A Comparison of Constitutive Promoters for Expression of Transgenes in Alfalfa (Medicago Sativa)

The activity of constitutive promoters was compared in transgenic alfalfa plants using two marker genes. Three promoters, the 35S promoter from cauliflower mosaic virus (CaMV), the cassava vein mosaic virus (CsVMV) promoter, and the sugarcane bacilliform badnavirus (ScBV) promoter were each fused to the beta-glucuronidase (gusA) gene. The highest GUS enzyme activity was obtained using the CsVMV promoter and all alfalfa cells assayed by in situ staining had high levels of enzyme activity. The 35S promoter was expressed in leaves, roots, and stems at moderate levels, but the promoter was not active in stem pith cells, root cortical cells, or in the symbiotic zones of nodules. The ScBV promoter was active primarily in vascular tissues throughout the plant. In leaves, GUS activity driven by the CsVMV promoter was approximately 24-fold greater than the activity from the 35S promoter and 38-fold greater than the activity from the ScBV promoter. Five promoters, the double 35S promoter, figwort mosaic virus (FMV) promoter, CsVMV promoter, ScBV promoter, and alfalfa small subunit Rubisco (RbcS) promoter were used to control expression of a cDNA from Trichoderma atroviride encoding an endochitinase (ech42). Highest chitinase activity in leaves, roots, and root nodules was obtained in plants containing the CsVMV:ech42 transgene. Plants expressing the endochitinase were challenged with Phoma medicaginis var. medicaginis, the causal agent of spring black stem and leaf spot of alfalfa. Although endochitinase activity in leaves of transgenic plants was 50- to 2650-fold greater than activity in control plants, none of the transgenic plants showed a consistent increase in disease resistance compared to controls. The high constitutive levels of both GUS and endochitinase activity obtained demonstrate that the CsVMV promoter is useful for high-level transgene expression in alfalfa.     

Expression and Characterization of Hepatitis B Surface Antigen in Transgenic Potato Plants

Transgenic potato plants expressing the gene of hepatitis B surface antigen (HBsAg) under the control of the double promoter of 35S RNA of cauliflower mosaic virus (CaMV 35SS) and the promoter of patatin gene of potato tubers have been obtained. Biochemical analysis of the plants was performed. The amount of HBsAg in leaves, microtubers, and tubers of transgenic potatoes growing in vitro and in vivo was 0.005-0.035% of the total soluble protein. HBsAg content reached 1 microg/g in potato tubers and was maximal in plants expressing the HBsAg gene under the control of CaMV 35SS promoter. In transgenic plants expressing HBsAg gene under the control of tuber-specific patatin promoter, HBsAg was found only in microtubers and tubers and was absent in leaves. Western blot analysis of HBsAg eluted from immunoaffinity protein A-Sepharose matrix has been performed. The molecular weight of HBsAg peptide was approximately 24 kD, which is in agreement with the size of the major protein of the envelope of hepatitis B virus. Using gel filtration, it was determined that the product of HBsAg gene expression in potato plants is converted into high-molecular-weight multimeric particles. Therefore, as well as in recombinant HBsAg-yeast cells, assembling of HBsAg monomers into immunogenic aggregates takes place in HBsAg-transgenic potato, which can be used as a source of recombinant vaccine against hepatitis B virus.     

Preferential expression of a plant cystatin at nematode feeding sites confers resistance to Meloidogyne incognita and Globodera pallida

The expression patterns of three promoters preferentially active in the roots of Arabidopsis thaliana have been investigated in transgenic potato plants in response to plant parasitic nematode infection. Promoter regions from the three genes, TUB-1, ARSK1 and RPL16A were linked to the GUS reporter gene and histochemical staining was used to localize expression in potato roots in response to infection with both the potato cyst nematode, Globodera pallida and the root-knot nematode, Meloidogyne incognita. All three promoters directed GUS expression chiefly in root tissue and were strongly up-regulated in the galls induced by feeding M. incognita. Less activity was associated with the syncytial feeding cells of the cyst nematode, although the ARSK1 promoter was highly active in the syncytia of G. pallida infecting soil grown plants. Transgenic potato lines that expressed the cystatin OcIDeltaD86 under the control of the three promoters were evaluated for resistance against Globodera sp. in a field trial and against M. incognita in containment. Resistance to Globodera of 70 +/- 4% was achieved with the best line using the ARSK1 promoter with no associated yield penalty. The highest level of partial resistance achieved against M. incognita was 67 +/- 9% using the TUB-1 promoter. In both cases this was comparable to the level of resistance achieved using the constitutive cauliflower mosaic virus 35S (CaMV35S) promoter. The results establish the potential for limiting transgene expression in crop plants whilst maintaining efficacy of the nematode defence.     

