Subtractive Hybridization Research Articles

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 .

Surface Quality and Mechanical Properties of 316L Stainless Steel Manufactured by Powder Feeding Laser Additive and Milling Subtractive Hybrid Manufacturing

Surface Quality and Mechanical Properties of 316L Stainless Steel Manufactured by Powder Feeding Laser Additive and Milling Subtractive Hybrid Manufacturing

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.

SARI inhibits growth and reduces survival of oral squamous cell carcinomas (OSCC) by inducing endoplasmic reticulum stress

AimsSARI (suppressor of activator protein (AP)-1, regulated by interferon (IFN) was identified as a novel tumor suppressor by applying subtraction hybridization to terminally differentiating human melanoma cells. The anti-tumor activity of SARI and the correlation between expression and cancer aggression and metastasis has been examined in multiple cancers, but its potential role in oral squamous cell carcinomas (OSCC) has not been explored. MethodsSARI expression was monitored in tumor tissues of OSCC patients by performing immunohistochemistry. Ectopic expression of SARI was achieved using a replication defective adenovirus expressing SARI (Ad.SARI). A nude mouse xenograft model was used to evaluate the in vivo efficacy of SARI. Endoplasmic reticulum (ER) stress was monitored in SARI infected OSCC cells by confocal microscopy. Key findingIn this study, we demonstrate that SARI expression is significantly lower in OSCC tumor tissue as compared to normal adjacent tissue. Ectopic expression of SARI induces cancer-specific cell death in human OSCC cell lines and in a paclitaxel plus cisplatin non-responder OSCC patient-derived (PDC1) cell line. Mechanistically, SARI inhibits zinc finger protein GLI1 expression through induction of endoplasmic reticulum (ER) stress. Using a nude mouse xenograft model, we show that intratumoral injections of Ad.SARI significantly reduce PDC1 tumor burden, whereas treatment with an ER stress inhibitor efficiently rescues tumors from growth inhibition. SignificanceOverall, our data provides a link between induction of ER stress and inhibition of the GLI1/Hedgehog signaling pathway and the tumor suppressive activity of SARI in the context of OSCC.

A Novel Deoxynivalenol-Activated Wheat Arl6ip4 Gene Encodes an Antifungal Peptide with Deoxynivalenol Affinity and Protects Plants against Fusarium Pathogens and Mycotoxins.

Deoxynivalenol (DON) is one of the most widespread trichothecene mycotoxins in contaminated cereal products. DON plays a vital role in the pathogenesis of Fusarium graminearum, but the molecular mechanisms of DON underlying Fusarium–wheat interactions are not yet well understood. In this study, a novel wheat ADP-ribosylation factor-like protein 6-interacting protein 4 gene, TaArl6ip4, was identified from DON-treated wheat suspension cells by suppression subtractive hybridization (SSH). The qRT-PCR result suggested that TaArl6ip4 expression is specifically activated by DON in both the Fusarium intermediate susceptible wheat cultivar Zhengmai9023 and the Fusarium resistant cultivar Sumai3. The transient expression results of the TaARL6IP4::GFP fusion protein indicate that TaArl6ip4 encodes a plasma membrane and nucleus-localized protein. Multiple sequence alignment using microscale thermophoresis showed that TaARL6IP4 comprises a conserved DON binding motif, 67HXXXG71, and exhibits DON affinity with a dissociation constant (KD) of 91 ± 2.6 µM. Moreover, TaARL6IP4 exhibited antifungal activity with IC50 values of 22 ± 1.5 µM and 25 ± 2.6 µM against Fusarium graminearum and Alternaria alternata, respectively. Furthermore, TaArl6ip4 interacted with the plasma membrane of Fusarium graminearum spores, resulting in membrane disruption and the leakage of cytoplasmic materials. The heterologous over-expression of TaArl6ip4 conferred greater DON tolerance and Fusarium resistance in Arabidopsis. Finally, we describe a novel DON-induced wheat gene, TaArl6ip4, exhibiting antifungal function and DON affinity that may play a key role in Fusarium–wheat interactions.

