Pung-Ling Huang

Immunogenicity of recombinant GP5 protein of porcine reproductive and respiratory syndrome virus expressed in tobacco plant.

The aim of the study was to evaluate the immunogenicity of the ORF5-encoded major envelop glycoprotein 5 (GP5) of porcine reproductive and respiratory syndrome virus (PRRSV) expressed in tobacco plant as a potential pig oral vaccine in protection against PRRSV infection. Six-week-old PRRSV-free pigs were fed four times orally with 50g of chopped fresh GP5 transgenic tobacco leaves (GP5-T) (GP5 reaching 0.011% of total soluble protein) or wild-type tobacco leaves (W-T) each on days 0, 14, 28, and 42. Samples of serum, saliva, and peripheral blood mononuclear cells (PBMCs) were collected on days -1, 6, 13, 20, 27, 34, 41, and 48 after the initial oral vaccination. A similar vaccination-dependent gradual increase in the responses of serum and saliva anti-PRRSV total IgG and IgA, respectively, and in the levels of PRRSV-specific blastogenic response of PBMCs was seen in GP5-T-treated pigs; all statistically significant elevations occurred after the 2nd vaccination and were revealed after 20 days post-initial oral vaccination (DPIOV). Pigs fed on GP5-T also developed serum neutralizing antibodies to PRRSV at a titer of 1:4-1:8 after the 4th vaccination by 48 DPIOV. No detectable anti-PRRSV antibody responses and PRRSV-specific blastogenic response were seen in W-T-treated pigs. The present study has demonstrated that pigs fed on GP5-T could develop specific mucosal as well as systemic humoral and cellular immune responses against PRRSV. The results also support that transgenic plant as GP5-T can be an effective system for oral delivery of recombinant subunit vaccines in pigs.

The immunogenicity of DNA constructs co-expressing GP5 and M proteins of porcine reproductive and respiratory syndrome virus conjugated by GPGP linker in pigs.

The heterodimer of glycoprotein 5 (GP5) and non-glycosylated matrix protein (M) is the leading target for the development of new generation of vaccines against porcine reproductive and respiratory syndrome virus (PRRSV) infection. It has been demonstrated that DNA vaccine co-expressing GP5 and M proteins as a fusion protein aroused better immunogenicity than that expressing GP5 or M alone, but it was no better than the DNA vaccine co-expressing GP5 and M proteins with two different promoters. Altered natural conformation of the co-expressed GP5 and M fusion protein was considered as the major cause. Glycine-proline-glycine-proline (GPGP) linker can minimize the conformational changes in tertiary structure and provide flexibility of the peptide chain. The objective of this study was to evaluate whether the immunogenicity of DNA constructs co-expressing GP5 and M proteins linked by GPGP could be enhanced in pigs. Three recombinant DNA constructs expressing GP5/M fusion protein without GPGP linker (pcDNA-56), GP5/M fusion protein conjugated by GPGP linker (pcDNA-5L6), and M/GP5 fusion protein conjugated by GPGP linker (pcDNA-6L5) were established. Sixteen PRRSV-free pigs were randomly assigned to four groups and inoculated intramuscularly with 3 consecutive doses of 500 μg of empty vector pcDNA3.1, pcDNA-56, pcDNA-5L6 or pcDNA-6L5 each at a 2-week interval followed by challenge with 5 × 10(5) TCID(50) PRRSV at 3 weeks after the final inoculation. All pcDNA-56-, pcDNA-5L6-, and pcDNA-6L5- but not pcDNA-3.1-inoculated pigs developed neutralizing antibodies (NAs) 3 weeks after the final inoculation and a gradual increase in NA titers after PRRSV challenge, indicating that pigs inoculated with these DNA constructs could establish a sufficient immune memory. The pcDNA-5L6- and pcDNA-6L5-inoculated pigs displayed lower level and shorter period of viremia and lower tissue viral load following PRRSV challenge than did the pcDNA-56-inoculated pigs. The strategy of co-expressing GPGP-linked GP5 and M fusion protein may be a promising approach for future PRRSV vaccine development, possibly via the improvement of natural conformation of the target fusion protein.

Evaluation of the immunogenicity of a transgenic tobacco plant expressing the recombinant fusion protein of GP5 of porcine reproductive and respiratory syndrome virus and B subunit of Escherichia coli heat-labile enterotoxin in pigs.

