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 β-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.     

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.     

Effect of an enhanced CaMV 35S promoter and a fruit-specific promoter on uida gene expression in transgenic tomato plants

Summary Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T₀ and T₁ plants, and assayed for β-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T₁ plants segregated in a 3∶1 Mendelian ratio.     

Expression of functional elements inserted into the 35S promoter region of infectious cauliflower mosaic virus replicons.

We describe experiments directed towards development of cauliflower mosaic virus (CaMV) replicons for propagation of functional elements during infection of plants. Modifications and inserts were introduced into replaceable domains associated with the 35S promoter. The 35S enhancer (-208 to -56) was found to potentiate promoter activity when in reverse orientation sufficient to establish systemic infection. However, replacement of the 35S enhancer with that from the nos promoter caused loss of infectivity. A 31 bp oligonucleotide containing a polypurine tract specifying initiation of CaMV plus strand DNA synthesis was inserted into a 35S enhancer deletion mutant and propagated in plants. Analysis of progeny DNA showed the presence of an additional discontinuity at its new location in the 35S enhancer, indicating that the artificial primer had functioned correctly in an ectopic site. An intron and flanking sequences from the RNA leader of the Arabidopsis phytoene desaturase (pds) gene, when inserted into the 35S enhancer in forward orientation was very efficiently spliced during infection. The CaMV replicon carrying the pds gene fragment produced unusual infection characteristics, with plants showing early symptoms and then recovering. We conclude that infectious CaMV replicons can be used to carry a variety of elements that target both viral and host functions.     

Relationship Between Ω as well as the Length of 3′poly (dA) and Efficiency of Gene Expression

Three plant high expression vectors harboring 25, 50 and 100 deoxyadenylate (dA) residues respectively in 3' untranslated region (3'-UTR) were constructed by inserting poly(dA) sequence into the primary vector containing CaMV 35S promoter doubled with region B and II which is a leader sequence derived from tobacco mosaic virus, within 5'-UTR. Transient expression of chimeric GUS gene in transgenic tobacco (Nicotiana tabacum L. ) mesophyll protoplasts showed that:doubled enhancer, Ω and poly (dA) increasd GUS expression. When both Ω and poly (dA) were present, the level of expression increased further, compared to that when only Ω was present. Moreover, when Ω was present, doubling the length of poly (dA) resulted in a further increase in GUS expression, which suggested a positive relationship between poly(dA) length and the level of expression.     

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     

A chemically inducible cucumber mosaic virus amplicon system for expression of heterologous proteins in plant tissues

A novel cucumber mosaic virus inducible viral amplicon (CMViva) expression system has been developed that allows for tightly regulated chemically inducible expression of heterologous genes in plant hosts. Transient production of recombinant α₁-antitrypsin (rAAT), a human blood protein, was demonstrated in Nicotiana benthamiana leaves. The highest production levels were obtained by co-infiltrating leaves with Agrobacterium tumefaciens cells containing CMViva carrying the AAT gene and A. tumefaciens cells carrying a binary vector constitutively expressing the gene silencing suppressor p19. Accumulation of up to thirty-fold more rAAT was observed in leaves (24 mg per 100 g leaf tissue) when compared with the expression levels observed using the cauliflower mosaic virus (CaMV) 35S promoter. Significantly, 70% of the rAAT produced using the CMViva expression system was found to be biologically active, a 170-fold increase in functional protein compared with the CaMV 35S expression system.     

Evidence for a Dual Strategy in the Expression of Cauliflower Mosaic Virus Open Reading Frames I and IV

Studies have indicated that cauliflower mosaic virus (CaMV) gene expression is mediated by the translation of polycistronic 35S pregenomic RNA, but the involvement of some minor subgenomic RNA species is also suspected. We examined the involvement of the 35S promoter in the expression of CaMV open reading frames (ORFs) I and IV using both 35S RNA-driven and promoter-less ORF I- and ORF IV-β-glucuronidase (GUS) fusion constructs. In addition to the 35S promoter-dependent expression of both ORF I- and IV-GUS fusions, we detected the 35S promoter-independent expression of both fusion genes via subgenomic mRNAs, which were detected by Northern blotting in the protoplasts transfected with the 35S promoter-driven constructs as well as in those transfected with the promoter-less constructs. These results suggest the involvement of subgenomic RNAs in the expression of CaMV ORFs I and IV, and the operation of a dual strategy in the expression of two viral genes.     

CaMV 35S promoter directs β-glucuronidase expression in Ganoderma lucidum and Pleurotus citrinopileatus

The cauliflower mosaic virus (CaMV) 35S promoter has been most commonly used in plant transformation studies, but its activity in mushrooms has not been reported. p301-b is a binary vector containing a bialaphos resistance gene driven by the promoter of Lentinus edodes glyceraldehyde-3-phosphate dehydrogenase (GPD) gene. CaMV 35S-GUS was inserted into p301-b, and the resulting construct p301-bG was transformed to protoplasts of Ganoderma lucidum and basidiospores of Pleurotus citrinopileatus. GUS activity was observed in the transformants, indicating that CaMV 35S promoter can direct expression of exogenous gene in the mushrooms. This is the first report on the application of CaMV 35S promoter in genetic modification of mushrooms.     

