Characterization of MicroRNAs and Gene Expression in ACC Oxidase RNA Interference-Based Transgenic Bananas.

Banana (Musa acuminata, AAA group) is a typically respiratory climacteric fruit. Previously, genes encoding ACC oxidase, one of the key enzymes in ethylene biosynthesis, Mh-ACO1 and Mh-ACO2 in bananas were silenced individually using RNAi interference technology, and fruit ripening of transgenic bananas was postponed. Here, the differential expression of miRNAs and their targeted mRNAs were analyzed in the transcriptomes of fruits at the third ripening stage, peel color more green than yellow, from the untransformed and RNAi transgenic bananas. Five significantly differentially expressed miRNAs (mac-miR169a, mac-miR319c-3p, mac-miR171a, mac-miR156e-5p, and mac-miR164a-5p) were identified. The predicted miRNA target genes were mainly enriched in six KEGG pathways, including 'sulfur relay system', 'protein digestion and absorption', 'histidine metabolism', 'pathogenic E. coli infection', 'sulfur metabolism', and 'starch and sucrose metabolism'. After ethylene treatment, the expression of ACC oxidase silencing-associated miRNAs was down-regulated, and that of their target genes was up-regulated along with fruit ripening. The evolutionary clustering relationships of miRNA precursors among 12 gene families related to fruit ripening were analyzed. The corresponding expression patterns of mature bodies were mainly concentrated in flowers, fruits, and leaves. Our results indicated that ethylene biosynthesis is associated with miRNAs regulating the expression of sulfur metabolism-related genes in bananas.

Near-Infrared Nanophosphor Embedded in Mesoporous Silica Nanoparticle with High Light-Harvesting Efficiency for Dual Photosystem Enhancement.

Light-harvesting and conversion ability is important to promote plant growth, and especially when resources are limited. A near-infrared (NIR) nanophosphor embedded with mesoporous silica nanoparticles (MSN), ZnGa2 O4 :Cr3+ ,Sn4+ (ZGOCS), was developed and its optical properties were harnessed to enhance the photosynthetic ability of Brassica rapa spp. chinensis. The broad excitation of ZGOCS from the ultraviolet to the visible region allowed the conversion of extra light into near-infrared light (650-800 nm) and thus promoted the dual photosystem via the Emerson effect. ZGOCS@MSN was spherical with a size of 65±10 nm and good dispersion. A light conversion ability of up to 75 % under different wavelengths was achieved. Moreover, the electron transfer rate of photosynthesis increased by 100 % with a suitable ZGOCS@MSN concentration. Plant and animal models were used to explore the effects of the nanophosphor. ZGOCS@MSN distribution was tracked by monitoring its NIR emission in plant and animal tissues, demonstrating that this nanophosphor can be potentially utilized in plant growth.

Expression Fluctuations of Genes Involved in Carbohydrate Metabolism Affected by Alterations of Ethylene Biosynthesis Associated with Ripening in Banana Fruit.

The banana is a typical climacteric fruit that undergoes ethylene dependent ripening. During fruit ripening, ethylene production triggers a developmental cascade that results in a series of physiological and biochemical changes. The fruit transcriptomes of untransformated wild-type (WT) and RNAi transgenic banana plants for Mh-ACO1 and Mh-ACO2 have been previously sequenced and analyzed, and most of the differentially expressed genes were enriched in 'carbon fixation in photosynthetic organism', 'cysteine and methionine metabolism', 'citrate cycle (tricarboxylic acid cycle, TCA cycle)', and 'starch and sucrose metabolism' based on Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation. In this research, we investigated the expression fluctuations of genes involved in carbohydrate metabolism affected by alterations of ethylene biosynthesis associated with ripening in banana fruits. Expression profiles of sucrose synthase, sucrose phosphate synthase, neutral invertase, and acidic invertase/ß-fructofuranosidase, as analyzed by Avadis and Trinity, showed that both analyses were complementary and consistent. The overall gene expression tendency was confirmed by the implementation of quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) with mRNAs of banana fruits in Mh-ACO1 and Mh-ACO2 RNAi transgenic plants. These results indicated that altered expression of genes associated with ethylene biosynthesis strongly influenced the expression levels of genes related to starch and sucrose metabolism, as well as the glycolysis pathway in ripening banana fruits.

