Double CaMV 35S Promoter Research Articles

A Model for Pharmaceutical Proteins Production from Arabidopsis Suspension Culture

Pharmaceutical proteins production in plant suspension culture has been of interest due to the ease of manipulation, low cost and low allergenic reaction in humans. In this work, the constitutive double CaMV 35S promoter and the abiotic stress-responsive promoter of the Rd29a locus were compared to produce Green Fluorescent Protein (GFP) used as a model for secreted protein in Arabidopsis thaliana suspension culture. Induction of Rd29a inducible promoter by plant hormone abscisic acid (ABA) was also studied. A signal sequence (SS) of a 33 kDa rice protein was added to the constructs to promote secretion. The secretion of GFP into the extracellular spaces of the transgenic seedling was detected under confocal microscope. The fl uorescence intensity of GFP in the suspension culture was measured by fl uorometry. A temporal maximum expression of GFP was observed from the CaMV 35S promoter on day 18. This model can be used as an example to produce pharmaceutical proteins in suspension culture in the future.

Expression of a sphingomyelinase‐coding gene from Trichoderma harzianum conferred bacterial tolerance in tobacco

AbstractDiseases caused by bacteria are an important and widespread constraint, occurring in almost all crops, including vegetables, pulses, cereals, ornamentals, fruit and forages. Although several strategies have been developed to obtain disease resistance in plants using genetic engineering, few studies have effectively demonstrated the control of bacterial diseases. It has previously been reported that a gene encoding a sphingomyelinase (ThSMase), identified in Trichoderma harzianum, is up‐regulated during biocontrol against phytopathogens, and this may have biotechnological applications. SMases are involved in multiple cellular functions, including the immune response against pathogens. We hypothesized that the expression of this gene from fungi in transgenic tobacco could generate resistance to plant pathogens. ThSMase was cloned under control of the double CaMV 35S promoter plus a leader sequence from alfalfa mosaic virus (AMV), and stably introduced and expressed in tobacco. Reverse transcription‐quantitative PCR analysis revealed that the ThSMase gene was expressed at similar levels in all transgenic lines tested. Our results showed no statistically significant difference in susceptibility after challenging transgenic and nontransgenic lines with the fungus Sclerotinia sclerotiorum. However, transgenic tobacco plants revealed a significant resistance to the bacterium Pseudomonas syringae pv. tabaci, and a tolerance to Xylella fastidiosa. Our results demonstrated the strong potential of ThSMase in biotechnological processes such as molecular breeding of the plants.

Effect of Calmodulin-like Gene (CML) Overexpression on Stilbene Biosynthesis in Cell Cultures of Vitis amurensis Rupr.

Stilbenes are plant phenolics known to rapidly accumulate in grapevine and other plants in response to injury or pathogen attack and to exhibit a great variety of healing beneficial effects. It has previously been shown that several calmodulin-like protein (CML) genes were highly up-regulated in cell cultures of wild-growing grapevine Vitis amurensis Rupr. in response to stilbene-modulating conditions, such as stress hormones, UV-C, and stilbene precursors. Both CML functions and stilbene biosynthesis regulation are still poorly understood. In this study, we investigated the effect of overexpression of five VaCML genes on stilbene and biomass accumulation in the transformed cell cultures of V. amurensis. We obtained 16 transgenic cell lines transformed with the VaCML52, VaCML65, VaCML86, VaCML93, and VaCML95 genes (3–4 independent lines per gene) under the control of the double CaMV 35S promoter. HPLC-MS analysis showed that overexpression of the VaCML65 led to a considerable and consistent increase in the content of stilbenes of 3.8–23.7 times in all transformed lines in comparison with the control calli, while biomass accumulation was not affected. Transformation of the V. amurensis cells with other analyzed VaCML genes did not lead to a consistent and considerable effect on stilbene biosynthesis in the cell lines. The results indicate that the VaCML65 gene is implicated in the signaling pathway regulating stilbene biosynthesis as a strong positive regulator and can be useful in viticulture and winemaking for obtaining grape cultivars with a high content of stilbenes and stress resistance.

Improved salinity tolerance and growth performance in transgenic sunflower plants via ectopic expression of a wheat antiporter gene (TaNHX2).