The Commelina yellow mottle virus promoter is a strong promoter in vascular and reproductive tissues.

Commelina yellow mottle virus (CoYMV) is a double-stranded DNA virus that infects the monocot Commelina diffusa. Although CoYMV and cauliflower mosaic virus (CaMV; another double-stranded DNA virus) probably replicate by a similar mechanism, the particle morphology and host range of CoYMV place it in a distinct group. We present evidence that a prompter fragment isolated from CoYMV confers a tissue-specific pattern of expression that is different from that conferred by the CaMV 35S promoter. When the CoYMV promoter is used to drive expression of the beta-glucuronidase reporter gene in stably transformed tobacco plants, beta-glucuronidase activity occurs primarily in the phloem, the phloem-associated cells, and the axial parenchyma of roots, stems, leaves, and flowers. Activity is also detected throughout the anther, with highest activity in the tapetum. In contrast, the CaMV 35S promoter is active in most cell types. The CoYMV promoter is a strong promoter, and when the activity of the CoYMV promoter is compared with that of a duplicated CaMV 35S promoter, it is 30% as active in tobacco suspension cells and up to 25% as active in maize suspension cells. These properties of the CoYMV promoter make it potentially useful for high-level expression of engineered genes in vascular cells.     

Plant selection principle based on xylose isomerase

Summary The xylose isomerase genes (xylA) from Thermoanaerobacterium thermosulfurogenes and Streptomyces rubiginosus were introduced and expressed in three plant species (potato, tobacco and tomato) and transgenic plants were selected on xylose-containing medium. The xylose isomerase genes were transferred to explants of the target plant by Agrobacterium-mediated transformation. The xylose isomerase genes were expressed under the control of the enhanced cauliflower mosaic virus 35S promoter and the Ω′ translation enhancer sequence from tobacco mosaic virus. In potato and tomato, xylose isomerase selection was more efficient than the established kanamycin selection. The level of enzyme activity in the regenerated transgenic plants selected on xylose was 5–25-fold higher than the enzyme activity in control plants selected on kanamycin. The xylose isomerase system enables transgenic cells to utilize xylose as a carbohydrate source. In contrast to antibiotic or herbicide resistance-based system where transgenic cells survive on a selective medium but nontransgenic cells are killed, the xylose system is an example of a positive selection system where transgenic cells proliferate while non-transgenic cells are starved but still survive. The results show that a new selection method, is established. The xylose system is devoid of the disadvantages of antibiotic or herbicide selection, and depends on an enzyme which is already being widely utilized in specific food processes and that is generally recognized as safe for use in the starch industry.     

Inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs

Summary Granule-bound starch synthase [GBSS; EC 24.1.21] determines the presence of amylose in reserve starches. Potato plants were transformed to produce antisense RNA from a gene construct containing a full-length granule-bound starch synthase cDNA in reverse orientation, fused between the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator. The construct was integrated into the potato genome by Agrobacterium rhizogenes-mediated transformation. Inhibition of GBSS activity in potato tuber starch was found to vary from 70% to 100%. In those cases where total suppression of GBSS activity was found both GBSS protein and amylose were absent, giving rise to tubers containing amylose-free starch. The variable response of the transformed plants indicates that position effects on the integrated sequences might be important. The results clearly demonstrate that in tubers of potato plants which constitutively synthesize antisense RNA the starch composition is altered.     

Synthesis and expression of the gene for human epidermal growth factor in transgenic potato plants

Summary The gene for human epidermal growth factor (hEGF) was chemically synthesized and used for expression in transgenic potato. The hEGF coding sequence was modified by PCR to introduce ATG start codon and fused either to the 35S cauliflower mosaic virus promoter or to the patatin class I promoter. The highest hEGF peptide content, 120 pg/mg soluble protein, was found in potato tubers when the chimeric gene was expressed under the control of patatin promoter.     

