Combination of viral promoter sequences to generate highly active promoters for heterologous therapeutic protein over-expression in plants

To develop strong promoters for protein over-expression in both dicots and monocots, we constructed a new family of chimeric promoters using sequences of the Commelina Yellow Mosaic Virus (CoYMV), of the Cassava Vein Mosaic Virus (CsVMV) and activating sequences from the CaMV 35S promoter. The chimeric promoters were cloned upstream from the gusA reporter gene. The constructs were used in transient expression experiments, via DNA-coated gold particle delivery to tobacco leaves and maize endosperms. The results showed that candidates among the chimeric promoters could drive expression of the reporter gene to very high levels in the dicot plant tobacco, and all chimeric promoters showed higher expression in maize endosperm than the maize γ-zein promoter used as reference for the monocot expression. Expression cassettes were then used in stable tobacco transformation. Determination of GUS activity throughout growth of the primary transformants showed that two promoters (MPr1163 and MPr1165) could drive expression three to five-fold higher than the highly efficient enhanced 35S promoter. The use of MPr1163 was additionally validated for successful heterologous protein production of human lactoferrin in tobacco via agroinfiltration.     

Spacer length-dependent protection of specific activity of pollen and/or embryo promoters from influence of CaMV 35S promoter/enhancer in transgenic plants

The influence of the CaMV 35S promoter/enhancer on expression profiles of four Arabidopsis thaliana pollen- and/or embryo-specific promoters, APRS, ESL, MXL, and DLL, was tested in transgenic tobacco plants. Individual promoters were fused to the gus reporter gene and cloned in head-to-head orientation with the CaMV 35S:hpt expression unit within the same T-DNA. With the exception of the TATA-less promoter DLL, all other combinations generated interactions between the promoter under investigation and 35S promoter/enhancer resulting in ectopic β-glucuronidase (GUS) expression in vegetative organs and tissues, the most susceptible being the stem, followed by callus, leaf, and root. To eliminate this crosstalk, DNA spacers of length 1, 2 and 5 kb were cloned between the interacting sequences. Ectopic GUS staining was dependent on the affected promoter as well as the distance between the 5′-end of the CaMV 35S promoter and the reporter gene translation start site. When this distance was less than 1 kb strong ectopic GUS staining was observed in all vegetative tissues, similar to the CaMV35S:gus expression profile in transgenic tobacco plants. Insertion of spacer DNA sequences of increasing length resulted in gradual reduction of ectopic GUS staining in tested plants. Of the tissues and organs related to plant reproduction, only anthers and seed coats in the early stages of seed development showed ectopic GUS staining. Developing pollen and embryos showed a pattern of GUS activity consistent with the predicted role of a developmental stage-specific promoter in transgenic tobacco plants.     

Transient gene expression in tobacco protoplasts: II. Comparison of the reporter gene systems for CAT,NPT II,and GUS

The reporter genes for Chloramphenicolacetyltransferase (CAT), Neomycinphosphotransferase-(NPT)-II and -Glucuronidase (GUS) were compared in transient gene expression experiments in tobacco mesophyll protoplasts. For this purpose, nearly identical chimeric genes controlled by the CaMV 35 S promoter were constructed. The detection level of each system was determined yielding the following order of relative sensitivity: CAT相似文献   

Functional Characterization of a Strong Bi-directional Constitutive Plant Promoter Isolated from Cotton Leaf Curl Burewala Virus

Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells.     