A novel method of generating RNAi libraries for high‐throughput gene function analysis of creeping bentgrass

AbstractRNA interference (RNAi) is a powerful tool for gene function analysis, particularly for outcrossing polyploid species where simultaneous mutations of multiple alleles of a gene with similar but nonidentical sequences may not be readily achieved with the novel clustered regularly interspaced short palindromic repeats/Cas9 gene editing tools. The most effective RNAi strategy in plants is to use long hairpin RNAi (lhRNAi) constructs containing inverted repeats (IRs) of target genes. Previously, we used a Phi29‐Amplified RNAi Construct method to create a single lhRNAi construct that was shown to be highly effective in gene silencing in the model grass Brachypodium distachyon (L.) P.Beauv. Here, we describe a method of creating a library of lhRNAi constructs ready for high‐throughput gene function analysis in creeping bentgrass (Agrostis stolonifera L.), an important turfgrass species. A cDNA population in which salt‐responsive genes were enriched by suppression subtractive hybridization was used as the source of IRs. Sequencing of 13 randomly selected colonies from the lhRNAi library showed that the size of IRs varied from 184 to 994 bp, which is appropriate for RNAi, and nine of the IRs were shown to be homologous to known genes in closely related grasses. In addition to the demonstration of the proof‐of‐concept of creating an expression‐ready library, the salt stress‐specific lhRNAi library represents a valuable resource for studying the functions of salt‐responsive genes in creeping bentgrass. The method described here is broadly applicable to all plant species where an efficient plant transformation platform is available.

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.

Deformation Prediction and Experimental Study of 316L Stainless Steel Thin-Walled Parts Processed by Additive-Subtractive Hybrid Manufacturing.

The hybrid process integrates two or more different processes, such as additive and subtractive manufacturing, which have gained appreciable consideration in recent years. The deformation of hybrid manufacturing is an essential factor affecting machining quality. The purpose of this paper is to study the effect of milling on stress release and surface deformation of additive manufacturing (AM) specimens in the process of additive and subtractive hybrid manufacturing (ASHM) of 316L stainless steel thin-walled parts, so as to effectively improve the forming quality of thin-walled parts manufactured by the combined processing of ASHM. To this end, a series of experiments were carried out to study the relationship between stress distribution and thermal stress deformation of 316L stainless steel thin-walled parts prepared by LMD, and the changes of stress and deformation of these thin-walled parts after subsequent milling. An infrared camera and laser distance sensor were used to record the temperature field data and deformation data to analyze the influence factors of temperature and stress on the machining results. Then, the finite element software was used to simulate the stress and deformation of the thin-walled parts in the additive manufacturing process and the subsequent milling process. Meanwhile, the model was verified through the experiments. In addition, the relationship between the milling force and the milling parameters of the AM parts was studied by orthogonal test and regression analysis.

Tomato chlorosis virus infection represses chloroplast related genes in tomato.

Tomato chlorosis virus (ToCV) causes mild to severe chlorosis on tomato leaves. Although it has been postulated that chlorosis is caused by changes in biosynthesis, degradation, function and/or structure of chlorophyll, mechanisms of virus-induced chlorosis has not been explored to date. To understand the mechanisms of virus-induced chlorosis, ToCV-tomato interaction was studied at transcriptome level using suppression subtractive hybridization (SSH) followed by macroarray hybridization and RT-qPCR. First, a susceptible tomato cultivar, MoneyMaker was graft-inoculated with ToCV. Next, cDNAs were synthesized from mRNAs of leaf samples from ToCV-inoculated and mock-inoculated control plants at 30-day of post inoculation (dpi). Then, a cDNA library enriched with ToCV-repressed genes was constructed using SSH. 250 randomly selected clones from library were sequenced and the expressions of 140 genes were determined by nylon macroarray hybridizations. The expression of 119 genes were significantly decreased by 1.5 or greater fold in response to ToCV infection. The expressions of many genes involved in transcription, defense response, metabolism, and photosynthesis, signaling and protein fate were repressed upon infection with ToCV. The genes repressed by ToCV in tomato were mostly associated with organelles, especially with the chloroplast. After expression analysis of 40 selected ToCV-repressed genes by RT-qPCR assay, the repression of 37 genes in response to ToCV infection was confirmed. Furthermore, a detail expression analysis of ferredoxin 2 (Fd2) gene between 0 dpi and 45 dpi in ToCV-inoculated and mock-inoculated control plants showed that the expression of Fd-2 gene was significantly repressed at 8 to 30 dpi in ToCV-infected plants.

Advances in identification methods of alien genomic components in plants

Plants with alien genomic components (alien chromosomes / chromosomal fragments / genes) are important materials for genomic research and crop improvement. To date, four strategies based on trait observation, chromosome analysis, specific proteins, and DNA sequences have been developed for the identification of alien genomic components. Among them, DNA sequence-based molecular markers are mainly used to identify alien genomic components. This review summarized several molecular markers for identification of alien genomic components in wheat, cabbage and other important crops. We also compared the characteristics of nine common molecular markers, such as simple sequence repeat (SSR), insertion-deletion (InDel) and single nucleotide polymorphism (SNP). In general, the accuracy of using a combination of different identification methods is higher than using a single identification method. We analyzed the application of different combination of identification methods, and provided the best combination for wheat, brassica and other crops. High-throughput detection can be easily achieved by using the new generation molecular markers such as InDel and SNP, which can be used to determine the precise localization of alien introgression genes. To increase the identification efficiency, other new identification methods, such as microarray comparative genomic hybridization (array-CGH) and suppression subtractive hybridization (SSH), may also be included.