伪狂犬病毒gD基因在转基因烟草中的表达

将猪伪狂犬病毒 (pseudorabiesvirus ,PRV)最主要的保护性抗原基因gD完整编码区亚克隆到修饰的植物双元表达载体pBI 35SL中 ,使其置于强启动子CaMV 35S doubleenhancer TEV 5′UTR下游 ,构建的转基因植物双元表达质粒经农杆菌介导转化烟草 .PCR检测叶片筛选阳性植株 ,Southern杂交进一步证实gD已整合到转基因烟草基因组中 .固相酶联斑点试验和Western印迹表明 ,gD在烟草获得正确表达并具有抗原性     

Effect of untranslated leader sequence of AMV RNA 4 and signal peptide of pathogenesis-related protein 1b on attacin gene expression, and resistance to fire blight in transgenic apple

A cDNA clone of the gene encoding attacin was used to construct three plasmid binary vectors in which attE was controlled by the cauliflower mosaic virus 35S promoter with duplicated upstream B domain (35S) (p35SAtt), 35S with the untranslated leader sequence of alfalfa mosaic virus RNA 4 (AMV) (p35SAMVAtt), and 35S with AMV and the signal peptide of pathogenesis-related protein 1b from tobacco (SP) (p35SAMVSPAtt), respectively. These plasmids and pLDB15 containing attE under the control of the potato proteinase inhibitor II (Pin2) promoter were used in Agrobacterium-mediated transformation of the apple scion cultivar `Galaxy' and the apple rootstock M.26 to enhance resistance to Erwinia amylovora, the bacterium that causes fire blight. The mean attacin content of transgenic lines containing attacin with AMV was three times higher than lines without AMV. Northern blots suggested that AMV functioned in apple as it does in other plant species by enhancing translation of attE mRNA. Transgenic `Galaxy' lines with attacin fused to SP had lower attacin content than lines without SP. In vitro assays indicated that attacin was partially degraded in the intercellular fluid of apple leaves. However, transgenic `Galaxy' lines transformed with attacin fused to SP had significantly less disease than those without SP suggesting that intercellularly secreted attacin is more effective in reducing E. amylovora infection than intracellularly localized attacin. A negative correlation was observed between attacin content and disease resistance in Pin2Att transgenic `Galaxy' lines following inoculation with E. amylovora, suggesting that attacin enhances resistance to fire blight.     

Comparing constitutive promoters using CAT activity in transgenic tobacco plants

The effectiveness of different promoters for use in transgenic tobacco was compared using a reporter gene expressing chloramphenicol acetyl transferase (CAT). Plasmids with CAT gene controlled by cauliflower mosaic virus 35S (CaMV 35S), rice actin1 (Ract1) and tobacco polyubiquitin (Tubi.u4) promoters were delivered into tobacco plants by Agrobacterium-mediated transformation. The Ract1 promoter, previously shown to be a strong promoter in rice and other monocots, failed to promote strong expression in tobacco. CAT expression was greatest from the vector carrying Tubi.u4 with a 5'UTR and leader intron without a ubiquitin monomer. In transgenic plants harboring the Tubi.u4 promoter, CAT expression was approximately twice that of the CaMV 35S promoter. Our results suggest that foreign genes under the control of a ubiquitin promoter devoid of monomer will be useful for high-level gene expression in tobacco.     

Transient gene expression after electroporation of protoplasts derived from embryogenic maize callus

Protoplasts isolated from embryogenic callus cultures derived from immature embryos ofZea mays L. are suitable for analysis of transient gene expression using electroporation-mediated DNA transfer. Expression of introduced genes is comparable to the levels obtained with protoplasts from Black Mexican Sweet suspension cultures. Two different promoters, that directing synthesis of the 35S RNA of cauliflower mosaic virus and the maizeAdh1 promoter were placed in front of the luciferase reporter gene to assess protoplast gene expression and the impact of an intron on expression level.Abbreviations 35S promoter isolated from CaMV - CaMV cauliflower mosaic virus - Adh1 maize gene encoding Alcohol dehydrogenase-1 enzyme - BMS suspension cultures of the Black Mexican Sweet maize variety     

Cauliflower mosaic virus 35 S promoter directs S phase specific expression in plant cells

Summary An experimental system to study cell cycle specific gene expression in plant cells was developed using protoplasts from tobacco cells synchronized by aphidicolin treatment. Chimeric plasmids consisting either of the chloramphenicol acetyltransferase (CAT) gene downstream of the cauliflower mosaic virus (CaMV) 35 S promoter or the nopaline synthase (nos) promoter were introduced into synchronized protoplasts of four cell cycle stages by electroporation. In the case of the CaMV 35 S promoter cyclic oscillation of CAT activity was observed which paralleled the cell cycle of the recipient cells. The peak of CAT activity was found in the S phase, while no such cyclic change was observed in the case of the nos promoter. This system clearly shows that it is feasible to search for a cell cycle specific promoter. The significance of these observations is discussed in relation to the study of plant cells.     

Transient gene expression in aleurone protoplasts isolated from developing caryopses of barley and wheat

Methods have been developed for the isolation of aleurone protoplasts from developing caryopses of Hordeum vulgare and Triticum aestivum in order to study transient expression of introduced genes. Chimaeric gene constructs were introduced into aleurone protoplasts by polyethylene glycol (PEG). Transient expression directed by the 35S promoter from cauliflower mosaic virus (CaMV) of the reporter gene encoding chloramphenicol acetyl transferase (CAT) was detected in aleurone protoplasts from developing barley and wheat grains. Using a similar construct, CAT activity increased when the alcohol dehydrogenase intron 1 fragment from maize was ligated between the 35S promoter and the CAT coding region. The demonstration of transient expression in protoplasts from developing aleurone layers indicates that they may be useful for investigating tissue and developmental control of genes coding for cereal seed proteins.