Epitope analysis of antibodies in Japanese to human cytomegalovirus phosphoprotein 150 with synthetic peptides

Serological detection of antibodies specific to human cytomegalovirus (HCMV) is not reliable because the assay uses the whole HCMV protein fraction. Antigenic materials composed of well-characterized viral proteins are being tried for serodiagnosis in Europe. Epitopes of antibodies to HCMV phosphoprotein 150 (pp150) encoded by UL32 in Japanese individuals were investigated for comparison with the results in Europe. The major epitopes on amino acid residues 496 to 652 of HCMV pp150 were identified and the detection of antibodies with an enzyme-linked immunosorbent assay (ELISA) of synthetic peptides against the main epitopes was established. Fifteen seropositive and five seronegative serum samples for the epitope mapping and 131 seropositive and 50 seronegative samples for ELISA were investigated. Overlapping 15-mer peptides moving by two amino acids through V496-H652 were synthesized. The main epitope regions were V508-D530, L526-Q544, S536-D554, T616-G634, S624-P642, and L632-H652. When each peptide was conjugated with bovine serum albumin for ELISA, 80.9% of the seropositive samples were judged to be positive. The results of this study are the same as those for European sera, so the antigenic materials developed in Europe might be used to replace the whole HCMV protein fraction in Japanese.     

人巨细胞病毒pp150蛋白片段与MDBP蛋白片段的融合表达及初步应用

人巨细胞病毒(HCMV)感染是临床上常见的一种病毒性传播疾病,正常人群常无明显的临床症状,而对器官移植患者、免疫力低下及孕妇等人可产生严重的危害。以HCMVAD169病毒株基因为模板,经PCR扩增了编码pp150蛋白片段的UL32基因和编码MDBP蛋白片段的UL57基因,目的基因转化入pMD18-T克隆载体后再经酶切与表达载体pET-11a连接构建出融合基因表达载体,然后转入大肠杆菌BL21,重组大肠杆菌经诱导表达融合蛋白pp150/MDBP。经SDS-PAGE分析,其相对分子量约为27kD,表达量约占菌体蛋白的17.45%,Westernblot鉴定为阳性,ELISA及蛋白芯片检测表明融合蛋白具有良好的抗原性,经过初步应用表明其对血清IgG及IgM的检出率与全抗原相比一致,具有进一步开发应用的价值。     

Bicaudal D1-Dependent Trafficking of Human Cytomegalovirus Tegument Protein pp150 in Virus-Infected Cells