Two Paralogous Genes Encoding Auxin Efflux Carrier Differentially Expressed in Bitter Gourd (Momordica charantia).

The phytohormone auxin regulates various developmental programs in plants, including cell growth, cell division and cell differentiation. The auxin efflux carriers are essential for the auxin transport. To show an involvement of auxin transporters in the coordination of fruit development in bitter gourd, a juicy fruit, we isolated novel cDNAs (referred as McPIN) encoding putative auxin efflux carriers, including McPIN1, McPIN2 (allele of McPIN1) and McPIN3, from developing fruits of bitter gourd. Both McPIN1 and McPIN3 genes possess six exons and five introns. Hydropathy analysis revealed that both polypeptides have two hydrophobic regions with five transmembrane segments and a predominantly hydrophilic core. Phylogenetic analyses revealed that McPIN1 shared the highest homology to the group of Arabidopsis, cucumber and tomato PIN1, while McPIN3 belonged to another group, including Arabidopsis and tomato PIN3 as well as PIN4. This suggests different roles for McPIN1 and McPIN3 in auxin transport involved in the fruit development of bitter gourd. Maximum mRNA levels for both genes were detected in staminate and pistillate flowers. McPIN1 is expressed in a particular period of early fruit development but McPIN3 continues to be expressed until the last stage of fruit ripening. Moreover, these two genes are auxin-inducible and qualified as early auxin-response genes. Their expression patterns suggest that these two auxin transporter genes play a pivotal role in fruit setting and development.

Novel therapeutic effects of sesamin on diabetes-induced cardiac dysfunction.

Diabetes is a risk factor that increases the occurrence and severity of cardiovascular events. Cardiovascular complications are the leading cause of mortality of 75% of patients with diabetes >40 years old. Sesamin, the bioactive compound extracted from Sesamum indicum, is a natural compound that has diverse beneficial effects on hypoglycemia and reducing cholesterol. The aim of this study is to investigate sesamin effects to diabetes-inducing cardiac hypertrophy. In the present study bioinformatics analysis demonstrated cardiac hypertrophy signaling may be the most important pathway for upregulating genes in sesamin-treated groups. To verify the bioinformatics prediction, sesamin was used as the main bioactive compound to attenuate the impact of diabetes induced by streptozotocin (STZ) on cardiac function in a rat model. The results revealed that oral administration of sesamin for 4 weeks (100 and 200 mg/kg body weight) marginally improved blood glucose levels, body weight and significantly ameliorated the effects on heart rate and blood pressure in rats with type 1 diabetes relative to control rats. The QT interval of sesamin was also reduced relative to the control group. The findings indicated that sesamin has potential cardioprotective effects in the STZ-induced diabetes model. This suggested that this can be used as a novel treatment for patients with diabetes with cardiac dysfunction complication.

Global Transcriptomic Analysis of Targeted Silencing of Two Paralogous ACC Oxidase Genes in Banana.

Among 18 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase homologous genes existing in the banana genome there are two genes, Mh-ACO1 and Mh-ACO2, that participate in banana fruit ripening. To better understand the physiological functions of Mh-ACO1 and Mh-ACO2, two hairpin-type siRNA expression vectors targeting both the Mh-ACO1 and Mh-ACO2 were constructed and incorporated into the banana genome by Agrobacterium-mediated transformation. The generation of Mh-ACO1 and Mh-ACO2 RNAi transgenic banana plants was confirmed by Southern blot analysis. To gain insights into the functional diversity and complexity between Mh-ACO1 and Mh-ACO2, transcriptome sequencing of banana fruits using the Illumina next-generation sequencer was performed. A total of 32,093,976 reads, assembled into 88,031 unigenes for 123,617 transcripts were obtained. Significantly enriched Gene Oncology (GO) terms and the number of differentially expressed genes (DEGs) with GO annotation were 'catalytic activity' (1327, 56.4%), 'heme binding' (65, 2.76%), 'tetrapyrrole binding' (66, 2.81%), and 'oxidoreductase activity' (287, 12.21%). Real-time RT-PCR was further performed with mRNAs from both peel and pulp of banana fruits in Mh-ACO1 and Mh-ACO2 RNAi transgenic plants. The results showed that expression levels of genes related to ethylene signaling in ripening banana fruits were strongly influenced by the expression of genes associated with ethylene biosynthesis.

