Temperature-Regulated Flowering Locus T-Like Gene Coordinates the Spike Initiation in Phalaenopsis Orchid.

Phalaenopsis aphrodite can be induced to initiate spike growth and flowering by exposure to low ambient temperatures. However, the factors and mechanisms responsible for spike initiation in P. aphrodite remain largely unknown. In this study, we show that a repressor Flowing Locus T-like (FTL) gene, FTL, can act as a negative regulator of spike initiation in P. aphrodite. The mRNA transcripts of PaFTL are consistently high during high ambient temperature, thereby preventing premature spike initiation. However, during low ambient temperature, PaFTL expression falls while FT expression increases, allowing for spike initiation. Knock-down of PaFTL expression through virus-inducing gene silencing promoted spike initiation at 30/28°C. Moreover, PaFTL interacts with FLOWERING LOCUS D in a similar manner to FT to regulate downstream flowering initiation genes. Transgenic P. aphrodite plants exhibiting high expression of PaFTL do not undergo spike initiation, even when exposed to low ambient temperatures. These findings shed light on the flowering mechanisms in Phalaenopsis and provide new insights into how perennial plants govern spike initiation in response to temperature cues.

TALE-based organellar genome editing and gene expression in plants.

KEY MESSAGE: TALE-based editors provide an alternative way to engineer the organellar genomes in plants. We update and discuss the most recent developments of TALE-based organellar genome editing in plants. Gene editing tools have been widely used to modify the nuclear genomes of plants for various basic research and biotechnological applications. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 editing platform is the most commonly used technique because of its ease of use, fast speed, and low cost; however, it encounters difficulty when being delivered to plant organelles for gene editing. In contrast, protein-based editing technologies, such as transcription activator-like effector (TALE)-based tools, could be easily delivered, expressed, and targeted to organelles in plants via Agrobacteria-mediated nuclear transformation. Therefore, TALE-based editors provide an alternative way to engineer the organellar genomes in plants since the conventional chloroplast transformation method encounters technical challenges and is limited to certain species, and the direct transformation of mitochondria in higher plants is not yet possible. In this review, we update and discuss the most recent developments of TALE-based organellar genome editing in plants.

Phalaenopsis flowering locus VE regulates floral organ maturation.

KEY MESSAGE: PaFVE is low ambient temperature-inducible and acts as a systemic regulator in the early stage of floral development in Phalaenopsis. Phalaenopsis aphrodite: subsp. formosana, a native orchid species of Taiwan, is an economically important ornamental crop that requires low ambient temperature for floral transition. Currently, limited genetic information about such orchid species hampers genetic manipulation for specific or improved floral traits, and the control of flowering time independent of temperature regulation. In this study, the sequence of the full-length of Phalaenopsis flowering locus VE (PaFVE) gene was determined. Spatial and temporal expression studies showed that mRNA transcripts of PaFVE were inducible by low ambient temperature, and high levels of expression occurred after spiking initiation and remained high throughout the early stage of floral development. Further investigation revealed that floral organ development was impeded in PaFVE-silenced P. aphrodite, but flowering time and floral organogenesis were not compromised. Analysis of the downstream flowering genes suggested that the delay in floral maturation is associated with a corresponding decrease in the expression of downstream flowering genes, PaSOC1, PaSOC1L and PaAGL24. The ectopic expression of PaFVE in Arabidopsis resulted in an accelerated flowering time, accompanied by an increase in the expression of AtSOC1, thus revealing the functional role of PaFVE as a floral regulator. Overall, our results demonstrate that PaFVE has evolutionarily diverged and conserved functions, and serves as a regulator of floral organ maturation in Phalaenopsis and a regulator of flowering time in Arabidopsis.

Genome-wide analysis of GDSL-type esterases/lipases in Arabidopsis.