Human cytomegalovirus (HCMV) virion assembly takes place in the nucleus and cytoplasm of infected cells. The HCMV virion tegument protein pp150 (ppUL32) is an essential protein of HCMV and has been suggested to play a role in the cytoplasmic phase of HCMV assembly. To further define its role in viral assembly and to identify host cell proteins that interact with pp150 during viral assembly, we utilized yeast two-hybrid analyses to detect an interaction between pp150 and Bicaudal D1 (BicD1), a protein thought to play a role in trafficking within the secretory pathway. BicD1 is known to interact with the dynein motor complex and the Rab6 GTPase. The interaction between pp150 and BicD1 was confirmed by coimmunoprecipitation and fluorescence resonance energy transfer. Depletion of BicD1 with short hairpin RNA (shRNA) caused decreased virus yield and a defect in trafficking of pp150 to the cytoplasmic viral assembly compartment (AC), without altering trafficking to the AC of another essential tegument protein, pp28, or the viral glycoprotein complex gM/gN. The C terminus of BicD1 has been previously shown to interact with the GTPase Rab6, suggesting a potential role for Rab6-mediated vesicular trafficking in HCMV assembly. Finally, overexpression of the N terminus of truncated BicD1 acts in a dominant-negative manner and leads to disruption of the AC and a decrease in the assembly of infectious virus. This phenotype was similar to that observed following overexpression of dynamitin (p50) and provided additional evidence that morphogenesis of the AC and virus assembly were dynein dependent.Human cytomegalovirus (HCMV) (human herpesvirus 5 [HHV-5]), the prototypical betaherpesvirus, is ubiquitous in humans and establishes a persistent infection in the host (19). HCMV also reinfects healthy seropositive individuals, suggesting another mechanism for maintaining persistence in a population (9). Intrauterine transmission and HCMV infection of the developing fetus constitute a leading viral cause of birth defects (32). HCMV is also a leading cause of opportunistic infections in immunocompromised patients, including transplant recipients and patients with AIDS (10, 20). HCMV infection has also been implicated as a cofactor in such diverse diseases as atherosclerosis and cancer (8, 17, 33, 66).HCMV replicates its genome in the nucleus, and acquisition of the final tegument and envelope is thought to occur in the cytoplasm of infected cells (73, 77). Envelopment of HCMV has been reported to occur by budding into cytoplasmic vacuoles that are composed of HCMV glycoproteins required for the assembly of infectious virions (37). The fully mature virus is released from the cell through either exocytosis or, possibly, lysis of the infected cells (56). The nucleic acid-containing capsid is embedded in a proteinaceous tegument layer that occupies the space between the nucleocapsid and the envelope. The tegument contains approximately 40% of the virion protein mass and approximately 20 to 25 known virion proteins, most of which are phosphorylated (40, 44). The assembly pathway and protein interactions required for formation of the tegument layer and the role of individual tegument proteins in the replication and assembly of infectious HCMV remain poorly understood. Deletion of viral genes encoding some tegument proteins results in varying levels of impairment in virus production (11-13, 35, 43, 45, 53, 68). Some tegument proteins, such as pp28 (pUL99) and ppUL25, are expressed only in the cytoplasm of infected cells during HCMV replication, whereas others, such as ppUL53 and pp65 (pUL83), are expressed in the nuclei of cells early in infection but are localized predominantly in the cytoplasm late in infection (68). Others, such as the tegument protein ppUL69, are expressed only in the nuclei of infected cells. Finally, the intracellular localization of other tegument proteins, such as pp150 (pUL32), is less well defined in that both nuclear and cytoplasmic localizations have been described (34, 68).HCMV pp150 (basic phosphoprotein [BPP], pUL32) is the 1,048-amino-acid product of the UL32 gene of HCMV and an abundant constituent of the HCMV virion. Homologues of pp150 are found in other betaherpesviruses, including chimpanzee CMV, rat CMV, mouse CMV, HHV-6, and HHV-7, but not in alpha- or gammaherpesviruses (2). It is expressed late in HCMV infection (15, 68). It comprises 9.1% of infectious virion mass and 2% of the mass of dense bodies, suggesting that it is preferentially incorporated into virions (87). It has an estimated molecular mass of 113 kDa and is posttranslationally modified by phosphorylation and glycosylation, resulting in a molecular mass of 150 kDa in purified virus preparations analyzed by SDS-PAGE (41, 42, 65). pp150 has been classified as a tegument protein based on its presence in virion preparation, noninfectious enveloped particles, and cytoplasmic nucleocapsids but not in immature nuclear capsids (27, 28, 40). It has been suggested that pp150 contacts the capsids through the distal end of the capsomeres or through the triplex subunits that interlink them (16, 86). It has been reported to bind HCMV capsids in vitro through its amino one-third (6). We have also noted association of pp150 with the virion capsid by cryo-immunoelectron microscopy (W. Britt and H. Zhou, UCLA, Los Angeles, CA, unpublished findings). In primary human foreskin fibroblast (HFF) cells infected with HCMV, pp150 accumulates in a juxtanuclear structure that is termed the assembly compartment (AC), which colocalizes with markers of the distal secretory pathway and with other tegument proteins, including pp28 and pp65 and envelope glycoproteins gB, gH, and gM/gN (68). The virus-induced AC appears to overlap with microtubules emanating from the microtubule-organizing center (MTOC) and is proposed to be a cytoplasmic site of virion assembly (37, 68).The function of pp150 is unknown, although its close association with the nucleocapsid suggests potential involvement in nuclear targeting during entry and in nuclear targeting of the encapsidated viral DNA, capsid tegumentation, and/or envelopment late in infection. It is essential for production of infectious virus, since the deletion of the UL32 open reading frame (ORF) leads to loss of virus replication and has been reported to be important in cytoplasmic maturation of HCMV, especially in viral egress (2, 22, 84, 91, 92). In cells infected with ΔUL32 virus, which lacks pp150, fewer virus particles accumulated in the cytoplasm, although nuclear steps in virus assembly were not affected (84). It was also observed that in the absence of pp150, nucleocapsids were present in the viral assembly compartment but failed to proceed further to vesicle transport-associated release (84). These observations, together with pp150 abundance in the virion, suggest a primary contribution for this structural protein in the morphogenesis and/or cytoplasmic transport of progeny virion particles to sites of virion envelopment.Since pp150 has no predicted intracellular trafficking signals, its localization to the AC in virus-infected cells has been postulated to be dependent on interactions with cellular and/or viral proteins. Using yeast two-hybrid (Y2H) screening experiments we identified the cellular protein Bicaudal D1 (BicD1) as an interacting cellular protein. Bicaudal D was originally defined as a Drosophila protein that is involved in establishing the asymmetric cytoplasm in the developing oocyte (82, 89). Two homologues of Bicaudal D, BicD1 and BicD2, have been reported in humans, and these proteins have been reported to be involved in dynein-mediated microtubule transport as well as in COPI-independent Golgi-endoplasmic reticulum (ER) transport (38, 39, 55). Microtubule-dependent transport is an energy-dependent active transport system that includes both positive-end (directed away from the MTOC) and negative-end (directed toward the MTOC) transport. The direction of transport depends on cargo interactions with the molecular motors directing this transport, with dynein being associated with negative-end transport and kinesin with positive-end transport. BicD1 colocalizes with Rab6a in the trans-Golgi network and on cytoplasmic vesicles that associate with Golgi membranes in a Rab6-dependent manner secondary to a Rab6 binding domain at the C terminus of BicD1, suggesting an important role for BicD1 as an adaptor for dynein-dependent transport in the cell (55). In addition to having a role in the Golgi-ER trafficking, BicD1 has been shown to regulate anchoring of microtubules to the centrosome, as BICD1/2 knockdown induced microtubule unfocusing, with microtubules no longer appearing to radiate from the centrosome (26). BicD1 binds to its cargo via its C-terminal domain and to the dynein motor via its N-terminal domain (38). In this study we demonstrated that pp150 and BicD1 interact and that this interaction was required for localization of pp150 to the AC in virus-infected cells. In addition, we demonstrated that inhibition of BicD1 expression by short hairpin RNA (shRNA) led to a reduction in the yield of infectious virus. Finally, we demonstrated that formation of the AC and the assembly of infectious virions were dynein dependent, suggesting a critical role in microtubules in the production of infectious HCMV. Together, these results argue that HCMV replication is dependent on efficient localization of pp150 to the AC through its interaction with BicD1 and that pp150 localization to the AC is dynein dependent.     