Sunflower (Helianthus annuus. L) is one of the principal oil seed crops affected by the salinity stress, which limits the oil content and crop yield of sunflower plants. The acclimatization of plants to abiotic stresses such as salinity tolerance is mainly mediated by the vacuolar Na+/H+ antiporters (NHX) by tagging Na+ into vacuoles from the cytosol. We show here that the over-expression of wheat TaNHX2 gene in transgenic sunflower conferred improved salinity stress tolerance and growth performance. Transgenic sunflower plants were produced by infecting the embryonic axis ex-plants with Agrobacterium tumefaciens strain EHA105 containing a pBin438-TaNHX2 binary vector that carried a wheat antiporter (TaNHX2) gene under the control of a double CaMV 35S promoter with NPT II gene as a selectable marker. PCR analysis of T0 and T1 transgenic plants confirmed the integration of TaNHX2 in sunflower genome. Stable integration and expression of TaNHX2 in sunflower genome was further verified by Southern hybridization and semi-quantitative RT-PCR analyses. As compared to the non-transformed plants, TaNHX2 expressing transgenic plants showed better growth performance and accumulated higher Na+, K+ contents in leaves and roots under salt stress (200mM NaCl). Transgenic sunflower plants displayed improved protection against cell damage exhibiting stable relative water content, chlorophyll content, increased proline accumulation and improved reactive oxygen species (ROS) scavenging because of higher activities of the antioxidant enzymes like superoxide dismutase and ascorbate peroxidase, along with decreased production of hydrogen peroxide, free oxygen radical and malondialdehyde (MDA) under salt stress (200mM NaCl). Taken together, our findings suggest that TaNHX2 expression in sunflower plants contributed towards improving growth performance under sodium chloride stress.

Cre-mediated marker gene removal for production of biosafe commercial oilseed rape

The Cre/loxP-based self-excision represents a promising strategy for removal of hazardous transgene(s) from genomes of targeted crops prior to their introduction into environment. Here, we applied the Cre/loxP self-excision strategy in which the cre recombinase gene is driven by the embryo-specific CRUC promoter from Arabidopsis thaliana. Besides, the Cre/loxP cassette, the T-DNA also consisted of the β-glucuronidase gene, controlled by the double dCaMV 35S promoter and the selectable neomycin phosphotransferase gene; the latter was aimed to be removed from the transgenic genome. The Cre/loxP self-excision cassette was introduced into genomes of four commercial oilseed rape cultivars Haydn, Heros, Hunter and Topas (Brassica napus L.) via Agrobacterium tumefaciens. Transgenic T0 plants were regenerated from all cultivars. Their detailed molecular analyses revealed premature activation of the CRUC promoter that resulted in partial excision of the nptII gene. Progenies of four self-pollinated T0 lines were further analysed, and the data confirmed complete excision event in 5 out of 105 (4.8%) of T1 transgenic oilseed rape plants. The excision efficiency does not seem to depend on the target cultivar. However, the poor transformation efficiency of rapeseed and the limited specificity of the CRUC promoter are clearly the bottleneck of this approach, and the feasibility of (tissue-specific) self-excision of selectable marker gene from genomes of each commercial rapeseed variety adds to their perspective to cope the increasing negative impacts of climate changes.

Development of Wolffia arrhiza as a Producer for Recombinant Human Granulocyte Colony-Stimulating Factor.

To date, the expression of recombinant proteins in transgenic plants is becoming a powerful alternative to classical expression methods. Special efforts are directed to the development of contained cultivation systems based on cell culture or rhyzosecretion, which reliably prevents the heterologous DNA releasing into the environment. A promising object for the development of such systems is the tiny aquatic plant of Wolffia arrhiza, which can be used as a dipped culture in bioreactors. Herein we have expressed the human granulocyte colony-stimulating factor (hG-CSF) in nuclear-transformed Wolffia. The nucleotide sequence of hG-CSF was optimized for expression in Wolffia and cloned into the vector pCamGCSF downstream of double CaMV 35S promoter. Wolffia plants were successfully transformed and 34 independent transgenic lines with hG-CSF gene were obtained, PCR and Southern blot analysis confirmed the transgenic origin of these lines. Western blot analysis revealed accumulation of the target protein in 33 transgenic lines. Quantitative ELISA of protein extracts from these lines showed hG-CSF accumulation up to 35.5 mg/kg of Wolffia fresh weight (0.194% of total soluble protein). This relatively high yield holds promise for the development of Wolffia-based expression system in strictly controlled format to produce various recombinant proteins.

