Suppression Subtractive Hybridization cDNA Library Research Articles

Osmopriming-associated genes in Poincianella pyramidalis

Osmopriming of seeds can increase seed vigour, allowing faster germination and field emergence, especially under adverse field conditions. The restricted imbibition during priming treatments reactivates metabolism although germination will be prevented. In order to identify and characterize genes that are involved in the improvement of vigour of Poincianella pyramidalis (Catingueira) seeds upon priming, we produced two different Suppression Subtractive Hybridization (SSH) cDNA libraries. These were a Forward and Reverse subtraction of cDNA samples isolated from untreated dry seeds (unprimed) and osmoprimed seeds. The two different libraries were sequenced by Illumina GAII next generation sequencing, resulting in almost 20 million reads that could be assigned to 5298 different contigs. Of these, 999 were only found in the unprimed seeds library and 2711 were specific for osmoprimed seeds. The contigs were annotated and subjected to gene set enrichment analysis and differential expression of several genes was confirmed by qRT-PCR analysis. The identified differentially expressed genes might play an important role in vigour improvement upon priming and may be potential markers for tolerance to water stress in P. pyramidalis seeds.

Identification of Haemaphysalis longicornis Genes Differentially Expressed in Response to Babesia microti Infection.

Haemaphysalis longicornis is a tick and a vector of various pathogens, including the human pathogenetic Babesia microti. The objective of this study was to identify female H. longicornis genes differentially expressed in response to infection with B. microti Gray strain by using a suppression subtractive hybridization (SSH) procedure. A total of 302 randomly selected clones were sequenced and analyzed in the forward subtracted SSH cDNA library related to Babesia infection, and 110 clones in the reverse cDNA library. Gene ontology assignments and sequence analyses of tick sequences in the forward cDNA library showed that 14 genes were related to response to stimulus or/and immune system process, and 7 genes had the higher number of standardized sequences per kilobase (SPK). Subsequent real-time PCR detection showed that eight genes including those encoding for Obg-like ATPase 1 (ola1), Calreticulin (crt), vitellogenin 1 (Vg1) and Vg2 were up-regulated in fed ticks. Compared to uninfected ticks, infected ticks had six up-regulated genes, including ola1, crt and Vg2. Functional analysis of up-regulated genes in fed or Babesia-infected ticks by RNA interference showed that knockdown of crt and Vg2 in infected ticks and knockdown of ola1 in uninfected ticks accelerated engorgement. In contrast, Vg1 knockdown in infected ticks had delayed engorgement. Knockdown of crt and Vg1 in infected ticks decreased engorged female weight. Vg2 knockdown reduced B. microti infection levels by 51% when compared with controls. The results reported here increase our understanding of roles of H. longicornis genes in blood feeding and B. microti infection.

Identification of salt-induced transcripts by suppression subtractive hybridization and their expression analysis under the combination of salt and elevated CO2 conditions in Salicornia brachiata

Soil salinity is a major abiotic stress that affects global agricultural productivity. Exploring the mechanisms that halophytes employ to thrive and flourish under saline environments is essential to increase the salt tolerance in sensitive crop species. Of the three halophytes used in this study Salicornia brachiata and Suaeda maritima belong to the same family Chenopodiaceae, while Sesuvium portulacastrum, a mangrove-associated halophyte, belongs to the family Aizoaceae. Assuming that halophytes of same family share similar salt tolerance mechanisms, we generated a suppression subtractive hybridization (SSH1) cDNA library from salt-treated leaf tissues of S. brachiata as tester and that of S. maritima as driver to identify salt-responsive genes unique to S. brachiata. To elucidate the difference in salt-tolerance mechanisms, and to identify salt-tolerance mechanisms amongst different families of halophytes, SSH2 library was generated from salt-treated leaf tissue of S. brachiata as tester and that of S. portulacastrum as driver. Totally, 87 and 49 EST clones representing unique genes were obtained from SSH1 and SSH2 libraries, respectively. Examination of the expression patterns of 17 (SSH1) and 15 (SSH2) differentially expressed genes using semi-quantitative RT-PCR confirmed up-regulation of these genes in shoots in response to salt treatment and elevated CO2 condition, but to a different extent. This study has provided insights into the molecular responses of S. brachiata to salt stress and elevated CO2 conditions.