Cytomegalovirus pUL96 is critical for the stability of pp150-associated nucleocapsids

Maturation of human cytomegalovirus (HCMV) initiates with nucleocapsids that egress from the nucleus and associate with a juxtanuclear cytoplasmic assembly compartment, where virion envelopment and release are orchestrated. Betaherpesvirus conserved proteins pp150 (encoded by UL32) and pUL96 are critical for HCMV growth in cell culture. pp150 is a capsid-proximal tegument protein that preserves the integrity of nucleocapsids during maturation. pUL96, although expressed as an early protein, acts late during virus maturation, similar to pp150, based on the comparable antigen distribution in UL96, UL32, or UL96/UL32 dual mutant virus-infected cells. pp150 associates with nuclear capsids prior to DNA encapsidation, whereas both pp150 and pUL96 associate with extracellular virus, suggesting that pUL96 is added after pp150. In the absence of pUL96, capsid egress from the nucleus continues; however, unlike wild-type virus infection, pp150 accumulates in the nuclear, as well as in the cytoplasmic, compartment. Ultrastructural evaluation of a UL96 conditional mutant revealed intact nuclear stages but aberrant nucleocapsids accumulating in the cytoplasm comparable to the known phenotype of UL32 mutant virus. In summary, pUL96 preserves the integrity of pp150-associated nucleocapsids during translocation from the nucleus to the cytoplasm.     