Enhanced salinity stress tolerance in transgenic chilli pepper (Capsicum annuum L.) plants overexpressing the wheat antiporter (TaNHX2) gene

Transgenic chilli pepper (Capsicum annuum L.) plants tolerant to salinity stress were produced by introducing the wheat Na+/H+ antiporter gene (TaNHX2) via Agrobacterium-mediated transformation. Cotyledonary explants were infected with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBin438 that contains a wheat antiporter (TaNHX2) gene driven by the double CaMV 35S promoter and NPT II gene as a selectable marker. PCR and semiquantitative RT-PCR analysis confirmed that the TaNHX2 gene had been integrated and expressed in the T1 generation of transgenic pepper plants as compared to the non-transformed plants. Southern blot analysis further verified the integration and presence of TaNHX2 gene in the genome of chilli pepper plants. Biochemical assays of these transgenic plants revealed enhanced levels of proline, chlorophyll, superoxide dismutase, ascorbate peroxidase, relative water content, and reduced levels of hydrogen peroxide (H2O2), malondialdehyde compared to wild-type plants under salt stress conditions. The present investigation clearly showed that overexpression of the TaNHX2 gene enhanced salt stress tolerance in transgenic chilli pepper plants.

Overexpression of Arabidopsis thaliana gibberellic acid 20 oxidase (AtGA20ox) gene enhance the vegetative growth and fiber quality in kenaf (Hibiscus cannabinus L.) plants.

Kenaf (Hibiscus cannabinus L.; Family: Malvaceae), is multipurpose crop, one of the potential alternatives of natural fiber for biocomposite materials. Longer fiber and higher cellulose contents are required for good quality biocomposite materials. However, average length of kenaf fiber (2.6 mm in bast and 1.28 mm in whole plant) is below the critical length (4 mm) for biocomposite production. Present study describes whether fiber length and cellulose content of kenaf plants could be enhanced by increasing GA biosynthesis in plants by overexpressing Arabidopsis thaliana Gibberellic Acid 20 oxidase (AtGA20ox) gene. AtGA20ox gene with intron was overexpressed in kenaf plants under the control of double CaMV 35S promoter, followed by in planta transformation into V36 and G4 varieties of kenaf. The lines with higher levels of bioactive GA (0.3–1.52 ng g−1 fresh weight) were further characterized for their morphological and biochemical traits including vegetative and reproductive growth, fiber dimension and chemical composition. Positive impact of increased gibberellins on biochemical composition, fiber dimension and their derivative values were demonstrated in some lines of transgenic kenaf including increased cellulose content (91%), fiber length and quality but it still requires further study to confirm the critical level of this particular bioactive GA in transgenic plants.

VaCPK20 gene overexpression significantly increased resveratrol content and expression of stilbene synthase genes in cell cultures of Vitis amurensis Rupr

Resveratrol, a naturally occurring plant phenol, has been reported to exhibit a wide range of valuable biological and pharmacological properties. In the present investigation, we show that transformation of a Vitis amurensis Rupr. cell suspension with the gene VaCPK20 for a calcium-dependent protein kinase (CDPK) under the control of double CaMV 35S promoter increased resveratrol production in five independently transformed cell lines in 9-68 times compared with control cells. The VaCPK20-transformed calli were capable of producing 0.04-0.42 % dry wt. of resveratrol, while the control calli produced up to 0.008 % dry wt. of resveratrol Also, we characterized expression of stilbene synthase (STS) genes in the five VaCPK20-transgenic cell lines of V. amurensis. In all VaCPK20-transgenic cell lines, expression of VaSTS7 increased; while expression of VaSTS1 decreased. We suggest that transformation of V. amurensis calli with the VaCPK20 gene induced resveratrol accumulation via enhancement of expression of the VaSTS7 gene involved in resveratrol biosynthesis. The obtained data first demonstrate that overexpression of a CDPK gene resulted in increased accumulation of a stilbenoid phytoalexine in transgenic plant cells. We propose that the VaCPK20 gene could play an important role in the regulation of resveratrol biosynthesis in grape cells.

Overexpression of a wheat Na+/H+ antiporter gene (TaNHX2) enhances tolerance to salt stress in transgenic tomato plants (Solanum lycopersicum L.)

