Successful production of human epidermal growth factor in tobacco chloroplasts in a biologically active conformation.

Human epidermal growth factor (hEGF) is an important therapeutic compound with multiple applications particularly in pharmaceutical industry. Human EGF has already been expressed in different expression systems, however, the production of hEGF with bioactivity in chloroplasts has not been successful so far. In this study, we expressed a 6 × His-tagged hEGF in tobacco chloroplasts in its native conformation for the potential of large-scale production of hEGF for industrial applications. Several transplastomic plant lines were obtained, which were screened by PCR (polymerase chain reaction) using primers specific to selectable gene aadA, hEGF- and GFP-coding sequences that were included in the chloroplast expression vector. The selected lines were confirmed to be homoplasmic by PCR verification and Southern blot analysis. Immunoblotting assays of homoplasmic lines using antibodies raised against hEGF confirmed the accumulation of hEGF in transplastomic plants and the ELISA results demonstrated the expression levels of hEGF were between 0.124% and 0.165% of the total soluble proteins (TSP), namely, 23.16-25.77 ng/g of the fresh weight. In terms of activity, the data from cell proliferation and elongation assays showed that the tobacco-derived recombinant hEGF was as bioactive as its commercial counterpart. To our knowledge, this is the first report of recombinant production of hEGF with native bioactivity form in the chloroplast stroma. Overall, our results demonstrate the potential of higher plant chloroplasts for the production of a human therapeutic, hEGF, in an active conformation.

Efficient expression of fusion human epidermal growth factor in tobacco chloroplasts.

BACKGROUND: Chloroplast transformation is a robust technology for the expression of recombinant proteins. Various types of pharmaceutical proteins including growth factors have been reported in chloroplasts via chloroplast transformation approach at high expression levels. However, high expression of epidermal growth factor (EGF) in chloroplasts with the technology is still unavailable. RESULTS: The present work explored the high-level expression of recombinant EGF, a protein widely applied in many clinical therapies, in tobacco chloroplasts. In this work, homoplastic transgenic plants expressing fusion protein GFP-EGF, which was composed of GFP and EGF via a linker, were generated. The expression of GFP-EGF was confirmed by the combination of green fluorescent observation and Western blotting. The achieved accumulation of the recombinant fusion GFP-EGF was 10.21 ± 0.27% of total soluble proteins (1.57 ± 0.05 g kg- 1 of fresh leaf). The chloroplast-derived GFP-EGF was capable of increasing the cell viability of the NSLC cell line A549 and enhancing the phosphorylation level of the EGF receptor in the A549 cells. CONCLUSION: The expression of recombinant EGF in tobacco chloroplasts via chloroplast transformation method was achieved at considerable accumulation level. The attempt gives a good example for the application of chloroplast transformation technology in recombinant pharmaceutical protein production.

Co-expression of four penaeidins in transgenic rice seeds: an alternative strategy for substitute antibiotic agricultural products.

The co-expression of multiple antimicrobial peptides (AMPs) in genetically modified (GM) crops can give plants a broader antibacterial spectrum and lower the pathogen risk of drug resistance. Therefore, four penaeidins (shrimp-derived AMPs) were fused and encoded in an artificial gene (PEN1234), driven by the seed-specific promoter Pzein, with the aim of co-expression in seeds of transgenic rice. The resistant rice plants, acquired via Agrobacterium-mediated transformation and glufosinate screening, were identified by PCR and the modified disk-diffusion method, and eight GM lines with high AMP content in the seeds were obtained. Among them, the PenOs017 line had the largest penaeidin content, at approximately 251-300 µg/g in seeds and 15-47 µg/g in roots and leaves. The AMPs in the seeds kept their antibacterial properties even after the seed had been boiled in hot water and could significantly inhibit the growth of methicillin-resistant Staphylococcus aureus, and AMPs in the leaves could effectively inhibit Xanthomonas oryzae pv. Oryzae. The results indicate that PenOs017 seeds containing AMPs are an ideal raw-material candidate for antibiotic-free food and feed, and may require fewer petrochemical fungicides or bactericides for disease control during cultivation than conventional rice.

Hyper-expression of GFP-fused active hFGF21 in tobacco chloroplasts.