KEY MESSAGE: In this present study, we introduce a fundamental framework and provide information regarding the possible roles of GDSL-type esterase/lipase gene family in Arabidopsis. GDSL-type esterases/lipases are hydrolytic enzymes with multifunctional properties such as broad substrate specificity, regiospecificity, and stereoselectivity. In this study, we identified 105 GDSL-type esterase/lipase genes in Arabidopsis thaliana by conducting a comprehensive computational analysis. Expression studies indicated that GDSL-type lipase proteins showed varied expression patterns. Phylogenetic tree analysis indicated that AtGELP (Arabidopsis thaliana GDSL-type esterase/lipase protein) gene family was divided into four clades. The phylogenetic analysis, combined with protein motif architectures, and expression profiling were used to predict the roles AtGELP genes. To investigate the physical roles of the AtGELP gene family, we successfully screened 88 AtGELP T-DNA knockout lines for 54 AtGELP genes from 199 putative SALK T-DNA mutants. Transgenic plants of AtGELP genes were used to elucidate the phenotypic characteristics in various developmental stages or stress conditions. Our results suggest that the AtGELP genes have diverse physical functions such as affecting the germination rate and early growth of seedlings subjected to high concentrations of glucose, or being involved in biotic stress responses.

The bHLH142 Transcription Factor Coordinates with TDR1 to Modulate the Expression of EAT1 and Regulate Pollen Development in Rice.

Male sterility plays an important role in F1 hybrid seed production. We identified a male-sterile rice (Oryza sativa) mutant with impaired pollen development and a single T-DNA insertion in the transcription factor gene bHLH142. Knockout mutants of bHLH142 exhibited retarded meiosis and defects in tapetal programmed cell death. RT-PCR and in situ hybridization analyses showed that bHLH142 is specifically expressed in the anther, in the tapetum, and in meiocytes during early meiosis. Three basic helix-loop-helix transcription factors, UDT1 (bHLH164), TDR1 (bHLH5), and EAT1/DTD1 (bHLH141) are known to function in rice pollen development. bHLH142 acts downstream of UDT1 and GAMYB but upstream of TDR1 and EAT1 in pollen development. In vivo and in vitro assays demonstrated that bHLH142 and TDR1 proteins interact. Transient promoter assays demonstrated that regulation of the EAT1 promoter requires bHLH142 and TDR1. Consistent with these results, 3D protein structure modeling predicted that bHLH142 and TDR1 form a heterodimer to bind to the EAT1 promoter. EAT1 positively regulates the expression of AP37 and AP25, which induce tapetal programmed cell death. Thus, in this study, we identified bHLH142 as having a pivotal role in tapetal programmed cell death and pollen development.

Transcriptome-wide analysis of the MADS-box gene family in the orchid Erycina pusilla.

Orchids exhibit a range of unique flower shapes and are a valuable ornamental crop. MADS-box transcription factors are key regulatory components in flower initiation and development. Changing the flower shape and flowering time can increase the value of the orchid in the ornamental horticulture industry. In this study, 28 MADS-box genes were identified from the transcriptome database of the model orchid Erycina pusilla. The full-length genomic sequences of these MADS-box genes were obtained from BAC clones. Of these, 27 were MIKC-type EpMADS (two truncated forms) and one was a type I EpMADS. Eleven EpMADS genes contained introns longer than 10 kb. Phylogenetic analysis classified the 24 MIKC(c) genes into nine subfamilies. Three specific protein motifs, AG, FUL and SVP, were identified and used to classify three subfamilies. The expression profile of each EpMADS gene correlated with its putative function. The phylogenetic analysis was highly correlated with the protein domain identification and gene expression results. Spatial expression of EpMADS6, EpMADS12 and EpMADS15 was strongly detected in the inflorescence meristem, floral bud and seed via in situ hybridization. The subcellular localization of the 28 EpMADS proteins was also investigated. Although EpMADS27 lacks a complete MADS-box domain, EpMADS27-YFP was localized in the nucleus. This characterization of the orchid MADS-box family genes provides useful information for both orchid breeding and studies of flowering and evolution.

NDH expression marks major transitions in plant evolution and reveals coordinate intracellular gene loss.