Synbindin Downregulation Participates in Slit Diaphragm Dysfunction

Introduction: Synbindin, originally identified as a neuronal cytoplasmic molecule, was found in glomeruli. The cDNA subtractive hybridization technique showed the mRNA expression of synbindin in glomeruli was downregulated in puromycin aminonucleoside (PAN) nephropathy, a mimic of minimal-change nephrotic syndrome. Methods: The expression of synbindin in podocytes was analyzed in normal rats and 2 types of rat nephrotic models, anti-nephrin antibody-induced nephropathy, a pure slit diaphragm injury model, and PAN nephropathy, by immunohistochemical analysis and RT-PCR techniques. To elucidate the function of synbindin, a gene silencing study with human cultured podocytes was performed. Results: Synbindin was mainly expressed at the slit diaphragm area of glomerular epithelial cells (podocytes). In both nephrotic models, decreased mRNA expression and the altered staining of synbindin were already detected at the early phase when proteinuria and the altered staining of nephrin, a key molecule of slit diaphragm, were not detected yet. Synbindin staining was clearly reduced when severe proteinuria was observed. When the cultured podocytes were treated with siRNA for synbindin, the cell changed to a round shape, and filamentous actin structure was clearly altered. The expression of ephrin-B1, a transmembrane protein at slit diaphragm, was clearly lowered, and synaptic vesicle-associated protein 2B (SV2B) was upregulated in the synbindin knockdown cells. Conclusion: Synbindin participates in maintaining foot processes and slit diaphragm as a downstream molecule of SV2B-mediated vesicle transport. Synbindin downregulation participates in slit diaphragm dysfunction. Synbindin can be an early marker to detect podocyte injury.

Hybrid manufactured dynamometer for cutting force measurement

This paper describes a two-step (additive + subtractive) hybrid manufacturing approach for a laser powder bed fusion 316L stainless steel constrained-motion cutting force dynamometer (PBF CMD). The purpose of the research is to produce a near-net shape metallic dynamometer via additive manufacturing (AM) with post-processing by machining and wire-EDM to achieve the desired surface finish and performance. A cutting force comparison is presented with a commercially-available dynamometer.

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.

Overexpression of differentially expressed AhCytb6 gene during plant-microbe interaction improves tolerance to N2 deficit and salt stress in transgenic tobacco

Stenotrophomonas maltophilia has plant growth-promoting potential, and interaction with Arachis hypogaea changes host-plant physiology, biochemistry, and metabolomics, which provides tolerance under the N2 starvation conditions. About 226 suppression subtractive hybridization clones were obtained from plant-microbe interaction, of which, about 62% of gene sequences were uncharacterized, whereas 23% of sequences were involved in photosynthesis. An uncharacterized SSH clone, SM409 (full-length sequence showed resemblance with Cytb6), showed about 4-fold upregulation during the interaction was transformed to tobacco for functional validation. Overexpression of the AhCytb6 gene enhanced the seed germination efficiency and plant growth under N2 deficit and salt stress conditions compared to wild-type and vector control plants. Results confirmed that transgenic lines maintained high photosynthesis and protected plants from reactive oxygen species buildup during stress conditions. Microarray-based whole-transcript expression of host plants showed that out of 272,410 genes, 8704 and 24,409 genes were significantly (p < 0.05) differentially expressed (> 2 up or down-regulated) under N2 starvation and salt stress conditions, respectively. The differentially expressed genes belonged to different regulatory pathways. Overall, results suggested that overexpression of AhCytb6 regulates the expression of various genes to enhance plant growth under N2 deficit and abiotic stress conditions by modulating plant physiology.

Cloning and physical localization of male-biased repetitive DNA sequences in Spinacia oleracea (Amaranthaceae)

Spinach (Spinacia oleracea Linnaeus, 1753) is an ideal material for studying molecular mechanisms of early-stage sex chromosome evolution in dioecious plants. Degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) technique facilitates the retrotransposon-relevant studies by enriching specific repetitive DNA sequences from a micro-dissected single chromosome. We conducted genomic subtractive hybridization to screen sex-biased DNA sequences by using the DOP-PCR amplification products of micro-dissected spinach Y chromosome. The screening yielded 55 male-biased DNA sequences with 30 576 bp in length, of which, 32 DNA sequences (12 049 bp) contained repeat DNA sequences, including LTR/Copia, LTR/Gypsy, simple repeats, and DNA/CMC-EnSpm. Among these repetitive DNA sequences, four DNA sequences that contained a fragment of Ty3-gypsy retrotransposons (SP73, SP75, SP76, and SP77) were selected as fluorescence probes to hybridization on male and female spinach karyotypes. Fluorescence in situ hybridization (FISH) signals of SP73 and SP75 were captured mostly on the centromeres and their surrounding area for each homolog. Hybridization signals primarily appeared near the putative centromeres for each homologous chromosome pair by using SP76 and SP77 probes for FISH, and sporadic signals existed on the long arms. Results can be served as a basis to study the function of repetitive DNA sequences in sex chromosome evolution in spinach.