Overexpression of Rice CBS Domain Containing Protein Improves Salinity, Oxidative, and Heavy Metal Tolerance in Transgenic Tobacco

We have recently identified and classified a cystathionine ??-synthase domain containing protein family in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa L.). Based on the microarray and MPSS data, we have suggested their involvement in stress tolerance. In this study, we have characterized a rice protein of unknown function, OsCBSX4. This gene was found to be upregulated under high salinity, heavy metal, and oxidative stresses at seedling stage. Transgenic tobacco plants overexpressing OsCBSX4 exhibited improved tolerance toward salinity, heavy metal, and oxidative stress. This enhanced stress tolerance in transgenic plants could directly be correlated with higher accumulation of OsCBSX4 protein. Transgenic plants could grow and set seeds under continuous presence of 150?mM NaCl. The total seed yield in WT plants was reduced by 80%, while in transgenic plants, it was reduced only by 15?C17%. The transgenic plants accumulated less Na⁺, especially in seeds and maintained higher net photosynthesis rate and Fv/Fm than WT plants under NaCl stress. Transgenic seedlings also accumulated significantly less H₂O₂ as compared to WT under salinity, heavy metal, and oxidative stress. OsCBSX4 overexpressing transgenic plants exhibit higher abiotic stress tolerance than WT plants suggesting its role in abiotic stress tolerance in plants.     

Improvement of protein quality in transgenic soybean plants

Glycinin is one of the abundant storage proteins in soybean seeds. A modified Gy1 (A1aB1b) proglycinin gene with a synthetic DNA encoding four continuous methionines (V3-1) was connected between the hpt gene and the modified green fluorescent protein sGFP(S65T) gene, and a resultant plasmid was introduced into soybean by particle bombardment in order to improve nutritional value of its seeds. After the selection with hygromycin, the efficiency of gene introduction was evaluated. More than 60 % of the regenerated plants tolerant to hygromycin yielded the hpt and V3-1 fragment by polymerase chain reaction (PCR) analysis, and the expression of sGFP was detected in about 50 % of putative transgenic soybeans. Southern hybridization confirmed the presence of transgenes in T0 plants and the transgenic soybeans hybridized with the hpt and V3-1 genes were analyzed showed different banding patterns. Most of the transgenic plants were growing, flowering normally and produced seeds. Analysis of seed obtained from transgenic soybean plants expressing hpt and V3-1 genes showed higher accumulation of glycinin compared with non-transgenic plants. In addition, protein expression in transgenic soybean plants was observed by using 2D-electrophoresis.     

Expression of the <Emphasis Type="Italic">Vicia faba VfPIP1</Emphasis> gene in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants improves their drought resistance

Plant aquaporins are believed to facilitate water transport across cell membranes. However, the relationship between aquaporins and drought resistance in plants remains unclear. VfPIP1, a putative aquaporin gene, was isolated from Vicia faba leaf epidermis, and its expression was induced by abscisic acid (ABA). Our results indicated that the VfPIP1 protein was localized in the plasma membrane, and its expression in V. faba was induced by 20% polyethylene glycol 6000. To further understand the function of VfPIP1, we obtained VfPIP1-expressing transgenic Arabidopsis thaliana plants under the control of the CaMV35S promoter. As compared to the wild-type control plants, the transgenic plants exhibited a faster growth rate, a lower transpiration rate, and greater drought tolerance. In addition, the stomata of the transgenic plants closed significantly faster than those of the control plants under ABA or dark treatment. These results suggest that VfPIP1 expression may improve drought resistance of the transgenic plants by promoting stomatal closure under drought stress.     

人巨细胞病毒大外膜磷蛋白pp150羧基末端的高效表达及初步应用

以基因工程抗原取代天然抗原用于人巨细胞病毒（ＨＣＭＶ）感染的诊断,具有广阔的前景。为此,分离ＨＣＭＶ大外膜磷蛋白ｐｐ１５０基因ＯＲＦ３端４３４ｂｐ（编码羧基末端ａａ９４３～ａａ１０４８）的ＤＮＡ片段,导入带有高水平转录启动子Ｐｔｒｃ和六个串联组氨酸的原核表达载体ｐＴｒｃＨｉｓ,在大肠杆菌中得到高效表达,ＳＤＳ ＰＡＧＥ显示一明显的分子量为１５６ｋＤ的融合蛋白条带,ＬＫＢ激光扫描占菌体总蛋白的５１％以上,并且以可溶性形式存在。表达产物经高效特异性纯化后,ＥＬＩＳＡ结果表明其能够与ＨＣＭＶＩｇＭ阳性血清呈特异反应,为建立新一代ＨＣＭＶＩｇＭ检测试剂盒打下了基础。     

Generation and immunogenicity of Japanese encephalitis virus envelope protein expressed in transgenic rice