BACKGROUND: Key innovations have facilitated novel niche utilization, such as the movement of the algal predecessors of land plants into terrestrial habitats where drastic fluctuations in light intensity, ultraviolet radiation and water limitation required a number of adaptations. The NDH (NADH dehydrogenase-like) complex of Viridiplantae plastids participates in adapting the photosynthetic response to environmental stress, suggesting its involvement in the transition to terrestrial habitats. Although relatively rare, the loss or pseudogenization of plastid NDH genes is widely distributed across diverse lineages of photoautotrophic seed plants and mutants/transgenics lacking NDH function demonstrate little difference from wild type under non-stressed conditions. This study analyzes large transcriptomic and genomic datasets to evaluate the persistence and loss of NDH expression across plants. RESULTS: Nuclear expression profiles showed accretion of the NDH gene complement at key transitions in land plant evolution, such as the transition to land and at the base of the angiosperm lineage. While detection of transcripts for a selection of non-NDH, photosynthesis related proteins was independent of the state of NDH, coordinate, lineage-specific loss of plastid NDH genes and expression of nuclear-encoded NDH subunits was documented in Pinaceae, gnetophytes, Orchidaceae and Geraniales confirming the independent and complete loss of NDH in these diverse seed plant taxa. CONCLUSION: The broad phylogenetic distribution of NDH loss and the subtle phenotypes of mutants suggest that the NDH complex is of limited biological significance in contemporary plants. While NDH activity appears dispensable under favorable conditions, there were likely sufficiently frequent episodes of abiotic stress affecting terrestrial habitats to allow the retention of NDH activity. These findings reveal genetic factors influencing plant/environment interactions in a changing climate through 450 million years of land plant evolution.

MSRB7 reverses oxidation of GSTF2/3 to confer tolerance of Arabidopsis thaliana to oxidative stress.

Methionine sulfoxide reductases (MSRs) catalyse the reduction of oxidized methionine residues, thereby protecting proteins against oxidative stress. Accordingly, MSRs have been associated with stress responses, disease, and senescence in a taxonomically diverse array of organisms. However, the cytosolic substrates of MSRs in plants remain largely unknown. Here, we used a proteomic analysis strategy to identify MSRB7 substrates. We showed that two glutathione transferases (GSTs), GSTF2 and GSTF3, had fewer oxidized methionine (MetO) residues in MSRB7-overexpressing Arabidopsis thaliana plants than in wild-type plants. Conversely, GSTF2 and GSTF3 were highly oxidized and unstable in MSRB7-knockdown plants. MSRB7 was able to restore the MetO-GSTF2M100/104 and MetO-GSTF3M100 residues produced during oxidative stress. Furthermore, both GSTs were specifically induced by the oxidative stress inducer, methyl viologen. Our results indicate that specific GSTs are substrates of MSRs, which together provide a major line of defence against oxidative stress in A. thaliana.

D-optimal Design Optimization of Solvent Mixture for Flavonoid Extraction from Phalaenopsis Leaves with Antioxidant Activity.

Aims: This study aimed to optimize the extraction of flavonoids and antioxidants from Phalaenopsis leaves by using solvent mixtures. Method: The total flavonoid content (TFC) and antioxidant activity were evaluated using the colorimetric method and ferric-reducing antioxidant power (FRAP), respectively. Maceration extracts from fresh leaves were used for the analysis. The study used the Design Expert 13.0 program to optimize the solvents (water, acetone, and methanol) and their combined ratio. Result: The results showed that 100% acetone was the best solvent for both responses, with a desirability value of 0.884, TFC of 0.434 mg QE/g fresh weight (FW) and FRAP of 713.53 µmol TE/g FW. Screening of the most potent Phalaenopsis genotypes for obtaining the most active leaf extract showed that P. amboinensis and P. pantherina were the best genotypes for TFC (0.786-0.797 mg QE/g FW) and FRAP activity (862.25-891.48 µmol TE/g FW). Conclusion: This study demonstrates an easy and useful way to obtain flavonoids and antioxidants from Phalaenopsis materials that can be used in the flower-based industry to make new functional ingredients.

Catalog of Erycina pusilla miRNA and categorization of reproductive phase-related miRNAs and their target gene families.