Escherichia coli heat-labile enterotoxin B subunit (LTB) can be used as an adjuvant for co-administered antigens. Our previous study showed that the expression of neutralizing epitope GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) in transgenic tobacco plant (GP5-T) could induce PRRSV-specific immune responses in pigs. A transgenic tobacco plant co-expressing LTB and PRRSV GP5 as a fusion protein (LTB-GP5-T) was further constructed and its immunogenicity was evaluated. Pigs were given orally three consecutive doses of equal concentration of recombinant GP5 protein expressed in leaves of LTB-GP5-T or GP5-T at a 2-week interval and challenged with PRRSV at 7 weeks post-initial immunization. Pigs receiving LTB-GP5-T or GP5-T developed PRRSV-specific antibody- and cell-mediated immunity and showed significantly lower viremia and tissue viral load and milder lung lesions than wild type tobacco plant (W-T). The LTB-GP5-T-treated group had relatively higher immune responses than the GP5-T-treated group, although the differences were not statistically significant.

Characterization and expression analysis of a banana gene encoding 1‐aminocyclopropane‐1‐carboxylate oxidase

A cDNA encoding the banana 1‐aminocyclopropane‐l‐carboxylate (ACC) oxidase has previously been isolated from a eDNA library that was constructed by extracting poly(A)+ RNA from peels of ripening banana. This cDNA, designated as pMAO2, has 1,199 bp and contains an open reading frame of 318 amino acids. In order to identify ripening‐related promoters of the banana ACC oxidase gene, pMAO2 was used as a probe to screen a banana genomic library constructed in the λEMBL3 vector. The banana ACC oxidase MA02 gene has four exons and three introns, with all of the boundaries between these introns and exons sharing a consensus dinucleotide sequence of GT‐AG. The expression of MA02 gene in banana begins after the onset of ripening (stage 2) and continues into later stages of the ripening process. The accumulation of MAO2 mRNA can be induced by 1 μl/l exogenous ethylene, and it reached steady state level when 100 μl/1 exogenous ethylene was present.

Differential expression and functional characterization of the NADPH cytochrome P450 reductase genes from Nothapodytes foetida.

Three unique NADPH:cytochrome P450 reductase (CPR) cDNAs have been isolated from a Nothapodytes foetida cDNA library and characterized. Phylogenetic analysis showed that NfCPR1 is a class I isoform, whereas NfCPR2 and NfCPR3 are class II isoforms. Both NfCPR1 and NfCPR2 transcripts were detected in all examined organs of N. foetida, with the highest level for NfCPR1 being in the seeds whereas for NfCPR2 predominantly in leaves. In contrast, NfCPR3 transcripts were only detected in flower buds and seeds at almost equal expression levels. Moreover, NfCPR1 expression did not change during wounding treatment, whereas NfCPR2 and NfCPR3 were induced in response to wounding. Microsomes isolated from insect cells co-expressing NfCPR2 and cytochrome P450 enzyme geraniol 10-hydroxylase (G10H) enhanced the production of eriodictyol from naringenin approximately 11-fold relative to control G10H-only insect cells, indicating the supportive role of NfCPR2 for G10H activity in insect cells.

Gene structure of PACO1, a petal senescence‐related related gene from Phalaenopsis encoding peroxisomal acyl‐CoA oxidase homolog

The putative peroxisomal acyl‐CoA oxidase gene PACO1 from Phalaenopsis was isolated from a genomic library constructed in the λEMBL3 vector by screening with PACO1 cDNA. This gene encompasses approximately 14 Kb and is divided into seven exons by six introns. The consensus GT‐AG dinucleotides situated at all the boundaries between exons and introns. According to the genomic Southern analysis with different fragments of the first intron, PACO1 gene possesses a large intron about 10 Kb interrupting the first and secondary exon.

Molecular Cloning and Characterization of A Novel 1-Aminocyclopropane-1-carboxylate Oxidase Gene Involved in Ripening of Banana Fruits

One novel banana fruit ripening related 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene quite different from ACC oxidase genes from other species was cloned. In contrast to other studies, the polypeptide encoded by this gene, named Mh-ACO1, lacks the putative leucine zipper motif which is conserved in all known ACC oxidases including the other previously reported banana ACC oxidase, Mh-ACO2. The locus consists of two nearly identical paralogous ACC oxidase genes arranged in opposite orientation and separated by a 3.1-kb intergenic region. The has only two introns, at positions identical to , which comprises a coding region interrupted by three introns. The predicted amino acid sequence of Mh-ACO1 shares less than 50% identity to those of ACC oxidase from other climacteric fruits, while that of Mh-ACO2 shows more than 65% homology. When expressed in Saccharomyces cerevisiae -encoded protein possessed the enzyme activity for ethylene conversion. The levels of mRNA corresponding to both and increased during fruit ripening and were induced by exogenous ethylene. We conclude that both and contribute to increased ethylene production in fruits and these two genes are differentially expressed in fruits and other organs in banana.