Evaluation of constitutive viral promoters in transgenic soybean roots and nodules

The efficiency of beta-glucuronidase (GUS) expression was evaluated with five viral promoters to identify the most suitable promoter or promoters for use in soybean hairy roots, including applications to study the symbiotic interaction with Bradyrhizobium japonicum. Levels of GUS activity were fluorimetrically and histochemically assayed when the GUS (uidA) gene was driven by the Cauliflower mosaic virus (CaMV) 35S promoter and enhanced 35S (E35S) promoter, the Cassava vein mosaic virus (CsVMV) promoter, the Figwort mosaic virus (FMV) promoter, and the Strawberry vein banding virus (SVBV2) promoter. We demonstrate that GUS activity was highest when driven by the FMV promoter and that the promoter activity of 35S and SVBV2 was significantly lower than that of the CsVMV and E35S promoters when tested in soybean hairy roots. In mature soybean root nodules, strong GUS activity was evident when the FMV, 35S, and CsVMV promoters were used. These results indicate that the FMV promoter facilitates the strong expression of target genes in soybean hairy roots and root nodules.     

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.     

Potato gene Y-1 is an N gene homolog that confers cell death upon infection with potato virus Y

ADG2 is a DNA sequence mapped to a resistance (R) gene-rich region at the distal end of chromosome XI in potato (Solanum tuberosum subsp. andigena). The gene, in which ADG2 represents the predicted nucleotide-binding domain (NBS), was cloned and characterized. The coding region of the gene (designated as Y-1) is 6,187 bp long and structurally similar to gene N that confers hypersensitive resistance to Tobacco mosaic virus in Nicotiana spp. Both belong to the TIR-NBS-LRR class of genes and show 57% identity at the amino acid sequence level. The introns of Y-1 were spliced as predicted from the sequence. Y-1 cosegregated with Ry(adg), a gene for extreme resistance to Potato virus Y (PVY) on chromosome XI, as tested in a potato-mapping population and with independent potato cultivars. Leaves of the transgenic potato plants expressing Y-1 under the control of Cauliflower mosaic virus 35S promoter developed necrotic lesions upon infection with PVY, but no significant resistance was observed, and plants were systemically infected with PVY.     

Transgenic Potato with PVY Coat Protein Gene and Its Small-Scale Field Test

Potato virus Y (PVY) infection may cause a severe yield depression up to 80%. To develop the potato (Solanum tuberosum L. ) cultivars that resist PVY infection is very crucial in potato production. The authors have been cloned the coat protein gene of PVY from its Chinese isolate. A chimaeric gene containing the cauliflower mosaic virus 35S promoter and PVY coat protein coding region was introduced into the potato cultivars “Favorita”, “Tiger head” and “K4” via Agrobacterium tumefaciens. Results from PCR and Southern blot analysis confirmed that the foreign gene has integrated into the potato chromosomes. These transgenic potato plants were mechanically inoculated with PVY virus (20 mg/L). The presence of the virus in the potato plants was determined by ELISA and method of back inoculation into tobacco. The authors observed a drastic reduction in the accumulation of virus in some transgenic potato lines. Furthermore, some transgenic potato lines produced more tubers per plant than the untransformed potato did, and the average weight of these transgenic plant tubers was also increased. In the field test, the morphology and development of these transgenic potato plants were normal, 3 transgenic lines of “Favorita” exhibited a higher yield than the untrasformed virus-free potato with an increase ranged from 20% to 30%. From these transgenic lines, it will be very hopeful to develop a potato cultivar which not only has a significant resistance to PVY infection, but also a good harvest in potato production.     