The orchid Erycina pusilla has a short life cycle and relatively low chromosome number, making it a potential model plant for orchid functional genomics. To that end, small RNAs (sRNAs) from different developmental stages of different organs were sequenced. In this miRNA mix, 33 annotated miRNA families and 110 putative miRNA-targeted transcripts were identified in E. pusilla. Fifteen E. pusilla miRNA target genes were found to be similar to those in other species. There were putative novel miRNAs identified by 3 different strategies. The genomic sequences of the four miRNAs that were identified using rice genome as the reference can form the stem loop structure. The t0000354 miRNA, identified using rice genome sequences and a Phalaenopsis study, had a high read count. The target gene of this miRNA is MADS (unigene30603), which belongs to the AP3-PI subfamily. The most abundant miRNA was E. pusilla miR156 (epu-miR156), orthologs of which work to maintain the vegetative phase by repressing the expression of the SQUAMOSA promoter-binding-like (SPL) transcription factors. Fifteen genes in the E. pusilla SPL (EpSPL) family were identified, nine of which contained the putative epu-miR156 target site. Target genes of epu-miR172, also a key regulator of developmental changes in the APETALA2 (EpAP2) family, were identified. Experiments using 5'RLM-RACE demonstrated that the genes EpSPL1, 2, 3, 4, 7, 9, 10, 14 and EpAP2-9, -10, -11 were regulated by epu-miR156 and epu-miR172, respectively.

Global transcriptome analysis and identification of a CONSTANS-like gene family in the orchid Erycina pusilla.

The high chromosome numbers, polyploid genomes, and long juvenile phases of most ornamental orchid species render functional genomics difficult and limit the discovery of genes influencing horticultural traits. The orchid Erycina pusilla has a low chromosome number (2n = 12) and flowers in vitro within 1 year, making it a standout candidate for use as a model orchid. However, transcriptomic and genomic information from E. pusilla remains limited. In this study, next-generation sequencing (NGS) technology was used to identify 90,668 unigenes by de novo assembly. These unigenes were annotated functionally and analyzed with regard to their gene ontology (GO), clusters of orthologous groups (COG), and KEGG pathways. To validate the discovery methods, a homolog of CONSTANS (CO), one of the key genes in the flowering pathway, was further analyzed. The Arabidopsis CO-Like (COL) amino acid sequences were used to screen for homologs in the E. pusilla transcriptome database. Specific primers to the homologous unigenes were then used to isolate BAC clones, which were sequenced to identify 12 E. pusilla CO-like (EpCOL) full-length genes. Based on sequence homology, domain structure, and phylogenetic analysis, these EpCOL genes were divided into four groups. Four EpCOLs fused with GFP were localized in the nucleus. Some EpCOL genes were regulated by light. These results demonstrate that nascent E. pusilla resources (transcriptome and BAC library) can be used to investigate the E. pusilla photoperiod-dependent flowering genes. In future, this strategy can be applied to other biological processes, marketable traits, and molecular breeding in this model orchid.

Optimal culture conditions of Piriformospora indica for volatile compound release to promote effective plant growth.

Piriformospora indica, which is an endophytic fungus that grows on various media in the absence of a host, emits plant growth promoting volatile organic compounds (VOCs). Kaefer medium (KF) has been shown to be the most suitable medium for P. indica growth; however, different media may differentially affect fungal metabolism which may in turn influence the VOC profiles of P. indica. To date, how the VOCs emitted from P. indica cultured on different media affect plant growth has not been well characterized. Here, we show that poor nutrient medium (PNM) promoted the growth of P. indica more effectively than potato dextrose agar (PDA) or KF medium. By contrast, plant total biomass and root fresh weight were increased 1.8-fold and 2.1-fold, when co-cultivated with P. indica cultured on PDA medium in comparison with KF or PNM medium, respectively. Furthermore, sucrose in the plant culture medium downregulated the fold-induction ratio of the plant growth promoted by P. indica VOCs.

Arabidopsis AtMSRB5 functions as a salt-stress protector for both Arabidopsis and rice.