Transgenic plants have become attractive as bioreactors to produce heterologous proteins that can be developed as edible vaccines. In the present study, transgenic rice expressing the envelope protein (E) of Japanese encephalitis virus (JEV), under the control of a dual cauliflower mosaic virus (CaMV 35S) promoter, was generated by Agrobacterium-mediated transformation. Southern blot, Northern blot, Western blot and ELISA analyses confirmed that the E gene was integrated into transgenic rice and was expressed in the leaves at levels of 1.1-1.9 μg/mg of total soluble protein. After intraperitoneal immunization of mice with crude protein extracts from transgenic rice plants, JEV-specific neutralizing antibody could be detected. Moreover, E-specific mucosal immune responses could be detected in mice after oral immunization with protein extracts from transgenic rice plants. These results show the potential of using a transgenic rice-based expression system as an alternative bioreactor for JEV subunit vaccine.     

A chimeric gene encoding the methionine-rich 2S albumin of the Brazil nut (Bertlrolletia excelsa H.B.K.) is stably expressed and inherited in transgenic grain legumes

The coding region of the 2S albumin gene of Brazil nut (Bertholletia excelsa H.B.K.) was completely synthesized, placed under control of the cauliflower mosaic virus (CaMV) 35S promoter and inserted into the binary vector plasmid pGSGLUC1, thus giving rise to pGSGLUC1-2S. This was used for transformation of tobacco (Nicotiana tabacum L. cv. Petit Havanna) and of the grain legume Vicia narbonensis L., mediated by the supervirulent Agrobacterium tumefaciens strain EHA 101. Putative transformants were selected by screening for neomycin phosphotransferase (NPT II) and β-glucuronidase (GUS) activities. Transgenic plants were grown until flowering and fruiting occurred. The presence of the foreign gene was confirmed by Southern analysis. GUS activity was found in all organs of the regenerated transgenic tobacco and legume plants, including the seeds. In the legume, the highest expression levels of the CaMV 35S promoter-controlled 2S albumin gene were observed in leaves and roots. 2S albumin was localized in the vacuoles of leaf mesophyll cells of transgenic tobacco. The Brazil nut protein was present in the 2S fraction after gel filtration chromatography of the legume seed proteins and could be clearly identified by immunoblotting. Analysis of seeds from the R₂ progenies of the legume and of transgenic tobacco plants revealed Mendelian inheritance of the foreign gene. Agrobacterium rhizogenes strain RifR 15834 harbouring the binary vector pGSGLUCl2S was also used to transform Pisum sativum L. and Vicia faba L. Hairy roots expressed the 2S albumin-specific gene. Several shoots were raised but they never completely rooted and no fertile plants were obtained from these transformants.     

Impact of Human Cytomegalovirus Infection and its Immune Response on Survival of Patients with Ovarian Cancer

Human cytomegalovirus (HCMV) has been detected in various types of tumors. We studied the prevalence of HCMV in ovarian cancer and its relation to clinical outcome. Paraffin-embedded tissues obtained prospectively from 45 patients with ovarian cancer and 30 patients with benign ovarian cystadenoma were analyzed for expression of HCMV immediate-early protein (IE) and HCMV tegument protein (pp65) by immunohistochemistry. Plasma was analyzed for HCMV serology. HCMV-IgG levels were higher in patients with ovarian cancer or benign cystadenoma than in age-matched controls (P?=?.002, P?<?.0001, respectively). HCMV IgM was detected in 12% of ovarian cancer patients and 3% of patients with benign tumors but was absent in controls. In patients with ovarian cancer, higher IgG levels were associated with better outcomes (P?=?.04). Extensive HCMV-IE protein expression was detected in 75% of ovarian cancers and 26% of benign tumors; pp65 was detected in 67% of ovarian cancers and 14% of benign tumors. A higher grade of HCMV infection was associated with higher stage of disease. Extensive HCMV-pp65 expression was associated with shorter median overall survival than focal expression (39 versus 42.5?months, P?=?.03). At study closure, 58% of ovarian cancer patients with focal pp65 expression were alive versus 27% of patients with extensive pp65 expression (P?=?.03). Thus, HCMV proteins are detected at different levels in ovarian tumors and benign cystadenomas. Ovarian cancer patients with focal HCMV-pp65 expression in their tumors and high IgG levels against HCMV lived longer, highlighting a need for in-depth studies of the oncomodulatory role of HCMV in ovarian cancer.     