Soil salinity is a major environmental stress limiting plant productivity. Vacuole Na+/H+ antiporters play important roles for the survival of plants under salt stress conditions. We have developed salt stress tolerant transgenic tomato plants (Solanum lycopersicum cv. PED) by overexpression of the wheat Na+/H+ antiporter gene TaNHX2 using Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBin438 that contains the TaNHX2 gene under the control of double CaMV 35S promoter and npt II as a selectable marker. PCR and Southern blot analysis confirmed that TaNHX2 gene has been integrated and expressed in the T1 generation transgenic tomato plants. When TaNHX2 expressing plants were exposed to 100 or 150 mM NaCl, they were found to be more tolerant to salt stress compared to wild type plants. Biochemical analyses also showed that transgenic plants have substantial amount of relative water content and chlorophyll content under salt stress conditions compared to wild type plants. The relative water content in transgenic and wild type plants ranged from 68 to 75 % and 46–73 % and chlorophyll content fall in between 1.8 to 2.4 mg/g fw and 1.0 to 2.4 mg/g fw, respectively, in all stress conditions. In the present study, we observed a better germination rate of T1 transgenic seeds under salt stress conditions compared with wild type plants. Our results indicated that TaNHX2-transgenic tomato plants coped better with salt stress than wild type plants.

Expression of anti-neuroexcitation peptide III of scorpion Buthus martensii Karsch BmK ANEP III in plants

Anti-neuroexcitation peptide III of Buthus martensii Karsch (BmK ANEP III) has better anti-epileptic and anticonvulsive effects in the test animal models. The present study is aimed at developing transgenic tomato and tobacco lines overproducing the ANEP III protein. Using the molecular cloning technique, the plant expression vector pBI-ANEP III was constructed successfully. The ANEP III expression cassette included a double CaMV 35S promoter with omega enhancers, the ANEP III gene with the Kozak sequence, the ER retention signal and the NOS terminator. Recombinant plasmids were transferred into Agrobacterium tumefaciens EHA105 by freeze-thaw transformation methods. By the Agrobacterium-mediated leaf disc transformation method, tobacco (Nicotiana tabacum) and tomato (Lycopersicum esculentum) lines were transformed. Transformants were screened and confirmed by PCR, RT-PCR and western blotting analysis. It was demonstrated that the ANEP III gene was successfully expressed in the genomic DNA of transgenic plants. The ANEP III protein was detected by immunofluorescence analysis, and the results confirmed the high amount of ANEP III protein, being 0.81 and 1.08% of total soluble proteins in transgenic tobacco and tomato. The study of plants with high expression levels of ANEP III has an important theoretical and practical significance and provides valuable information for establishing a new, economical and effective system for industrial protein production.

TRANSGENIC BANANA PLANTS RESISTANT TO BANANA BUNCHY TOP VIRUS INFECTION

Embryogenic cell suspensions (ECS) initiated from immature male flowers of banana cultivar 'Dwarf Brazilian' (AAB, Pome subgroup) were transformed using Agrobacterium tumefaciens containing one of four constructs derived from the replicase-associated protein (Rep) gene of the Hawaiian isolate of Banana bunchy top virus (BBTV). Each construct was engineered under control of a double CaMV 35S promoter and the AMV enchancer sequence in the binary plasmid pBI121. Constructs were transferred into A. tumefaciens strain AGLO and used to transform banana ECS. Plantlets that survived antibiotic selection were acclimated to greenhouse conditions and challenged with viruliferous banana aphids (Pentalonia nigronervosa). Ten adult or late instar aphids were allowed to feed for 2-4 weeks on test plants. All test plants were kept in the greenhouse and monitored for symptom expression for a period of 6 months. Control plants transformed with empty vector pBI121 only were included in all tests. A total of 270 test plants and 63 control plants were screened for BBTV resistance using this approach. One of 32 test plants transformed with the M1 (mutant Rep gene) construct, 5 of 74 test plants transformed with the AS1 (antisense Rep gene) construct, 5 of 38 test plants transformed with the PR1 (partial Rep gene) construct, and 10 of 126 test plants transformed with the R/PR1 (full-length Rep gene fused to antisepses partial Rep gene) construct were found to be resistant to BBTV challenge and showed no bunchy top symptoms. All of the control plants became infected with BBTV under these experimental conditions. Plants that survived BBTV challenge were analysed by quantitative PCR (per) and Southern hybridisations to determine the number of transgenes that were present in their genomes. Results from these analyses indicated that the resistant plants contained from 2 to more than 9 copies of the NPTII (kanamycin resistance) transgene carried on the pBI121 plasmid.