Human fibroblast growth factor 21 (hFGF21) is a promising candidate for metabolic diseases. In this study, a tobacco chloroplast transformation vector, pWYP21406, was constructed that consisted of codon-optimized encoding gene hFGF21 fused with GFP at its 5' terminal; it was driven by the promoter of plastid rRNA operon (Prrn) and terminated by the terminator of plastid rps16 gene (Trps16). Spectinomycin-resistant gene (aadA) was the marker and placed in the same cistron between hFGF21 and the terminator Trps16. Transplastomic plants were generated by the biolistic bombardment method and proven to be homoplastic by Southern blotting analysis. The expression of GFP was detected under ultraviolet light and a laser confocal microscope. The expression of GFP-hFGF21 was confirmed by immunoblotting and quantified by enzyme-linked immunosorbnent assay (ELISA). The accumulation of GFP-hFGF21 was confirmed to be 12.44 ± 0.45% of the total soluble protein (i.e., 1.9232 ± 0.0673 g kg-1 of fresh weight). GFP-hFGF21 promoted the proliferation of hepatoma cell line HepG2, inducing the expression of glucose transporter 1 in hepatoma HepG2 cells and improving glucose uptake. These results suggested that a chloroplast expression is a promising approach for the production of bioactive recombinant hFGF21.

Stable and efficient expression of human brain-derived neurotrophic factor in tobacco chloroplasts.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is an intensively studied neurotrophin that promotes various physiological processes, such as acceleration of cell proliferation and differentiation, and is, therefore widely used in clinical applications. METHODS AND RESULTS: In this study, an expression vector with a codon-optimized hBDNF gene was constructed and transferred into chloroplasts of tobacco by gene-gun. After three or four rounds of selection with optimal spectinomycin concentration, hBDNF was integrated into the chloroplast genome of homoplastomic plants, as confirmed by PCR and Southern hybridization. ELISA indicated that hBDNF fused with GFP represented approximately 15.72% ± 0.33% of total soluble protein in the leaves of transplastomic plants. Moreover, the chloroplast-derived hBDNF displayed biological activity similar to the commercial product. CONCLUSIONS: This is the first case report of hBDNF expression by chloroplast transformation in the plant model, providing an additional pathway for the production of chloroplast-expressed therapeutic proteins.

Production of active human FGF21 using tobacco mosaic virus-based transient expression system.

Fibroblast growth factor (FGF) family has a wide range of metabolic processes. FGF21 exerts critical physiological functions in clinical application. This study aimed to explore a convenient and highly efficient approach for rhFGF21 expression using TMV-TES. Firstly, the vector pTTEV-GFP was constructed, followed by optimisation of the expression parameters in Nicotiana benthamiana. Then, the rhFGF21 encoding gene harbouring vector pTTEV-rhFGF21 was constructed. Agrobacterium-mediated vacuum infiltration was performed with the optimised parameters and the expression of rhFGF21 was confirmed by the immunoblotting analysis. ELISA revealed that the protein accumulation of rhFGF21 accounts for 0.11% of total soluble proteins. The biological activity was evaluated and the results suggested that tobacco-expressed rhFGF21 could stimulate the glucose uptake in swiss 3T3-L1 adipocytes, which was similar to the activity of commercial products, suggesting its native biological activity. Therefore, using TMV-TES to express rhFGF21 will be a feasible approach for the mass production of rhFGF21.

Stable plastid transformation of rice, a monocot cereal crop.

Rice is one of the most important cereal crops and its biotechnology has been pursued to meet the food demand of ever-growing global population. Rice plastid transformation has been a great challenge to achieve homoplastomic plants due to its low efficiency of regeneration. In this experiment, Japonica rice line 19 was chosen to be the receptor for plastid transformation. A vector harboring smGFP gene was constructed and transferred into rice plastid genome by bombardment. The resistant callus was obtained after long-lasting multiple selections and proved to be in homoplastomic status by molecular testing. The plantlet was regenerated from homoplastomic callus and grown to seeding stage. This is the first case so far to achieve the homoplastomic rice and will be helpful to transform plastid genome of monocotyledonous crops with recalcitrant nature.

Potential large scale production of meningococcal vaccines by stable overexpression of fHbp in the rice seeds.