The effects of promoter on transient expression in conifer cell lines

Summary Protoplasts from suspension cultures of somatic embryos of white spruce (Picea glauca Moench Voss) were electroporated with plasmids containing the chimeric genes for chloramphenicol acetyl transferase (CAT) or -glucuronidase (GUS), under control of one of three promoters. Transient CAT gene expression of approximately equal magnitude resulted when the CAT gene was fused to either the cauliflower mosaic virus (CaMV) 35S promoter or the nopaline synthase (NOS) promoter. When the CAT gene was fused to a tandem repeat CaMV 35S promoter (pPBI-363), CAT enzyme activity compared to NOS or 35S promoters increased up to eightfold (cell line WS-34), and were up to 100-fold greater than control (electroporated without plasmid). Comparatively, protoplasts of black spruce (Picea mariana Mill) and jack pine (Pinus banksiana Lamb.), electroporated with pPBI-363, produced increases in CAT activity compared to control of 90-fold and 70-fold, respectively. White spruce (WS-34) protoplasts were subsequently electroporated with the GUS gene fused to the tandem repeat CaMV 35S promoter. Comparatively, GUS enzyme activity increased up to tenfold compared to GUS fused to a CaMV 35S promoter. The results indicated that transient expression of the CAT and GUS genes was influenced by the type of promoter and cell line used, as well as by electroporation conditions.NRCC No. 30498     

Cloning of a new LEA1 gene promoter from soybean and functional analysis in transgenic tobacco

LEA1 gene from Glycine max can be expressed in late-embryo stage of plants, and respond to salinity and dehydration stress. To elucidate the mechanism for stress tolerance and high expression in seeds, we isolated and characterized the promoter of LEA1 gene (EQ, 1997 bp) starting the 5′LEA1 coding region. A deletion mutant of EQ promoter (ED) and the full length promoter (EQ) were fused to GUS reporter gene and transformed into the tobacco leaf discs. The results indicated that expression of the reporter gene (GUS) could be regulated by EQ promoter, and was stronger than the mutant under the stress conditions. Also, the expression level of GUS gene driven by EQ promoter in transgenic tobacco seeds was significantly higher than that by the mutant promoter, which meant that it had a better tissue-specificity. Therefore, the active domain for the promoter was located between ?1997 and ?1000 bp. Additionally, the activity of EQ promoter was 2.1-, 3.3- and 0.4- times stronger than the activity of promoter CaMV35S under salt (24 h), drought (10 h) or ABA (24 h), respectively. Meanwhile, the GUS activity of EQ promoter in seeds was 1.8-fold stronger compared to the promoter CaMV35S. In summary, the new promoter (EQ) is bi-functional, stress-inducible and seed-specific. These findings provide a further understanding for the regulation of LEA1gene expression, and suggest a new way for improving seed quality under saline and alkaline land.     

Promoter cassettes, antibiotic-resistance genes, and vectors for plant transformation

We have constructed a set of plant transformation vectors, promoter cassettes, and chimeric antibiotic-resistance genes for the transformation and expression of foreign genes in plants sensitive to Agrobacterium infection. The different vectors allow for either concurrent or consecutive selection for kanamycin and hygromycin resistance and have a number of unique restriction sites for the insertion of additional DNA. The promoter cassettes utilize the CaMV 19S and CaMV 35S promoters and are constructed to allow for the easy insertion of foreign genes. The cloned gene can then easily be inserted into the transformation vectors. We have utilized the promoter cassettes to express the hygromycin-resistance gene either from the CaMV 35S or the CaMV 19S promoters, with both chimeric resistance genes allowing for the selection of hygromycin-resistant tobacco plants.     

Activity of promoters of carnation etched ring virus and dahlia mosaic virus in tobacco protoplasts and transgenic plants

Promoters of carnation etched ring virus (CERV) and dahlia mosaic virus (DMV) were cloned into binary vectors pCambia 1304, pCambia 1281Z, and pCambia 1291Z with reporter GFP and GUS genes. Activities of these promoters in tobacco protoplasts and transgenic plants were determined using these constructs. Histochemical GUS analysis demonstrated the absence of tissue-specificity in transgenic plants transformed with these promoters. The quantitative analysis of these promoter activities in transgenic tobacco plants, using 4-methylumbelliferone as a substrate, showed that 35S CaMV, CERV, and DMV promoters displayed approximately similar activities in transgenic tobacco plants.     