Integrated miRNA-mRNA analysis reveals regulatory pathways underlying the curly fleece trait in Chinese tan sheep

BackgroundTan sheep is an indigenous Chinese breed well known for its beautiful curly fleece. One prominent breed characteristic of this sheep breed is that the degree of curliness differs markedly between lambs and adults, but the molecular mechanisms regulating the shift are still not well understood. In this study, we identified 49 differentially expressed (DE) microRNAs (miRNAs) between Tan sheep at the two stages through miRNA-seq, and combined the data with that in our earlier Suppression Subtractive Hybridization cDNA (SSH) library study to elucidate the mechanisms underlying curly fleece formation.ResultsThirty-six potential miRNA-mRNA target pairs were identified using computational methods, including 25 DE miRNAs and 10 DE genes involved in the MAPK signaling pathway, steroid biosynthesis and metabolic pathways. With the differential expressions between lambs and adults confirmed by qRT-PCR, some miRNAs were already annotated in the genome, but some were novel miRNAs. Inhibition of KRT83 expression by miR-432 was confirmed by both gene knockdown with siRNA and overexpression, which was consistent with the miRNAs and targets prediction results.ConclusionOur study represents the comprehensive analysis of mRNA and miRNA in Tan sheep and offers detailed insight into the development of curly fleece as well as the potential mechanisms controlling curly hair formation in humans.

A novel white spot syndrome virus protein WSSV164 controls prophenoloxidases, PmproPOs in shrimp melanization cascade

Melanization, mediated by the prophenoloxidase (proPO)-activating system, is an important innate immune response in invertebrates. The implication of the proPO system in antiviral response and the suppression of host proPO activation by the viral protein have previously been demonstrated in shrimp. However, the molecular mechanism of viral-host interactions in the proPO cascade remains largely unexplored. Here, we characterized the viral protein, namely, WSSV164, which was initially identified from the forward suppression subtractive hybridization (SSH) cDNA library of the PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon that was challenged with white spot syndrome virus (WSSV). Using the yeast two-hybrid system, WSSV164 was found to interact with the PmproPO2 protein. The subsequent validation assay by co-immunoprecipitation revealed that WSSV164 directly bound to both PmproPO1 and PmproPO2. The gene silencing experiment was carried out to explore the role of WSSV164 in the control of the proPO pathway in shrimp, and the results showed that suppression of WSSV164 can restore PO activity in WSSV-infected shrimp hemolymph. The recombinant proteins of PmproPO1 and PmproPO2 were produced in Sf-9 cells and were shown to be successfully activated by exogenous trypsin and endogenous serine proteinases from shrimp hemocyte lysate supernatant (HLS), yielding PO activity in vitro. Moreover, the activated PO activity in shrimp HLS was dose-dependently reduced by the recombinant WSSV164 protein, suggesting that WSSV164 may interfere with the activation of the proPO system in shrimp. Taken together, these results suggest an alternative infection route of WSSV through the encoded viral protein WSSV164 that binds to the PmproPO1 and PmproPO2 proteins, interfering with the activation of the melanization cascade in shrimp.

Isolation and characterization of peroxiredoxin 1 gene of Dunaliella salina

Peroxiredoxin 1 (Prdx1) is a ubiquitously expressed protein in eukaryotic cells, and plays an important role in cell proliferation, differentiation, apoptosis, and redox signaling. Although Prdx1 has been better studied in yeasts and humans, only few Prdx1 genes have been cloned in green algae. The microalga Dunaliella salina (D. salina) is a model for the study of a variety of human cilia-related diseases. In this study, a suppression subtractive hybridization cDNA library of D. salina was constructed, and 6 flagellum-associated genes including D. salina Prdx1 (DsPrdx1) were isolated and identified. A 956bp full-length cDNA of DsPrdx1 was cloned using rapid amplification of cDNA end (RACE). The open reading frame (ORF) of this DNA sequence encodes a polypeptide of 201 amino acids with a predicted molecular weight of 22kDa and a theoretical isoelectric point (pI) of 5.27. Sequence comparison showed that Prdx1 is highly evolutionarily conserved from the unicellular green alga D. salina to human. To our knowledge, this is the first reported full-length sequence of Prdx1 in D. salina. Interestingly, the protein expression of DsPrdx1 was obviously increased during flagellar disassembly in D. salina. Additionally, a yeast two-hybrid assay showed interaction between Prdx1 and RNA, and suggested that DsPrdx1 can protect RNA from degradation by RNase. Taken together, DsPrdx1 not only participates in flagellar disassembly, but also protects RNA from degradation.