Salinity, drought and low temperature are major environmental factors that adversely affect crop productivity worldwide. In this study we adopted an activation tagging approach to identify salt tolerant mutants of Arabidopsis. Thousands of tagged Arabidopsis lines were screened to obtain several potential mutant lines resistant to 150 mM NaCl. Transcript analysis of a salt-stress tolerance 1 (sst1) mutant line indicated activation of AtMSRB5 and AtMSRB6 which encode methionine sulfoxide reductases. Overexpression of AtMSRB5 in Arabidopsis (B5OX) showed a similar salt tolerant phenotype. Furthermore, biochemical analysis indicated stability of the membrane protein, H+-ATPase 2 (AHA2) through regulation of Na+/K+ homeostasis which may be involved in a stress tolerance mechanism. Similarly, overexpression of AtMSRB5 in transgenic rice demonstrated a salt tolerant phenotype via the modulation of Na+/K+ homeostasis without a yield drag under salt and oxidative stress conditions.

Transcriptome-wide characterization of miRNA-directed and non-miRNA-directed endonucleolytic cleavage using Degradome analysis under low ambient temperature in Phalaenopsis aphrodite subsp. formosana.

Plant microRNAs (miRNAs) regulate gene expression through post-transcriptional gene silencing. Phalaenopsis aphrodite subsp. formosana is an orchid species native to Taiwan, which has high economic value and a high frequency of floral polymorphism. To date, few studies have focused on the regulatory roles of miRNAs and functional small RNAs (sRNAs) in orchids although understanding the regulation of flower development and flowering time is potentially important. Here, we combined analyses of the transcriptome, sRNAs and the degradome to identify sRNA-directed transcript cleavages in Phalaenopsis. Degradome analysis provided large-scale evidence of conserved and novel miRNA-directed cleavage of target transcripts, and 46 abundant sRNA groups and their target transcripts were identified. Low temperature-responsive sRNAs were validated with normalized reads from an sRNA library and quantitative stem-loop reverse transcription-PCR (RT-PCR) analysis. According to gene ontology (GO) categorization, target transcripts of the novel miRNAs and sRNAs are functionally involved in metabolic processes or responses to stress. One particular homologous gene, Allcontig28452, which encodes digalactosyldiacylglycerol synthase 2 (DGD2), was found to be targeted by natural antisense transcripts (NATs) unique to Phalaenopsis. In summary, comprehensive analyses of the transcriptome, sRNAs and degradome using deep sequencing technology provided a useful platform for investigating miRNA-directed and non-miRNA-directed endonucleolytic cleavage in a non-model plant, the orchid Phalaenopsis.

Arabidopsis root-abundant cytosolic methionine sulfoxide reductase B genes MsrB7 and MsrB8 are involved in tolerance to oxidative stress.

Excess reactive oxygen species (ROS) accumulation under various environmental stresses can damage intracellular polysaccharides, DNA, lipids and proteins. Methionine sulfoxide reductase (MSR) participates in a protein repair system that is one of the defensive mechanisms that diminishes oxidative destruction. In Arabidopsis, cytosolic MsrB7 and MsrB8 are oxidative stress-inducible protein repair enzymes that are abundant in the root. Here methyl viologen (MV) treatment was demonstrated to increase greatly the accumulation of H(2)O(2) in MsrB7-knockdown, MsrB8-knockdown and wild-type Arabidopsis, but not in transgenic plants overexpressing MsrB7 or MsrB8. The reduction in H(2)O(2) level under MV treatment in these overexpressing plants coincided with increased activity of glutathione S-transferase (GST), a herbicide-detoxifying enzyme. MsrB7 and MsrB8 are suggested to play an important role in defense against oxidative stress. Transgenic plants overexpressing MsrB7 or MsrB8 were viable and survived after MV and H(2)O(2) treatment. Ectopic expression of specific cytosolic MsrB genes may be useful for application in crop improvement.

Tomato RAV transcription factor is a pivotal modulator involved in the AP2/EREBP-mediated defense pathway.