Cropping as a Phytoremediation Practice for Oily Desert Soil with Reference to Crop Safety as Food

Broad beans (Vicia faba)could tolerate up to 10% (w)w) crude oil in desert soil (sand), therefore, the potential of this crop for cleaning oily desert soil via rhizosphere technology was investigated. The amounts of hydrocarbons recovered from oily desert soil samples supporting V. faba plants were less than the amounts extracted from uncultivated oily soil samples. Excised fresh V. faba roots with their intact rhizospheres resulted in the attenuation of n-octadecane, phenanthrene, and crude oil when shaken into sterile desert soil extract containing these hydrocarbons. The amounts of hydrocarbons eliminated were greater with roots of plants previously raised in oily soil than with roots of plants raised in clean soil. Similar hydrocarbon attenuation effects were recorded when, instead of excised roots, whole plants were used with their roots submerged in the hydrocarbon containing soil extract. The various parts of plants raised in oily desert soil contained more linolenic acid in their total lipids than did the same parts of plants raised in clean desert soil. This was much more pronounced for the roots than for shoots and seeds. The hydrocarbons of roots and shoots of V. faba plants were not as affected by oil pollution as were those of seeds, in which the proportions of very long chain hydrocarbons increased with increasing oil concentration in the soil. Those hydrocarbons are not recommended for human and animal nutrition.     

Immunochemical behaviour of legumin and vicilin from Vicia faba: a survey of related Proteins in the leguminosae subfamily faboideae

An antiserum specific for the legumin and vicilin of Vicia faba was used to examine extracts of seeds of taxa of the Fabeae and Trifolieae for the presence of related storage proteins, Proteins related to legumin were found to be widely distributed, indicating considerable conservation of the genetic information for this protein. Only Pisum sativum contained a protein immunochemically identical with the vicilin of V. faba; the equivalent proteins of all other genera tested here were immunochemically different from vicilin.     

Expression and Subcellular Targeting of Human Complement Factor C5a in Nicotiana species

We evaluated transgenic tobacco plants as an alternative to Escherichia coli for the production of recombinant human complement factor 5a (C5a). C5a has not been expressed in plants before and is highly unstable in vivo in its native form, so it was necessary to establish the most suitable subcellular targeting strategy. We used the strong and constitutive CaMV 35S promoter to drive transgene expression and compared three different subcellular compartments. The yields of C5a in the T₀ transgenic plants were low in terms of the proportion of total soluble protein (TSP) when targeted to the apoplast (0.0002% TSP) or endoplasmic reticulum (0.0003% TSP) but was one order of magnitude higher when targeted to the vacuole (0.001% TSP). The yields could be increased by conventional breeding (up to 0.014% TSP in the T₂ generation). C5a accumulated to the same level in seeds and leaves when targeted to the apoplast but was up to 1.7-fold more abundant in the seeds when targeted to the ER or vacuole, although this difference was less striking in the better-performing lines. When yields were calculated as an amount per gram fresh weight of transgenic plant tissue, the vacuole targeting strategy was clearly more efficient in seeds, reaching 35.8 µg C5a per gram of fresh seed weight compared to 10.62 µg C5a per gram fresh weight of leaves. Transient expression of C5aER and C5aVac in N. benthamiana, using MagnICON vectors, reached up to 0.2% and 0.7% of TSP, respectively, but was accompanied by cytotoxic effects and induced leaf senescence. Western blot of the plant extracts revealed a band matching the corresponding glycosylated native protein and the bioassay demonstrated that recombinant C5a was biologically active.     