AtCPK6, a functionally redundant and positive regulator involved in salt/drought stress tolerance in Arabidopsis

The calcium-dependent protein kinase (CDPK) family is needed in plant signaling during various physiological pathways. The Arabidopsis AtCPK6 gene belongs to the subclass of stress-inducible CDPKs, which is stimulated by salt and osmotic stress. To elucidate the physiological function of AtCPK6, transgenic Arabidopsis plants under the control of double CaMV 35S promoter were obtained. AtCPK6 over-expressing plants showed enhanced tolerance to salt/drought stresses. The elevated tolerance of the AtCPK6 over-expressing plants was confirmed by the change of proline and malondialdehyde (MDA). Real-time PCR analyses revealed that the expression levels of several stress-regulated genes were altered in AtCPK6 over-expressing plants. However, cpk6 mutant displayed no obvious difference with control. These results are likely to indicate that AtCPK6 is functionally redundant and a positive regulator involved in the tolerance to salt/drought stress in Arabidopsis.

Agrobacterium tumefaciens-mediated genetic transformation and plant regeneration from a complex tetraploid hybrid citrus rootstock

Agrobacterium-mediated genetic transformation of a tetraploid “tetrazyg” citrus rootstock selection ‘Orange #16’ [Nova mandarin ( Citrus reticulata Blanco) + Hirado Buntan pummelo ( Citrus grandis L. Osbeck)] × [Cleopatra mandarin ( C. reticulata Blanco) + Argentine trifoliate orange ( Poncirus trifoliata (L.) Raf.)] was performed. Juvenile epicotyl segments were transformed with a construct containing a bifunctional egfp– nptII fusion gene under the control of an enhanced double CaMV 35S promoter. Our protocol resulted in a reasonable transformation efficiency of 18%. Stable integration of the transgene was confirmed by visual observation of EGFP expression, PCR and Southern blot hybridization. The purpose of this work was to investigate the amenability of novel citrus rootstock germplasm being developed for improved tree size control, soil adaptation, and disease resistance, to existing transformation technologies. Seed trees of such transgenic tetraploids also have potential as trap plants containing potent insecticidal transgenes, due to their inedible fruit and inherent crossing barriers with conventional commercial diploid scion cultivars, and could be planted around producing citrus groves.

Agrobacterium-mediated transformation of embryogenic tissues of hybrid firs (Abies spp.) and regeneration of transgenic emblings

A genetic transformation system has been developed for selected embryogenic cell lines of hybrids Abies alba x A. cephalonica (cell lines AC2, AC78) and Abies alba x A. numidica (cell line AN72) using Agrobacterium tumefaciens. The cell lines were derived from immature or mature zygotic embryos on DCR medium containing BA (1 mg l(-1)). The T-DNA of plant transformation vector contained the beta-glucuronidase reporter gene under the control of double dCaMV 35S promoter and the neomycin phosphotransferase selection marker gene driven by the nos promoter. The regeneration of putative transformed tissues started approximately 1 week after transfer to the selection medium containing 10 mg geneticin l(-1). GUS activity was detected in most of the geneticin-resistant sub-lines AN72, AC2 and AC78, and the transgenic nature of embryogenic cell lines was confirmed by PCR approach. Plantlet regeneration from PCR-positive embryogenic tissues has been obtained as well. The presence of both gus and nptII genes was confirmed in 11 out of 36 analysed emblings.

Agrobacterium-mediated genetic transformation of mint with E. coli glutathione synthetase gene

Morphologically identical transgenic mint (Mentha arvensis L.) with bacterial glutathione synthetase gene has been developed. Transformed plants were obtained by co-cultivation of leaf disks with Agrobacterium tumefaciens strain LBA 4404 harbouring a binary vector pCAMBIA-CpGS that carried E. coli glutathione synthetase (GS), β-glucuronidase as reporter gene and nptII as selective marker gene for kanamycin resistance. Using a constitutive double CaMV 35S promoter and an rbcS transit peptide, we successfully addressed CpGS to the chloroplasts through pJIT 117 vector. Preculture and the presence of AS in the co-cultivation medium played a significant role in enhancing transformation frequency. The highest transformation frequency was achieved with MS selection medium supplemented with 25% coconut water, 1.12 mg l−1 BAP, 0.2 mg l−1 NAA, 50 mg l−1 kanamycin and 125 mg l−1 cefotaxime. Robust rooting of regenerated shoots was obtained in half-strength liquid MS medium containing 0.2 mg l−1 NAA and 50 mg l−1 kanamycin. The presence and expression of transgenes in transgenics (T0) was evidenced by GUS histoenzymatic assay, PCR and RT-PCR analysis of nptII and the gene of interest, i.e., GS of putative transgenic leaves. Chromosomal integration of GS gene was confirmed by Southern blot analysis. Transgenic plants were successfully acclimatized in the greenhouse. An overall transformation frequency of 15% was achieved in approximately 3 months of time period. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications.