Factor H binding protein (fHbp) is the most promising vaccine candidate against serogroup B of Neisseria meningitidis which is a major cause of morbidity and mortality in children. In order to facilitate large scale production of a commercial vaccine, we previously used transgenic Arabidopsis thaliana, but plant-derived fHbp is still far away from a commercial vaccine due to less biomass production. Herein, we presented an alternative route for the production of recombinant fHbp from the seeds of transgenic rice. The OsrfHbp gene encoding recombinant fHbp fused protein was introduced into the genome of rice via Agrobacterium-mediated transformation. The both stable integration and transcription of the foreign OsrfHbp were confirmed by Southern blotting and RT-PCR analysis respectively. Further, the expression of fHbp protein was measured by immunoblotting analysis and quantified by ELISA. The results indicated that fHbp was successfully expressed and the highest yield of fHbp was 0.52 ±â€¯0.03% of TSP in the transgenic rice seeds. The purified fHbp protein showed good antigenicity and immunogenicity in the animal model. The results of this experiment offer a novel approach for large-scale production of plant-derived commercial vaccine fHbp.

Production of Bioactive Recombinant Bovine Chymosin in Tobacco Plants.

Chymosin (also known as rennin) plays an essential role in the coagulation of milk in the cheese industry. Chymosin is traditionally extracted from the rumen of calves and is of high cost. Here, we present an alternative method to producing bovine chymosin from transgenic tobacco plants. The CYM gene, which encodes a preprochymosin from bovine, was introduced into the tobacco nuclear genome under control of the viral 35S cauliflower mosaic promoter. The integration and transcription of the foreign gene were confirmed with Southern blotting and reverse transcription PCR (RT-PCR) analyses, respectively. Immunoblotting analyses were performed to demonstrate expression of chymosin, and the expression level was quantified by enzyme-linked immunosorbent assay (ELISA). The results indicated recombinant bovine chymosin was successfully expressed at an average level of 83.5 ng/g fresh weight, which is 0.52% of the total soluble protein. The tobacco-derived chymosin exhibited similar native milk coagulation bioactivity as the commercial product extracted from bovine rumen.

Stable Expression of Basic Fibroblast Growth Factor in Chloroplasts of Tobacco.

Basic fibroblast growth factor (bFGF) is a multifunctional factor in acceleration of cell proliferation, differentiation and transference, and therefore widely used in clinical applications. In this study, expression vector pWX-Nt03 harboring a codon-optimized bFGF gene was constructed and introduced into the tobacco chloroplasts by particle bombardment. After four rounds of selection, bFGF was proved to integrate into the chloroplast genome of regenerated plants and two of four transgenic plants were confirmed to be homoplastomic by PCR and Southern hybridization. ELISA assay indicated that bFGF represented approximately 0.1% of total soluble protein in the leaves of transplastomic tobacco plants. This is the first report of bFGF expression via chloroplast transformation in model plant, providing an additional option for the production of chloroplast-produced therapeutic proteins.

Chloroplast-expressed MSI-99 in tobacco improves disease resistance and displays inhibitory effect against rice blast fungus.

Rice blast is a major destructive fungal disease that poses a serious threat to rice production and the improvement of blast resistance is critical to rice breeding. The antimicrobial peptide MSI-99 has been suggested as an antimicrobial peptide conferring resistance to bacterial and fungal diseases. Here, a vector harboring the MSI-99 gene was constructed and introduced into the tobacco chloroplast genome via particle bombardment. Transformed plants were obtained and verified to be homoplastomic by PCR and Southern hybridization. In planta assays demonstrated that the transgenic tobacco plants displayed an enhanced resistance to the fungal disease. The evaluation of the antimicrobial activity revealed that the crude protein extracts from the transgenic plants manifested an antimicrobial activity against E. coli, even after incubation at 120 °C for 20 min, indicating significant heat stability of MSI-99. More importantly, the MSI-99-containing protein extracts were firstly proved in vitro and in vivo to display significant suppressive effects on two rice blast isolates. These findings provide a strong basis for the development of new biopesticides to combat rice blast.

Dramatic genotypic difference in, and effect of genetic crossing on, tissue culture-induced mobility of retrotransposon Tos17 in rice.