Ralstonia solanacearum is the causal agent of bacterial wilt (BW), one of the most important bacterial diseases worldwide. We used cDNA microarray to survey the gene expression profile in transgenic tomato (Solanum lycopersicum) overexpressing Arabidopsis (Arabidopsis thaliana) CBF1 (AtCBF1), which confers tolerance to BW. The disease-resistant phenotype is correlated with constitutive expression of the Related-to-ABI3/VP1 (RAV) transcription factor, ethylene-responsive factor (ERF) family genes, and several pathogenesis-related (PR) genes. Using a transient assay system, we show that tomato RAV2 (SlRAV2) can transactivate the reporter gene driven by the SlERF5 promoter. Virus-induced gene silencing of SlERF5 and SlRAV2 in AtCBF1 transgenic and BW-resistant cultivar Hawaii 7996 plants gave rise to plants with enhanced susceptibility to BW. Constitutive overexpression of SlRAV2 in transgenic tomato plants induced the expression of SlERF5 and PR5 genes and increased BW tolerance, while knockdown of expression of SlRAV2 inhibited SlERF5 and PR5 gene expression under pathogen infection and significantly decreased BW tolerance. In addition, transgenic tomato overexpressing SlERF5 also accumulated higher levels of PR5 transcripts and displayed better tolerance to pathogen than wild-type plants. From these results, we conclude that SlERFs may act as intermediate transcription factors between AtCBF1 and PR genes via SlRAV in tomato, which results in enhanced tolerance to BW.

Ethylene Response Factor109 Attunes Immunity, Photosynthesis, and Iron Homeostasis in Arabidopsis Leaves.

Iron (Fe) is an essential micronutrient element for all organisms including plants. Chlorosis of young leaves is a common symptom of Fe deficiency, reducing the efficiency of photosynthesis, and, ultimately, crop yield. Previous research revealed strong responsiveness of the putative key transcription factor ERF109 to the Fe regime. To elucidate the possible role of ERF109 in leaf Fe homeostasis and photosynthesis, we subjected Arabidopsis thaliana erf109 knockout lines and Col-0 wild-type plants to transcriptome profiling via RNA-seq. The transcriptome profile of Fe-sufficient erf109 leaves showed a 71% overlap with Fe-deficient Col-0 plants. On the other hand, genes that were differentially expressed between Fe-deficient and Fe-sufficient Col-0 plants remained unchanged in erf109 plants under conditions of Fe deficiency. Mutations in ERF109 increased the expression of the clade Ib bHLH proteins bHLH38, bHLH39, bHLH101, the nicotianamine synthase NAS4, and the Fe storage gene FER1. Moreover, mutations in ERF109 led to significant down-regulation of defense genes, including CML37, WRKY40, ERF13, and EXO70B2. Leaves of erf109 exhibited increased Fe levels under both Fe-sufficient and Fe-deficient conditions. Reduced Fv/Fm and Soil Plant Analysis Development (SPAD) values in erf109 lines under Fe deficiency indicate curtailed ability of photosynthesis relative to the wild-type. Our findings suggest that ERF109 is a negative regulator of the leaf response to Fe deficiency. It further appears that the function of ERF109 in the Fe response is critical for regulating pathogen defense and photosynthetic efficiency. Taken together, our study reveals a novel function of ERF109 and provides a systematic perspective on the intertwining of the immunity regulatory network and cellular Fe homeostasis.

Integration of molecular biology tools for identifying promoters and genes abundantly expressed in flowers of Oncidium Gower Ramsey.

BACKGROUND: Orchids comprise one of the largest families of flowering plants and generate commercially important flowers. However, model plants, such as Arabidopsis thaliana do not contain all plant genes, and agronomic and horticulturally important genera and species must be individually studied. RESULTS: Several molecular biology tools were used to isolate flower-specific gene promoters from Oncidium 'Gower Ramsey' (Onc. GR). A cDNA library of reproductive tissues was used to construct a microarray in order to compare gene expression in flowers and leaves. Five genes were highly expressed in flower tissues, and the subcellular locations of the corresponding proteins were identified using lip transient transformation with fluorescent protein-fusion constructs. BAC clones of the 5 genes, together with 7 previously published flower- and reproductive growth-specific genes in Onc. GR, were identified for cloning of their promoter regions. Interestingly, 3 of the 5 novel flower-abundant genes were putative trypsin inhibitor (TI) genes (OnTI1, OnTI2 and OnTI3), which were tandemly duplicated in the same BAC clone. Their promoters were identified using transient GUS reporter gene transformation and stable A. thaliana transformation analyses. CONCLUSIONS: By combining cDNA microarray, BAC library, and bombardment assay techniques, we successfully identified flower-directed orchid genes and promoters.

