation of conidiation and antagonistic properties of the soil-borne plant beneficial fungus Trichoderma virens by a novel proline-, glycine-, tyrosine-rich protein and a GPI-anchored cell wall protein.

Trichoderma spp. are widely used as commercial biofungicides, and most commercial formulations are conidia based. Identification of genes that regulate conidiation would thus be of help in genetic reprogramming of these species to optimize sporulation. In this study, we constructed an SSH (suppression subtractive hybridization) library from RNA samples of the wild type strain and a non-conidiating mutant, M7, grown under constant illumination for 2 days. We identified several genes that are underexpressed in the mutant. Some of these genes are related to secondary metabolism, and a few could be associated with conidiation. Genes coding for the following proteins, among others, were identified: O-methyl transferase, ATPase, alpha/beta-hydrolase, WD repeat containing protein, dehydrogenase, thioesterase, translationally controlled tumour protein, and a proline-glycine-tyrosine-rich protein (PGYRP) that has been annotated in T.reesei as a signalling protein. Two of these genes, encoding Pgy1, a novel PGYRP, and Ecm33, a GPI-anchored cell wall protein, were further studied in detail by generation of deletion mutants. We demonstrate here that both these genes not only regulate radial growth and conidiation in Trichoderma virens, but are also involved in antagonism against soil-borne wide host range plant pathogens. Furthermore, deletion of ecm33 affected hydrophobicity and cell wall integrity.

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.