Effect of immune gene silencing in WSSV infected tiger shrimp Penaeus monodon

White spot syndrome virus, continues to cause huge economic loss to aquaculture industry. In the absence of effective therapeutics to control WSSV, it is important to understand the host pathogen interaction at the molecular level. Suppression subtractive hybridization (SSH) cDNA library was constructed which led to identification of several differentially expressed genes in response to WSSV infection in Penaeus monodon. The genes expressed in SSH cDNA library of shrimp gill and gut tissues belonged to a wide range of biological functions. The three differentially expressed genes, Single von Willebrand factor type C domain protein (pmSVC), P53 protein gene (pmP53) and ADP ribosylation factor (pmArf) were up-regulated against WSSV infection and were further characterized by gene silencing to study the role of these shrimp immune genes on WSSV multiplication. The sequence-specific knock down of pmSVC, pmP53 and pmArf using the dsRNA revealed that in pmSVC-dsRNA inoculated shrimps WSSV replication was more with increased viral copy numbers when compared with pmP53-dsRNA and pmArf -dsRNA inoculated shrimps. The varied response of immune genes to WSSV infection, indicated that host genes may either inhibit virus replication to some extent or might act as a target to facilitate viral pathogenesis.

Characteristics and Expression Profile of KRT71 Screened by Suppression Subtractive Hybridization cDNA Library in Curly Fleece Chinese Tan Sheep.

As an important commercial trait for sheep, curly fleece has a great economic impact on production costs and efficiency in sheep industry. To identify genes that are important for curly fleece formation in mammals, a suppression subtractive hybridization analysis was performed on the shoulder skin tissues exposed to two different growth stages of Chinese Tan sheep with different phenotypes (curly fleece and noncurling fleece). BLAST analysis identified 67 differentially expressed genes, of which 31 were expressed lower and 36 were expressed higher in lambs than in adult sheep. Differential expressions of seven randomly selected genes were verified using quantitative real-time polymerase chain reaction (qRT-PCR). KRT71 gene was selected for further study due to its high correlation with the curly hair phenotype in various mammal species. Semi-qPCR showed distinctively high expression of KRT71 in skin tissues. Moreover, qPCR result showed a significantly higher expression of KRT71 in curly fleece than noncurling Tan sheep. The luciferase assay and electrophoresis mobility shift assay showed that there were transcription factor binding sites in the promoter region of KRT71 related to the differential expression of KRT71 at the two growth stages of Tan sheep. Online bioinformation tools predicted MFZ1 as a transcriptional factor that regulates the expression of KRT71. These studies on KRT71 gene revealed some mechanisms underlying the relationship between the KRT71 gene and the curly fleece phenotype of Tan sheep.

Refinements to Hevea rubber breeding

Hevea brasiliensis (Willd. Ex. A. de. Juss. Muell-Arg.) is the prime source of natural rubber. Domestication of rubber began since 1876 with Wickham collecting 70,000 seeds from Upper Amazon and transported them to Kew Botanic Gardens. Somehow, rubber trees covering millions of hectares are believed to be derived from “22 seedlings” of Wickham’s original stock. Improving dry rubber yield is the exclusive and ultimate objective of Hevea breeding with consistent yield of 70 to 80 g/tree/tapping. Ultimately, in a small holding, a planter must gain an average yield of around 2200 to 2400 kg/ha from his stand (under optimal conditions), after accommodating tree-to-tree variations due to stock-scion interactions and soil heterogeneity. This is arduous, but achievable. Initial production of high-yielding clones gave 1600 kg/ha against 496 kg/ha of unselected seedlings. Adaptation and yielding potential of clones to specific environments are optimized through localized experimentation. Studies on adaptation of clones to new environments, especially to sub-optimal or marginal areas, are gaining momentum. As this extension happens, demand for new clones is on the rise. Possibilities of using rubber trees for reforestation, carbon sequestration and application of genomics in deriving climate resilient clones may come up in future, which breeders may have to take up with required priority. Five major methodologies followed are (a) primary clones and seed gardens, (b) derivation of recombinants and clone selection, (c) genetic analysis and variability management (d) early selection and estimation of genetic value and (e) application of genomics. Primary clones have immensely contributed in exploiting heterosis and production of new clones. For evaluation of recombinants, families are to be raised in closer spacing (2 or 3 m) and allowed to attain tappable girth for evaluation. While a normal breeding cycle takes 35 to 40 years, through skipping SSCTs and LSCTs, the scheme proposed can derive a clone in 17 years. Recent advances like transcriptome sequencing of bark and EST sequences generated from suppression subtractive hybridization-cDNA libraries could facilitate marker-assisted selection that could very well be used for selecting high-yielding genotypes at juvenile stage. Paternity identification can be done through breeding without breeding (BwB) in half-sibs and poly-cross (open pollinated) seedlings. Transcriptome studies have to come a long way to yield meaningful results to tag vivid genes responsible for QTLs, resistance and other quality traits, especially markers for cold/drought stress. It is opined that instead of subjecting plants for artificial cold/drought conditions, plants continuously exposed to such stress conditions must be used for analyses that can give a comprehensive indication of stress tolerance. The application of genotyping-by-sequencing (GBS) technique to simultaneously discover and delineate single nucleotide polymorphism (SNP) markers is a robust and cost-effective approach for generating a common set of genome-wide SNP data suitable for constructing integrated linkage maps from multiple populations. Studies on mitochondrial and chloroplast DNAs are welcome steps towards understanding ATP efficiency of accessions that need to be augmented further, so that clones with higher ATP efficiency can be used for breeding. Such innovative techniques shall govern breeding Hevea rubber in the future, only when breeders and genomic specialists are working in tandem.