Cloning and physical localization of male-biased repetitive DNA sequences in Spinacia oleracea (Amaranthaceae).

Spinach (Spinacia oleracea Linnaeus, 1753) is an ideal material for studying molecular mechanisms of early-stage sex chromosome evolution in dioecious plants. Degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) technique facilitates the retrotransposon-relevant studies by enriching specific repetitive DNA sequences from a micro-dissected single chromosome. We conducted genomic subtractive hybridization to screen sex-biased DNA sequences by using the DOP-PCR amplification products of micro-dissected spinach Y chromosome. The screening yielded 55 male-biased DNA sequences with 30 576 bp in length, of which, 32 DNA sequences (12 049 bp) contained repeat DNA sequences, including LTR/Copia, LTR/Gypsy, simple repeats, and DNA/CMC-EnSpm. Among these repetitive DNA sequences, four DNA sequences that contained a fragment of Ty3-gypsy retrotransposons (SP73, SP75, SP76, and SP77) were selected as fluorescence probes to hybridization on male and female spinach karyotypes. Fluorescence in situ hybridization (FISH) signals of SP73 and SP75 were captured mostly on the centromeres and their surrounding area for each homolog. Hybridization signals primarily appeared near the putative centromeres for each homologous chromosome pair by using SP76 and SP77 probes for FISH, and sporadic signals existed on the long arms. Results can be served as a basis to study the function of repetitive DNA sequences in sex chromosome evolution in spinach.

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.

Overexpression of an expansin-like gene, GhEXLB2 enhanced drought tolerance in cotton

Expansins are nonenzymatic cell wall proteins that play significant role in plant development as well as stress responses. Hereby, an expansin-like gene, GhEXLB2 was isolated from a cotton (Gossypium hirsutum L.) protoplast with suppression subtractive hybridization to characterize and study its responses against abiotic stresses. GhEXLB2 is the cell-wall localized protein. The expression of GhEXLB2 level was significantly high under polyethylene glycol and salt treatments. GhEXLB2 was further characterized in vitro by cloning and transformation into cotton. Cotton plants overexpressing GhEXLB2 showed enhanced drought tolerance at germination, seedling and flowering stages. After polyethylene glycol (PEG) treatment at germination stage, the length of main root and hypocotyl of overexpressing lines was significantly longer than YZ1 (wild type) and RNAi lines. In addition, H2O2 and malondialdehyde (MDA) contents were lower, while superoxide dismutase (SOD) and peroxidase (POD) activity was detected higher in overexpressing seedlings. On the other hand, higher SOD and POD activity was detected in overexpressing lines than WT plants in soil. In addition, water use efficiency (WUE), soluble sugar, and chlorophyll contents were also significantly greater in overexpressing plants. The present study revealed that GhEXLB2 play crucial role in enhancing drought resistivity in cotton.

Ecotoxicogenomic analysis of zebrafish embryos exposed to triclosan and mixture triclosan and methyl triclosan using suppression subtractive hybridization and next-generation sequencing

Triclosan (TCS) and methyl-triclosan (MTCS), an environmental transformation product of biocide of TCS, have been detected in water, sediment, fish, and invertebrates. In this study, the key pathway perturbation in zebrafish (Danio rerio) embryos exposed to TCS (300 μg/L) and TCS/MTCS mixture (300 μg/L TCS + 30 μg/L MTCS) was assessed by integrating the metabolomic and transcriptomic dysregulation. The differential expressed genes (DEGs) were obtained from the subtracted cDNA libraries by using the suppression subtractive hybridization and next-generation sequencing approach. The dysregulation of twenty-eight GO terms and four KEGG pathways, including oxidative phosphorylation and cardiac muscle contraction, were shown in the TCS treatment group, indicating that TCS could disrupt the mitochondrial inner membrane function by downshifting the electrochemical gradient. Meanwhile, the addition of MTCS in the exposure would cause fourteen additional significant KEGG pathway changes, demonstrating the different effects between two exposure. A pathway-based analysis using the identified DEGs and the altered metabolites in zebrafish embryos treated with TCS and TCS/MTCS mixture, collectively, has been applied. This study demonstrated that the integration of SSH-NGS and metabolomics could reveal toxic effects and potential diseases associated with the exposures of TCS and MTCS in aquatic environments.