Agrobacterium-mediated transformation of commercial melon (Cucumis melo L., cv. Amarillo Oro)

Cotyledon explants of muskmelon (Cucumis melo L., cv. Amarillo Oro) seedlings were co-cultivated with disarmed Agrobacterium tumefaciens strain LBA4404 that contained the binary vector plasmid pBI121.1. The T-DNA region of this binary vector contains the Nopaline synthase/neomycin phosphotransferase II (NPTII) chimeric gene for kanamycin resistance and the Cauliflower Mosaic Virus 35S/-glucuronidase (GUS) chimeric gene. After infection, the cotyledon pieces were placed in induction medium containing 100 mg/l kanamycin. Putative transformed shoots were obtained, followed by the development of morphologically normal plantlets. The transgenic nature of regenerants was demonstrated by polymerase chain reaction, Southern blot analysis, plant growth on medium selective for the transgene (NPTII) and expression of the co-transformed GUS gene. Factors affecting the transformation procedure are discussed.Abbreviations CaMV Cauliflower Mosaic Virus - Cf Cefotaxime - GUS -glucuronidase - Km Kanamycin - MS Murashige and Skoog - NOS nopaline synthase - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction     

Promoters pro-SmAMP1 and pro-SmAMP2 from Wild Plant <Emphasis Type="Italic">Stellaria media</Emphasis> for the Biotechnology of Dicotyledons

Comparative results of the studied effectiveness of two new promoters, pro-SmAMP1 and pro- SmAMP2, from chickweed (Stellaria media L.) in various types of cultivated plants with transient expression and in stable transformants are given. The effectiveness of the promoters was evaluated through the expression of the reporter uidA gene by measuring the activity of its GUS protein product. It was found that the deletion variant (442 bp) of the pro-SmAMP1 promoter was significantly stronger in plants of Nicotiana benthamiana (Domin) with transient expression than the deletion variant (455 bp) of the pro-SmAMP2 promoter. The effectiveness of these short deletion variants of both promoters under transient expression in the plants of rapeseed (Brassica napus L.) and sugar beet (Beta vulgaris L.) was comparable with that of the viral CaMV35S promoter. The functionality of the pro-SmAMP2 promoter in the calluses of common flax plants (Linum usitatissimum L.) was shown. In the homozygous lines of transgenic tobacco plants (Nicotiana tabacum L.), all deletion variants of the pro-SmAMP1 promoter and the shortest version of pro-SmAMP2 were twice as strong as the CaMV35S viral promoter. The effectiveness of short variants of both promoters from the chickweed in controlling the gene encoding neomycin phosphotransferase II in the transgenic plants of tobacco and arabidopsis (Arabidopsis thaliana L.) growing on media supplemented with recommended concentrations of kanamycin are not inferior to the duplicated 2хCaMV35S viral promoter. The obtained experimental data show that short deletion variants of pro-SmAMP1 (442 bp) and pro-SmAMP2 (455 bp) promoters may be recommended as strong constitutive promoters for use in the biotechnology of crop plants.     

The integrative expression of GUS gene driven by FCP promoter in the seaweed Laminaria japonica (Phaeophyta)

In this study, the background activity of β-glucuronidase (GUS) was analyzed histochemically and fluorometrically in the negative control of Laminaria japonica (Phaeophyta) thalli, showing low level of activity. GUS gene transformation without selectable gene in L. japonica was performed using four different promoters, i.e., Cauliflower mosaic virus 35S promoter (CaMV35S) from cauliflower mosaic virus, ubiquitin promoter (UBI) from maize, adenine-methyl transfer enzyme gene promoter (AMT) from virus in green alga Chlorella, and fucoxanthin chlorophyll a/c-binding protein gene promoter (FCP) from diatom Phaeodactylum tricornutum. The GUS transient activity was determined fluorometrically after bombarding sliced parthenogenetic sporophytes explants, and it was found that the activity resulting from CaMV35S and FCP promoters (in 114.3 and 80.6 pmol MU min⁻¹ (mg protein)⁻¹, respectively) was higher than for the other two promoters. The female gametophytes were bombarded and regenerated parthenogenetic sporophytes. FCP was the only promoter that resulted in detectable GUS chimeric expression activity during histochemical staining and polymerase chain reaction. Results of Southern blot showed that GUS gene was integrated with the L. japonica genome.