Silencing of the SlNAP7 gene influences plastid development and lycopene accumulation in tomato.

Ripening is an important stage of fruit development. To screen the genes associated with pigment formation in tomato fruit, a suppression subtractive hybridization (SSH) cDNA library was constructed by using tomato fruit in the green ripe and break ripe stages, and 129 differential genes were obtained. Using redness as a screening marker, virus-induced gene silencing (VIGS) of the differential genes was performed with a sprout vacuum-infiltration system (SVI). The results showed that silencing the SlNAP7 gene affected the chloroplast development of tomato leaves, manifesting as a photo-bleaching phenotype, and silenced fruit significantly affected the accumulation of lycopene, manifested as a yellow phenotype. In our study, we found that silencing the SlNAP7 gene downregulates the expression of the POR and PORA genes and destroys the normal development of the chloroplast. The expression of related genes included in the lycopene biosynthesis pathway was not significantly changed, but lycopene accumulation was significantly reduced in tomato fruit. Perhaps it was caused by the destruction of the chromoplast, which leads to the oxidation of lycopene. The results show that the SlNAP7 gene influences chloroplast development and lycopene accumulation in tomato.

Screening and identification of genes associated with flower senescence in tree peony (Paeonia x suffruticosa Andrews) using suppression subtractive hybridization

ABSTRACTA short blossom period is a major obstacle for the tree peony industry. In order to preferentially isolate genes associated with flower senescence in the tree peony variety ‘Lu He Hong’, forward and reverse suppression subtractive hybridization cDNA libraries were constructed using senescent and blooming petals as testers, respectively. A total of 712 high-quality expressed sequence tags, representing 211 unigenes, were obtained, and 181 unigenes were annotated on the basis of sequence similarity with data in the National Center for Biotechnology Information’s non-redundant database. In addition, 168 unigenes were functionally annotated using Kyoto Encyclopedia of Genes and Genomes databases, and 44 unigenes were classified into 14 functional categories using the Clusters of Orthologous Groups. Most of the unigenes appear to be involved in cellular components, biological processes, and molecular functions, according to Gene Ontology analysis. Analysis of 23 chosen genes using quantitative reverse transcription PCR confirmed that their transcription levels were consistent with the results of the suppression subtractive hybridization libraries.

Branchial transcriptional responses of spotted scat, Scatophagus argus, to acute hypo-osmotic stress