KEY MESSAGE : We show for the first time that intraspecific crossing may impact mobility of the prominent endogenous retrotransposon Tos17 under tissue culture conditions in rice. Tos17, an endogenous copia retrotransposon of rice, is transpositionally active in tissue culture. To study whether there exists fundamental genotypic difference in the tissue culture-induced mobility of Tos17, and if so, whether the difference is under genetic and/or epigenetic control, we conducted this investigation. We show that dramatic difference in tissue culture-induced Tos17 mobility exists among different rice pure-line cultivars sharing the same maternal parent: of the three lines studied that harbor Tos17, two showed mobilization of Tos17, which accrued in proportion to subculture duration, while the third line showed total quiescence (immobility) of the element and the fourth line did not contain the element. In reciprocal F1 hybrids between Tos17-mobile and -immobile (or absence) parental lines, immobility was dominant over mobility. In reciprocal F1 hybrids between both Tos17-mobile parental lines, an additive or synergistic effect on mobility of the element was noticed. In both types of reciprocal F1 hybrids, clear difference in the extent of Tos17 mobility was noted between crossing directions. Given that all lines share the same maternal parent, this observation indicates the existence of epigenetic parent-of-origin effect. We conclude that the tissue culture-induced mobility of Tos17 in rice is under complex genetic and epigenetic control, which can be either enhanced or repressed by intraspecific genetic crossing.

Transformation of alfalfa chloroplasts and expression of green fluorescent protein in a forage crop.

The ability to transform chloroplasts in multiple species is important for improving agricultural traits. Chloroplast transformation of alfalfa (Medicago sativa L.), a useful forage plant with high market value, was achieved using a vector carrying aadA and gfp genes being introduced into the chloroplasts of alfalfa via particle bombardment using leaves and calli as explants. Resistant somatic embryos were generated and developed into plantlets from explants. The transformation efficiency was 1.3% for callus explants and 2.7% for leaf explants. PCR and Southern blotting analyses revealed that the foreign genes were integrated into the transformed chloroplast genome. The occurrence of GFP was further confirmed by fluorescence microscopy. Expression of foreign genes in alfalfa chloroplasts is therefore possible, and provides a novel means for genetic improvement of agronomically important traits and production of value-added proteins.

Induced Pib Expression and Resistance to Magnaporthe grisea are Compromised by Cytosine Demethylation at Critical Promoter Regions in Rice.

Pib is a well-characterized rice blast-resistance gene belonging to the nucleotide binding site (NBS) and leucine-rich repeat (LRR) superfamily. Expression of Pib was low under non-challenged conditions, but strongly induced by the blast-causing fungal pathogen Magnaporthe grisea, thereby conferring resistance to the pathogen. It is generally established that cytosine methylation of the promoter-region often plays a repressive role in modulating expression of the gene in question. We report here that two critical regions of the Pib promoter were heavily CG cytosine-methylated in both cultivars studied. Surprisingly, induced expression of Pib by M. grisea infection did not entail its promoter demethylation, and partial demethylation by 5-azacytidine-treatment actually reduced Pib expression relative to wild-type plants. Accordingly, the blast disease-resistance was compromised in the 5'-azaC-treated plants relative to wild-type. In contrast, the disease susceptibility was not affected by the 5'-azaC treatment in another two rice cultivars that did not contain the Pib gene, ruling out effects of other R genes and non-specific genotoxic effects by the drug-treatment as a cause for the compromised Pib-conditioned blast-resistance. Taken together, our results suggest that promoter DNA methylation plays a novel enhancing role in conditioning high-level of induced expression of the Pib gene in times of M. grisea infection, and its conferred resistance to the pathogen.

20(S)-Ginsenoside Rg3 Inhibits Lung Cancer Cell Proliferation by Targeting EGFR-Mediated Ras/Raf/MEK/ERK Pathway.

Lung cancer is the leading cause of cancer death in the world and classified into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). As tyrosine kinase inhibitors (TKIs), several triterpenoid saponins can target to epidermal growth factor receptor (EGFR), a widely used molecular therapeutic target, to exhibit remarkable anti-proliferative activities in cancer cells. As one of triterpenoid saponins, 20([Formula: see text])-ginsenoside Rg3 [20([Formula: see text])-Rg3] was confirmed to be an EGFR-TKI in this work. According to the quantitative real-time reverse transcription-PCR (qRT-PCR) and immunoblotting analysis, 20([Formula: see text])-Rg3 was certified to play a key role on EGFR/Ras/Raf/MEK/ERK signal pathway regulation. Our data demonstrated that 20([Formula: see text])-Rg3 might block the cell cycle at the G0/G1 phase by downregulating CDK2, Cyclin A2, and Cyclin E1. Molecular docking suggested that the combination of both hydrophobic and hydrogen-bonding interactions may help stabilizing the 20([Formula: see text])-Rg3-EGFR binding. Furthermore, their binding stability was assessed by molecular dynamics simulation. Taken together, these data provide the evidence that 20([Formula: see text])-Rg3 could prohibit A549 cell proliferation, probably by arresting the cell cycle at the G0/G1 phase via the EGFR/Ras/Raf/MEK/ERK pathway.