Construction of hybrid promoters of caulimoviruses and analysis of their activity in transgenic plants

By the techniques of DNA shuffling, PCR, and restriction-ligation, chimeric forms of cauliflower (Brassica oleracea) mosaic virus (CaMV), dahlia (Dahlia pinnata) mosaic virus (DMV), and carnation (Dianthus caryophillus) etching ring virus (CERV) promoters were obtained at various combinations. Twelve chimeric promoters were cloned into pCambia binary vectors comprising the reporter GUS gene, and their activities in transgenic tobacco (Nicotiana tabacum) plants were determined fluorimetrically. 35S promoter and those of DMV (442 bp) and CERV (371 and 501 bp) were used as controls. Seven of analyzed promoters displayed higher and seven promoters lower activity in transgenic tobacco plants than 35S promoter. The highest activity was characteristic of natural DMV promoter, and the least one — natural CERV promoter 501 bp in size. The CERV promoter 371 bp in size was approximately similar in strength to 35S promoter.     

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.     

Construction of phosphomannose isomerase (PMI) transformation vectors and evaluation of the effectiveness of vectors in tobacco (Nicotiana tabacum L)

Phosphomannose isomerase (pmi) gene isolated from Escherichia coli allows transgenic plants carrying it to convert mannose-6- phosphate (from mannose), a carbon source that could not be naturally utilized by plants into fructose-6-phosphate which can be utilized by plants as a carbon source. This conversion ability provides energy source to allow the transformed cells to survive on the medium containing mannose. In this study, four transformation vectors carrying the pmi gene alone or in combination with the β-glucuronidase (gusA) gene were constructed and driven by either the maize ubiquitin (Ubi1) or the cauliflower mosaic virus (CaMV35S) promoter. Restriction digestion, PCR amplification and sequencing were carried out to ensure sequence integrity and orientation. Tobacco was used as a model system to study the effectiveness of the constructs and selection system. PMI11G and pMI3G, which carry gusA gene, were used to study the gene transient expression in tobacco. PMI3 construct, which only carries the pmi gene driven by CaMV35S promoter, was stably transformed into tobacco using biolistics after selection on 30 g 1(-1) mannose without sucrose. Transgenic plants were verified using PCR analysis. ABBREVIATIONS: PMI/pmi - Phosphomannose isomerase, Ubi1 - Maize ubiquitin promoter, CaMV35S - Cauliflower mosaic virus 35S promoter, gusA - β-glucuronidase GUS reporter gene.     

棉花曲叶病毒反式作用因子AC2的功能初探

有关非洲木薯花叶病毒（ACMV）、番茄金色花叶病毒（TGMV）的研究表明,双生病毒编码的反式作用因子AC2反式激活病毒链基因启动子的瞬时表达。以棉花曲叶病毒（CLCuV)侵染的烟草叶片组织总的DNA为模板,通过聚合酶反应扩增CLCuV的AC2基因片段并插入克隆载体。将AC2置于CaMV35S启动子下构建了瞬时表达载体。通过基因他法将质粒载体导入烟草（Nicotiana tabacumL.)和棉花（Gossypium hirstumL.)叶片细胞中进行瞬时表达,结果表明,在反式作用因子AC2的激活下,病毒链基因启动子驱动的GUS活性明显增强,然而激活后的病毒链基因启动子的活性仍低于互补链基因方向启动子;其表达方式与互补链基因启动子相似,即在叶肉及叶脉维管组织均有较高的活性。还探讨了AC2在土壤杆菌介导的转基因植物中的表达行为。     

Preliminary Functional Studies on AC2, a Novel Trans-acting Factor from Cotton Leaf Curl Virus

Studies on tomato golden mosaic virus and African cassava mosaic virus suggested that virion sense promoter was trans -activated in transient expression by A C2 encoded by geminivirus. The AC2 gene fragment of cott on leaf curl virus (CLCuV) was obtained from total DNA of CLCuV infected tobacco leaves by polymerase chain reaction, and the amplified DNA fragment was cloned into vector. Transient expres sion vectors were constructed by fusing the AC2 gene fra gment with CaMV 35S prom oter and nopaline terminator. These constructs were delivered into tobacco [ WT(Nicotiana tabacum L.) and cotton ( Gossypium hirsutum L.) leaf cells for transient expression by particle bombardment. Results indicated that activity of virion sense promoter was activated by AC2 and increased remarkably. However, the activity of trans-activated virion sense promoter was still lower than that of complementary sense promoter. Expression pattern of transactivated virion sense promoter was similar to that of complementary sense promoter with the high activity in both mesophyll and vascular of leaf vein. In this paper, the expression behavior of AC2 in Agrobacterium -mediated transgenic plants was also discussed. 