The spotted scat (Scatophagus argus) is a euryhaline marine fish with the capability to withstand different salinity levels and can tolerate abrupt transfer from salt water (SW) to fresh water (FW) or vice versa. Differentially expressed genes were identified by constructing suppression subtractive hybridization (SSH) cDNA libraries to explore branchial osmoregulatory mechanisms affected by salinity challenge. After trimming and blasting, 105 uniquely expressed sequence tags were identified. Among them, 24 candidate genes involved in the stress response, metabolism, and the respiratory chain were chosen for further validation by real-time polymerase chain reaction. More than two-fold expression differences were observed in ADP-ribosylation factor 1 (arf1), cyclic AMP-responsive element-binding protein 3-like 4 (creb3l4), EVI5-like protein (evi5l), protocadherin fat 2 (fat2), transferrin receptor protein 2 (tfr2), C-X-C chemokine receptor type 4 (cxcr4), aquaporin-3 (aqp3), sodium/potassium-transporting ATPase subunit beta-233 (nka β233), serum and glucocorticoid-regulated kinase 1 (sgk1), and ras GTPase-activating-like protein IQGAP1 (iqgap1). Among these genes, aqp and nka are important osmoregulatory factors. AQP3 protein was observed being localized to the membranes of mitochondrion-rich cells (MRCs) and pillar cells of the gill of S. argus, and NKA β233 subunit isoform was only found in MRC membranes by immunostaining. Significant differences in aqp3 and nka β233 expression occurred within 24h after being transferring into the freshwater, and nka β233 expression level continued to highly upregulated 2 and 7days post-transfer (dpt). These results suggest that branchial aqp3 and nka β233 play important roles in response to hypo-osmotic stress in S. argus.

Alterations in gene expression during fasting-induced atresia of early secondary ovarian follicles of coho salmon, Oncorhynchus kisutch

Molecular processes that either regulate ovarian atresia or are consequences of atresia are poorly understood in teleost fishes. We hypothesized that feed restriction that perturbs normal ovarian growth and induces follicular atresia would alter ovarian gene expression patterns. Previtellogenic, two-year old coho salmon (Oncorhynchus kisutch) were subjected to prolonged fasting to induce atresia or maintained on a normal feeding schedule that would promote continued ovarian development. To identify genes that were specifically up- or down-regulated during oocyte growth in healthy, growing fish compared to fasted fish, reciprocal suppression subtractive hybridization (SSH) cDNA libraries were generated using ovaries from fed and fasted animals. Differential expression of genes identified by SSH was confirmed with quantitative PCR. The SSH library representing genes elevated in ovaries of fed fish relative to those of fasted fish contained steroidogenesis-related genes (e.g., hydroxy-delta-5-steroid dehydrogenase), Tgf-beta superfamily members (e.g., anti-Mullerian hormone) and cytoskeletal intermediate filament proteins (e.g., type I keratin s8). Overall, these genes were associated with steroid production, cell proliferation and differentiation, and ovarian epithelialization. The library representing genes elevated in ovaries of fasted fish relative to fed fish contained genes associated with apoptosis (e.g., programmed cell death protein 4), cortical alveoli (e.g., alveolin), the zona pellucida (e.g., zona pellucida protein c), and microtubules (e.g., microtubule associated protein tau). Elevated expression of this suite of genes was likely associated with the initiation of atresia and/or a reduced rate of follicle development in response to fasting. This study revealed ovarian genes involved in normal early secondary oocyte growth and potential early markers of atresia.

Development and mapping of functional expressed sequence tag-derived simple sequence repeat markers in a rubber tree RRIM600 × PB217 population

Sets of polymorphic expressed sequence tag–simple sequence repeat (EST-SSR) markers from the rubber tree (Hevea brasiliensis) have been published by many researchers, but none has been specifically developed to study latex and wood yield traits. In this study, a total 10,321 rubber tree EST sequences, generated from suppression subtractive hybridization-cDNA libraries of bark and latex of high- and low-yielding clones, were used as sources for SSR searching. A total of 432 EST-SSR loci were identified and it was possible to design primer pairs for a subset of 298 EST-SSRs. The highest proportion of EST-SSRs was represented by dinucleotide repeats (46.6 %), followed by trinucleotide repeats (44.3 %). Based on BLASTX analysis, 234 ESTs (80 %) showed similarity to genes in NCBI databases and could be divided into 120 putative proteins with known function and 114 unknown proteins. To enhance the resolution of an existing linkage map from previous work on a rubber tree RRIM600 × PB217 population, 69 EST-SSR markers from the above set were tested to be integrated into the reference genetic map. The enriched map of 18 linkage groups spanned 2054.2 cM in length, showed an average genetic distance of 4.3 cM between adjacent markers, and included 63 new EST-SSR markers. The enhanced map from this study provides a basis for comparative mapping using PCR-based markers and identification of expressed genes possibly affecting important traits of interest.