Over-expression of fHbp in Arabdopsis for development of meningococcal serogroup B subunit vaccine.

Due to lack of commercial vaccine against the serogroup B (MenB) of Neisseria meningitides, the incidence of meningococcal disease remains high. To solve the issue, transgenic plants are used as bioreactors to produce a plant-derived fHbp subunit vaccine. In this study, the fHbp gene was optimized according to the codon usage bias of Arabidopsis thaliana, synthesized artificially, cloned into an expression vector, driven by a seed-specific promoter, and introduced into A. thaliana by Agrobacterium-mediated floral-dip transformation. Transgenic plants were identified by glufosinate selection, quickstix strips for PAT/bar tests and PCR analysis. The five plants showing higher expression of recombinant fHbp were screened through indirect ELISA. Southern blot analysis showed that the transgenic line rHF-22 had a single-copy integration and the highest expression of fHbp. Recombinant fHbp was purified from seeds of rHF-22 by nitrilotriacetic acid-mediated affinity chromatography, and the purity was 82.5%. BALB/c mice were tested for fHbp vaccine protection from lethal MenB infection, and the relative percent survival was found to be 80%. This study indicates that the recombinant fHbp produced from seeds of rHF-22 is a potential candidate for commercial MenB vaccine. It also provides a reference for safe, cheap and large-scale production of other plant-made vaccines.

Integration and Expression of gfp in the plastid of Medicago sativa L.

Here we describe a protocol of alfalfa (Medicago sativa L.) plastid transformation by which gfp, a gene encoding the green fluorescent protein (GFP), is inserted into plastid genome via particle bombardment and homoplastomic plant is obtained. Plastid engineering is likely to make a significant contribution to the genetic improvement of this crop and the production of vaccines and therapeutic proteins.

Sequencing of the chloroplast genomes of cytoplasmic male-sterile and male-fertile lines of soybean and identification of polymorphic markers.

The RN-type cytoplasmic male sterility (CMS) system used to develop Hybsoy-1, the first commercial hybrid soybean, has been subsequently applied to generate nearly all released soybean hybrids. Although more than 3 years are needed to classify sterile (S) and normal male-fertile (F) cytoplasms by conventional crossing, such classifications can be performed rapidly using organellar DNA-based molecular markers. Except for fertility, the agronomic traits of CMS hybrid soybean sterile and maintainer lines are identical. Consequently, it is difficult to distinguish them by routine visual inspection in the mixture arising in the course of field planting and harvesting during breeding. In this study, we performed next-generation sequencing of chloroplast DNAs of F- and S-cytoplasmic soybeans, assembled and annotated the genomes, and identified polymorphisms distinguishing them. Chloroplast DNAs of F and S cytoplasms were very similar in size (152,215 and 152,222 base pairs) and GC contents (35.37%). Among 23 shared SNPs in gene coding regions, we identified four that could be used in conjunction with restriction endonucleases to distinguish S and F cytoplasms. Although CMS is likely associated with mitochondrial DNA, maternal transmission of mitochondrial and chloroplast DNAs allows polymorphisms in either genome to be used to classify soybean cytoplasms, aiding hybrid soybean cultivar development.

A new approach for obtaining rapid uniformity in rice (Oryza sativa L.) via a 3x x 2x cross.

A triploid (2n = 3x = 36) rice plant was obtained by screening a twin seedling population in which each seed germinated to two or three sprouts that were then crossed with diploid plants. One diploid plant was chosen among the various F(1) progenies and developed into an F (2) population via self-pollination. Compared with the control variety Shanyou 63, this F (2) population had a stable agronomical performance in field trials, as confirmed by the F-test. The stability of the F (2) population was further substantiated by molecular analysis with simple sequence repeat markers. Specifically, of 160 markers assayed, 37 (covering all 12 chromosomes) were polymorphic between the parental lines. Testing the F (1) hybrid individually with these markers showed that each PCR product had only a single band instead of two bands from each parent. The bands were identical to either maternal (23 markers) or paternal (eight markers) bands or distinct from both parents (six markers). The amplified bands of all 60 randomly selected F (2) plants were uniform and identical to those of the F (1) hybrid. These results suggest that the F (1) plant is a non-segregating hybrid and that a stable F (2) population was obtained. This novel system provides an efficient means for shortening the cycle of hybrid rice seed production.