Expression profiles of a MhCTR1 gene in relation to banana fruit ripening

The banana (Musa spp.) is a typical climacteric fruit of high economic importance. The development of bananas from maturing to ripening is characterized by increased ethylene production accompanied by a respiration burst. To elucidate the signal transduction pathway involved in the ethylene regulation of banana ripening, a gene homologous to Arabidopsis CTR1 (constitutive triple response 1) was isolated from Musa spp. (Hsien Jin Chiao, AAA group) and designated as MhCTR1. MhCTR1 spans 11.5 kilobases and consists of 15 exons and 14 introns with consensus GT-AG nucleotides situated at their boundaries. MhCTR1 encodes a polypeptide of 805 amino acid residues with a calculated molecular weight of 88.6 kDa. The deduced amino acid sequence of MhCTR1 demonstrates 55%, 56% and 55% homology to AtCTR1, RhCTR1, and LeCTR1, respectively. MhCTR1 is expressed mostly in the mature green pulp and root organs. During fruit development MhCTR1 expression increases just before ethylene production rises. Moreover, MhCTR1 expression was detected mainly in the pulps at ripening stage 3, and correlated with the onset of peel yellowing, while MhCTR1 was constitutively expressed in the peels. MhCTR1 expression could be induced by ethylene treatment (0.01 μL L(-1)), and MhCTR1 expression decreased in both peel and pulp 24 h after treatment. Overall, changes observed in MhCTR1 expression in the pulp closely related to the regulation of the banana ripening process.

NIR-assisted orchid virus therapy using urchin bimetallic nanomaterials in phalaenopsis

The use of nanoparticles has drawn special attention, particularly in the treatment of plant diseases. Cymbidium mosaic virus (CymMV) and Odontoglossum ring spot virus (ORSV) are the most prevalent and serious diseases that affect the development of the orchid industry. In this study we treated nanoparticles as a strategy for enhancing the resistance of orchids against CymMV and ORSV. After chitosan-modified gold nanoparticles (Au NPs) were injected into Phalaenopsis leaves, the injected leaves were exposed to 980 nm laser for light–heat conversion. To evaluate virus elimination in the treated Phalaenopsis leaves, the transcripts of coat protein genes and the production of viral proteins were assessed by reverse transcription-Polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The expression of coat protein genes for both CymMV and ORSV was significantly lower in the chitosan-modified Au NP-treated Phalaenopsis leaves than in the control. Similarly, the amount of coat proteins for both viruses in the Phalaenopsis leaves was lower than that in the control (without nanoparticle injection). We propose that the temperature increase in the chitosan-modified Au NP-treated Phalaenopsis tissues after laser exposure reduces the viral population, consequently conferring resistance against CymMV and ORSV. Our findings suggest that the application of chitosan-modified Au NPs is a promising new strategy for orchid virus therapy.

cDNA cloning and functional characterization of ETHYLENE INSENSITIVE 3 orthologs from Oncidium Gower Ramsey involved in flower cutting and pollinia cap dislodgement

The cDNAs encoding ETHYLENE INSENSITIVE3 (EIN3) transcription factor, OgEIL1 and OgEIL2 of Oncidium were cloned, sequenced and characterized. The deduced amino acid sequences of OgEIL1 and OgEIL2 of identified cDNA clones contain all structural features found in the Arabidopsis EIN3, such as an amino terminal acidic domain, a proline-rich region, and five basic conserved domains. Complementation test for OgEIL1 in Arabidopsis ein3 mutant indicate that function of OgEIL1 is the same as Arabidopsis EIN3. RNA gel blot analysis indicated that OgEIL1 and OgEIL2 expressed differentially in the roots, stem, leaves and flower buds of Oncidium. OgEIL1 and OgEIL2 mRNA levels in fully opened flowers increased as time progressed after cutting and reached a maximum in the fifth day and decreased on seventh day, which is consistent with the hypothesis that flowers initiated to wilt when ethylene raised abruptly. In de-capped flowers, OgEIL2 mRNA showed a decrease, while OgEIL1 mRNA exhibited an increase. Exogenous application of ethylene increased the mRNA levels of OgEIL1 and OgEIL2 in flower buds and flowers after cutting compared prior to ethylene treatment, however, in pollinia de-capped flowers, both OgEIL1 and OgEIL2 mRNA levels responded to a decline to exogenous ethylene immediately after treatment. Collectively, it is suggested that the main functions of OgEIL1 and OgEIL2 are to modulate the senescence of Oncidium flowers.