Cloning and characterization of metallothionein gene (HcMT) from Halostachys caspica and its expression in E. coli

Halostachys caspica is a short shrub distributed in the semi-arid and saline–alkali area, which evolved various mechanisms for modulating salt and metal level. In the present study, a Type 2 metallothionein (HcMT) gene was cloned from the salt induced suppression subtractive hybridization (SSH) cDNA library of H.caspica. Quantitative real time PCR (qRT-PCR) analysis indicated that HcMT gene was up-regulated under the stress of Cu2+, Zn2+ and Cd2+, and the tolerance of E. coli strain harboring with the recombinant HcMT (pET-32a-HcMT) to Cu2+, Zn2+ and Cd2+ was enhanced compared to strain with control vector (pET-32a). Moreover, the purified TrxA-HcMT fusion protein from E. coli cells grown in the presence of 0.3mM CuSO4, 0.3mM ZnSO4, or 0.1mM CdCl2 could bind more metal ions than TrxA alone. The predicted 3D structure showed that HcMT could form a single metal–thiolate cluster, which confers the ability to bind five divalent metal ions through fourteen cysteine residues. These data indicate that HcMT may be involved in processes of metal tolerance in H. caspica and could be employed as a potential candidate for heavy metal phytoremediation.

Profiling of Up-Regulated Genes Response to Acute Hypo-Osmotic Stress in Hepatopancreas and Gill of the Pacific White Shrimps (Litopenaeus vannamei)

<p class="1Body">Suppression subtractive hybridization (SSH) was applied to screen responsively up-regulation genes in hepatopancreas and gill of Litopenaeus vannamei induced by acute hypo-osmotic stress. In the hepatopancreas, 196 clones were randomly selected and sequenced. 131 non-redundant transcripts, corresponding to 41 genes, were found with elevated expressions. They were functionally clustered into eight biological processes which were protein synthesis and processing, carbohydrate metabolism and energy production, transport, cell growth, apoptosis, cytoskeletal component, cell defense and homeostasis, signal transduction, accounting for 26.2%, 14.3%, 16.7%, 4.7%, 4.7%, 2.3%, 11.9% and 7.1%, respectively. When confirmed by real time qRT-PCR, the gene expression levels of MnSOD, glutathione and glutamine synthetase showed significant increases (2.64-folds, 3.44-folds and 2.16-folds, respectively) in hepatopancreas (<em>P</em><0.05). Totally 81 expressed sequence tags (ESTs) were obtained by random sequencing from the gill SSH cDNA library, and 52 unigenes, including 15 contigs and 37 singlets, after CP3 online matching were gained by clustering and assembling. The sequence alignment results revealed that 30 of them had significant homology to previously identified Genbank database sequences, while 22 unigenes did not match any sequence in the datebase and presumably represented unidentified cDNAs. Functionally, the unigenes were classified into six clusters, including ion channels and transport proteins (1); protein synthesis, translation and transcription factor (2); stress resistance and antioxidant factors (3); energy metabolism (4); signal receptor and transduction (5); cell fibrin and cytoskeletal proteins (6) with proportion of 23.3%, 20.0%, 20.0%, 16.7%, 10.0% and 10.0% respectively. Real time qRT-PCR confirmation revealed significant elevation of Arginine kinase, Carbonic anhydrase and NaK-ATPase-α-subunit expressions (3.73-folds, 2.55-folds and 5.83-folds, respectively) in the gill after acute low-osmotic stress (P < 0.05). These results provided insight into critical physiology processes and pathways constituting the hypo-osmotic stress adaption program in hepatopancreas and gill of <em>L. vannamei</em>.</p>

Cloning and expression analysis of UpPDS gene in Ulva pertusa

Phytoene desaturase (PDS) is an essential constituent of several carotenoid biosynthetic enzymes, which catalyzes phytoene into ζ-carotenoids. Here, a cDNA encoding PDS gene named UpPDS in Ulva pertusa was cloned by the approaches of expressed sequence tag sequencing of suppression subtractive hybridization cDNA library and rapid amplification of cDNA ends technique. The results showed that the full-length cDNA of UpPDS was 2029 bp, containing 1599 bp open reading frame and encoding 532 amino acids. Homology analysis showed that the deduced UpPDS protein was highly homologous to the PDS proteins from some algal species. Phylogenetic analysis indicated that the UpPDS was clustered together with PDS of Ectocarpus siliculosus firstly and was more close to PDS proteins from Chlamydomonas reinhardtii and Volvox carteri. The RT-qPCR analysis indicated that the transcript level of UpPDS was upregulated during the period of emersion.