Expression of recombinant human interferon-γ with antiviral activity in the bi-cistronic baculovirus-insect/larval system.

A bi-cistronic baculovirus-insect/larval system containing a polyhedron promoter, an internal ribosome entry site (IRES), and an egfp gene was developed as a cost-effective platform for the production of recombinant human interferon gamma (rhIFN-γ). There was no significant difference between the amounts of rhIFN-γ produced in the baculovirus-infected Spodoptera frugiferda 21 cells grown in serum-free medium and the serum-supplemented medium, while the Trichoplusia ni (T. ni) and Spodoptera exigua (S. exigua) larvae afforded rhIFN-γ amounting to 1.08±0.04 and 9.74±0.35 µg/mg protein respectively. The presence of non-glycosylated and glycosylated rhIFN-γ was confirmed by immunoblot and lectin blot. The immunological activity of purified rhIFN-γ, with 96% purity by Nickel (II)-nitrilotriacetic acid (Ni-NTA) affinity chromatography, was similar to that commercially available. Moreover, the rhIFN-γ protein from T. ni had more potent antiviral activity. These findings suggest that this IRES-based expression system is a simple and inexpensive alternative for large-scale protein production in anti-viral research.

Complete chloroplast genome of Oncidium Gower Ramsey and evaluation of molecular markers for identification and breeding in Oncidiinae.

BACKGROUND: Oncidium spp. produce commercially important orchid cut flowers. However, they are amenable to intergeneric and inter-specific crossing making phylogenetic identification very difficult. Molecular markers derived from the chloroplast genome can provide useful tools for phylogenetic resolution. RESULTS: The complete chloroplast genome of the economically important Oncidium variety Onc. Gower Ramsey (Accession no. GQ324949) was determined using a polymerase chain reaction (PCR) and Sanger based ABI sequencing. The length of the Oncidium chloroplast genome is 146,484 bp. Genome structure, gene order and orientation are similar to Phalaenopsis, but differ from typical Poaceae, other monocots for which there are several published chloroplast (cp) genome. The Onc. Gower Ramsey chloroplast-encoded NADH dehydrogenase (ndh) genes, except ndhE, lack apparent functions. Deletion and other types of mutations were also found in the ndh genes of 15 other economically important Oncidiinae varieties, except ndhE in some species. The positions of some species in the evolution and taxonomy of Oncidiinae are difficult to identify. To identify the relationships between the 15 Oncidiinae hybrids, eight regions of the Onc. Gower Ramsey chloroplast genome were amplified by PCR for phylogenetic analysis. A total of 7042 bp derived from the eight regions could identify the relationships at the species level, which were supported by high bootstrap values. One particular 1846 bp region, derived from two PCR products (trnHGUG -psbA and trnFGAA-ndhJ) was adequate for correct phylogenetic placement of 13 of the 15 varieties (with the exception of Degarmoara Flying High and Odontoglossum Violetta von Holm). Thus the chloroplast genome provides a useful molecular marker for species identifications. CONCLUSION: In this report, we used Phalaenopsis. aphrodite as a prototype for primer design to complete the Onc. Gower Ramsey genome sequence. Gene annotation showed that most of the ndh genes inOncidiinae, with the exception of ndhE, are non-functional. This phenomenon was observed in all of the Oncidiinae species tested. The genes and chloroplast DNA regions that would be the most useful for phylogenetic analysis were determined to be the trnHGUG-psbA and the trnFGAA-ndhJ regions. We conclude that complete chloroplast genome information is useful for plant phylogenetic and evolutionary studies in Oncidium with applications for breeding and variety identification.