Transgene expression for Gladiolus plants grown outdoors and in the greenhouse

Transgene expression was evaluated for Gladiolus plants transformed with either the CaMV 35S, double CaMV 35S, rolD, or Arabidopsis UBQ3 promoter controlling the uidA or bean yellow mosaic virus coat protein gene in either the sense or antisense orientation to determine differences in expression for plants grown in the greenhouse and outdoors for two years. There was more variability in GUS expression when plants were grown outdoors than in the greenhouse for two years. Four of the six transformed plant lines with the UBQ3, rolD, and CaMV 35S promoters grown outdoors showed significant differences in GUS expression from year to year as compared to two of the six lines with the UBQ3 and rolD promoters grown in the greenhouse. When grown the same year, two plant lines with the CaMV 35S and one line with the rolD promoter showed 2–16× higher levels of GUS expression outdoors than in the greenhouse, and one plant line with the UBQ3 promoter had 31× higher GUS expression in the greenhouse instead of outdoors. Three of six plant lines transformed with the bean yellow mosaic virus coat protein gene in either the sense or antisense orientation under control of the double CaMV 35S promoter showed obvious transgene expression as compared to three lines that did not show expression or negligible expression for both years when plants were grown both outdoors and in the greenhouse. This study verified long-term gene expression, rather than silencing, for Gladiolus plants when grown outdoors and in the greenhouse from year to year.

Expression of active hBMP2 in transgenic tobacco plants

Bone morphogenetic protein 2 (BMP2) is important for bone tissue repair. The goal of this research is to construct a high level human BMP2 (hBMP2) expression system using transgenic tobacco plants as a bioreactor. Cauliflower mosaic virus (CaMV) 35S promoter, alfalfa mosaic virus (AMV) enhancer, tobacco mosaic virus (TMV) enhancer, matrix attachment regions (MARs) sequence, and "Kozak" sequence were used to construct recombinant expression vectors and the high-expression vectors were screened out through GUS-fusions assay. The promoter is the most important factor; double-CaMV 35S promoter is more effective than single promoter. The AMV or TMV enhancer is able to promote the foreign protein expression. After four-step purification, the activated hBMP2 (0.02% total soluble protein) was obtained. Our results suggested that the transgenic tobacco has great potential to be used as a bioreactor to produce hBMP2.

Effects of codon modification on human BMP2 gene expression in tobacco plants

Bone morphogenetic protein 2 (BMP2) has great potential in therapeutic applications. We are working on generating transgenic plants as a bioreactor to produce BMP2. We have studied the effects of codon optimization on the expression of human BMP2 (hBMP2) in tobacco plants. Three modified hBMP2 genes were transformed into tobacco under the control of either cauliflower mosaic virus 35S (CaMV35S) promoter or double-CaMV35S promoter plus alfalfa mosaic virus (AMV) enhancer. The fused beta-glucuronidase (GUS) reporter gene was used to facilitate the assay of protein expression. The results indicated that codon optimization could increase the protein expression level obviously under CaMV35S promoter. However, under relatively stronger initiation condition (double-CaMV35S promoter plus AMV enhancer), only the gene with the lowest degree of codon optimization could increase the protein expression level. Our findings suggest that the action of codon optimization may be influenced by the factors of promoter strength and A+T content in tobacco plants.

A putative CENTRORADIALIS/ TERMINAL FLOWER 1-like gene, Ljcen1, plays a role in phase transition in Lotus japonicus

CENTRORADIALIS/TERMINAL FLOWER 1 (CEN/TFL1) genes play an important role in the phase transition of plant flowering. Here we characterized the expression pattern of a CEN/TFL1-like gene, Ljcen1, from Lotus japonicus. Sequence analysis revealed that Ljcen1 shared 67-76% identity to its homologs from a variety of plant species. Ljcen1 transcripts could be detected at the young root tip and reproductive shoot apical meristem of L. japonicus. RNA in situ hybridization analysis revealed that Ljcen1 was continuously expressed in the sub-domain of the primary inflorescence meristem and transiently expressed in the secondary inflorescence meristem. The ectopic expression of Ljcen1 in Arabidopsis driven by double CaMV 35S promoter delayed the flowering. These results suggested that Ljcen1 gene was involved in a conserved CEN/TFL1 pathway that functions in phase transition of shoot apical meristem in L. japonicus.