Analysis of genes that are differentially expressed during the Sclerotinia sclerotiorum-Phaseolus vulgaris interaction.

The fungus Sclerotinia sclerotiorum (Lib.) de Bary, one of the most important plant pathogens, causes white mold on a wide range of crops. Crop yield can be dramatically decreased due to this disease, depending on the plant cultivar and environmental conditions. In this study, a suppression subtractive hybridization cDNA library approach was used for the identification of pathogen and plant genes that were differentially expressed during infection of the susceptible cultivar BRS Pérola of Phaseolus vulgaris L. A total of 979 unigenes (430 contigs and 549 singletons) were obtained and classified according to their functional categories. The transcriptional profile of 11 fungal genes related to pathogenicity and virulence were evaluated by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Additionally, the temporal expression profile obtained by RT-qPCR was evaluated for the following categories of plant defense-related genes: pathogenesis-related genes (PvPR1, PvPR2, and PvPR3), phenylpropanoid pathway genes (PvIsof, PvFPS1, and 4CL), and genes involved in defense and stress-related categories (PvLox, PvHiprp, PvGST, PvPod, and PvDox). Data obtained in this study provide a starting point for achieving a better understanding of the pathosystem S. sclerotiorum–P. vulgaris.

Rice–gall midge interactions: Battle for survival

Gall midges are insects specialized in maneuvering plant growth, metabolic and defense pathways for their benefit. The Asian rice gall midge and rice share such an intimate relationship that there is a constant battle for survival by either partner. Diverse responses by the rice host against the midge include necrotic hypersensitive resistance reaction, non-hypersensitive resistance reaction and gall-forming compatible interaction. Genetic studies have revealed that major R (resistance) genes confer resistance to gall midge in rice. Eleven gall midge R genes have been characterized so far in different rice varieties in India. In addition, no single R gene confers resistance against all the seven biotypes of the Asian rice gall midge, and none of the biotypes is virulent against all the resistance genes. Further, the interaction of the plant resistance gene with the insect avirulence gene is on a gene-for-gene basis. Our recent investigations involving suppressive subtraction hybridization cDNA libraries, microarray analyses, gene expression assays and metabolic profiling have revealed several molecular mechanisms, metabolite markers and pathways that are induced, down-regulated or altered in the rice host during incompatible or compatible interactions with the pest. This is also true for some of the pathways studied in the gall midge. Next generation sequencing technology, gene expression studies and conventional screening of gall midge cDNA libraries highlighted molecular approaches adopted by the insect to feed, survive and reproduce. This constant struggle by the midge to overcome the host defenses and the host to resist the pest has provided us with an opportunity to observe this battle for survival at the molecular level.

Molecular Characterization of Differentially Expressed ADP Ribosylation Factor from WSSV Infected Black Tiger Shrimp Penaeus monodon

Suppression subtractive hybridization (SSH) cDNA library was constructed using hepatopancreas of the WSSV infected black tiger shrimp, Penaeus monodon. The differentially expressed ESTs of SSH cDNA library represented different functional categories belonging to energy and metabolism, osmoregulation, cytoskeletal and structural proteins, membrane traffic and organelle structure, immune genes, protein folding, transport and synthesis, ovarian and oocyte development, nucleic acid processing, cell cycle and heat shock protein. The ADP ribosylation factor (Arf) was one of the differentially expressed gene which was identified from the SSH cDNA library. The full length cDNA of P. monodon Arf (pmArf) was cloned by 3′- and 5′-rapid amplification of cDNA ends (3′/5′ RACE). The complete nucleotide sequence of pmArf encoded protein sequence of 180 amino acids revealed the characteristic conserved features of the Arf family proteins represented by the P-loop, the switch regions and interswitch region. The sequence homology indicated that the pmArf belonged to class II of Arf family. The gene expression analysis of pmArf and other immune related genes (penaeidin, anti-lipopolysaccharide factor and QM) in the gills, hepatopancreas and gut tissues of the WSSV infected shrimp was carried out by Real-Time PCR. The pmArf gene was up-regulated with highest gene expression levels at 24 h (1.33-fold) in the gill tissues and at 48 h in the gut tissues (1.44-fold) and hepatopancreas (1.46-fold) of WSSV infected shrimp, indicating its potential role in immune response against WSSV infection.