High-Level Transient Expression of ER-Targeted Human Interleukin 6 in Nicotiana benthamiana

Tobacco plants can be used to express recombinant proteins that cannot be produced in a soluble and active form using traditional platforms such as Escherichia coli. We therefore expressed the human glycoprotein interleukin 6 (IL6) in two commercial tobacco cultivars (Nicotiana tabacum cv. Virginia and cv. Geudertheimer) as well as the model host N. benthamiana to compare different transformation strategies (stable vs. transient expression) and subcellular targeting (apoplast, endoplasmic reticulum (ER) and vacuole). In T₀ transgenic plants, the highest expression levels were achieved by ER targeting but the overall yields of IL6 were still low in the leaves (0.005% TSP in the ER, 0.0008% in the vacuole and 0.0005% in the apoplast). The apoplast variant accumulated to similar levels in leaves and seeds, whereas the ER-targeted variant was 1.2-fold more abundant in seeds and the vacuolar variant was 6-fold more abundant in seeds. The yields improved in subsequent generations, with the best-performing T₂ plants producing the ER-targeted IL6 at 0.14% TSP in both leaves and seeds. Transient expression of ER-targeted IL6 in leaves using the MagnICON system resulted in yields of up to 7% TSP in N. benthamiana, but only 1% in N. tabacum cv. Virginia and 0.5% in cv. Geudertheimer. Although the commercial tobacco cultivars produced up to threefold more biomass than N. benthamiana, this was not enough to compensate for the lower overall yields. The recombinant IL6 produced by transient and stable expression in plants was biologically active and presented as two alternative bands matching the corresponding native protein.     

Expression of neutralizing epitope of porcine epidemic diarrhea virus in potato plants

Transgenic plants expressing recombinant proteins from pathogenic microorganisms provide an inexpensive edible vaccine for induction of local immunity. A neutralizing epitope of porcine epidemic diarrhea virus (PEDV) gene containing SEKDEL was expressed in potato using Agrobacterium-mediated transformation system. Putative transgenic plants were regenerated, and genomic PCR confirmed the presence of PEDV epitope gene in the potato plants. Based on the ELISA results, epitope of PEDV protein made up approximately 0.1% of the total soluble tuber protein.     

Knockdown of the 7S globulin subunits shifts distribution of nitrogen sources to the residual protein fraction in transgenic soybean seeds

Key message

A platform of gene silencing by amiRNA had been established in fertile transgenic soybean. We demonstrated that knockdown of storage protein shifted the distribution of nitrogen sources in soybean seeds.

Abstract

Artificial microRNAs (amiRNAs) were designed using the precursor sequence of the endogenous soybean (Glycine max L. Merrill) miRNA gma-miR159a and expressed in transgenic soybean plants to suppress the biosynthesis of 7S globulin, which is one of the major storage proteins. Seed-specific expression of these amiRNAs (amiR-7S) resulted in a strong suppression of 7S globulin subunit genes and decreased accumulation of the 7S globulin subunits in seeds. Thus, the results demonstrate that a platform for gene silencing by amiRNA was first developed in fertile transgenic soybean plants. There was no difference in nitrogen, carbon, and lipid contents between amiR-7S and control seeds. Four protein fractions were collected from defatted mature seeds on the basis of solubility at different pH to examine the distribution of nitrogen sources and compensatory effects. In the whey and lipophilic fractions, nitrogen content was similar in amiR-7S and control seeds. Nitrogen content was significantly decreased in the major soluble protein fraction and increased in the residual fraction (okara) of the amiR-7S seeds. Amino acid analysis revealed that increased nitrogen compounds in okara were proteins or peptides rather than free amino acids. Our study indicates that the decrease in 7S globulin subunits shifts the distribution of nitrogen sources to okara in transgenic soybean seeds.     

Modification of catalase and MAPK in Vicia faba cultivated in soil with high natural radioactivity and treated with a static magnetic field

The effects of a static magnetic field (SMF) and high natural radioactivity (HR) on catalase and MAPK genes in Vicia faba were investigated. Soil samples with high natural radioactivity were collected from Ramsar in north Iran where the annual radiation absorbed dose from background radiation is higher than 20 mSv/year. The specific activity of the radionuclides of ²³²Th, ²³⁶Ra, and ⁴⁰K was measured using gamma spectrometry. The seeds were planted either in the soil with high natural radioactivity or in the control soils and were then exposed to a SMF of 30 mT for 8 days; 8 h/day. Levels of expression of catalase and MAPK genes, catalase activity and H₂O₂ content were evaluated. The results demonstrated significant differences in the expression of catalase and MAPK genes in SMF- and HR-treated plants compared to the controls. An increase in catalase activity was accompanied by increased expression of its gene and accumulation of H₂O₂. Relative expression of the MAPK gene in treated plants, however, was lower than those of the controls. The results suggest that the response of V. faba plants to SMF and HR may be mediated by modification of catalase and MAPK.