Oral immunogenicity of porcine reproductive and respiratory syndrome virus antigen expressed in transgenic banana.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a persistent threat of economically significant influence to the swine industry worldwide. Recombinant DNA technology coupled with tissue culture technology is a viable alternative for the inexpensive production of heterologous proteins in planta. Embryogenic cells of banana cv. 'Pei chiao' (AAA) have been transformed with the ORF5 gene of PRRSV envelope glycoprotein (GP5) using Agrobacterium-mediated transformation and have been confirmed. Recombinant GP5 protein levels in the transgenic banana leaves were detected and ranged from 0.021%-0.037% of total soluble protein. Pigs were immunized with recombinant GP5 protein by orally feeding transgenic banana leaves for three consecutive doses at a 2-week interval and challenged with PRRSV at 7 weeks postinitial immunization. A vaccination-dependent gradational increase in the elicitation of serum and saliva anti-PRRSV IgG and IgA was observed. Furthermore, significantly lower viraemia and tissue viral load were recorded when compared with the pigs fed with untransformed banana leaves. The results suggest that transgenic banana leaves expressing recombinant GP5 protein can be an effective strategy for oral delivery of recombinant subunit vaccines in pigs and can open new avenues for the production of vaccines against PRRSV.

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.

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.

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.

Functional expression of geraniol 10-hydroxylase reveals its dual function in the biosynthesis of terpenoid and phenylpropanoid.

Geraniol 10-hydroxylase (G10H), a cytochrome P450 monooxygenase, has been reported to be involved in the biosynthesis of terpenoid indole alkaloids. The gene for Catharanthus roseus G10H (CrG10H) was cloned and heterologously expressed in baculovirus-infected insect cells. A number of substrates were subjected to assay the enzyme activity of CrG10H. As reported in a previous study, CrG10H hydroxylated the monoterpenoid geraniol at the C-10 position to generate 10-hydroxygeraniol. Interestingly, CrG10H also catalyzed 3'-hydroxylation of naringenin to produce eriodictyol. Coexpression of an Arabidopsis NADPH P450 reductase substantially increased the ability of CrG10H to hydroxylate naringenin. The catalytic activity of CrG10H was approximately 10 times more efficient with geraniol than with naringenin, judged by the k(cat)/K(m) values. Thus, G10H also plays an important role in the biosynthetic pathway of flavonoids, in addition to its previously described role in the metabolism of terpenoids.

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.

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.

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.

Genomic organization of a diverse ACC synthase gene family in banana and expression characteristics of the gene member involved in ripening of banana fruits.

The banana is one of the typical climacteric fruits with great economic importance in agriculture. To understand the basic mechanism underlying banana ripening, gene clones for banana ACC synthase (EC 4.4.1.14), a key regulatory enzyme in the ethylene biosynthetic pathway, were characterized. Genomic clones were analyzed by restriction mapping, and the data in conjunction with sequence comparisons with the previously isolated PCR fragments indicated that at least nine ACC synthase genes (MACS1-9) exist in the banana genome. Southern blot analysis showed they are located in different regions of the banana genome. Three lambda genomic clones (GMACS-1, -9, and -12) were completely sequenced, and gene structures of MACS1 (corresponding to alleles of GMACS-9 and -12) and MACS2 (corresponding to GMACS-1) were elucidated. The coding regions of these three genes were all interrupted by three introns. The size and location of introns are similar to the ACC synthase genes from tomato and Arabidopsis. Northern analysis showed that only MACS1 expressed during fruit ripening and was inducible by exogenous ethylene treatment, which indicates MACS1 is a significant member of the ACC synthase gene family related to ripening in banana fruit. The transcription initiation site of GMACS-12 containing MACS1 was defined. There is a TATTAAT sequence located at position -31 to -25 that qualifies as a TATA box. The delineation of transcription unit in MACS1 will facilitate the promoter studies for this gene and allow its specific functions involved in fruit ripening to be determined.

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.