Sorting of Glycoprotein B from Human Cytomegalovirus to Protein Storage Vesicles in Seeds of Transgenic Tobacco

As part of ongoing studies into the use of plant expression systems for making human therapeutic proteins, we have successfully expressed the major glycoprotein, gB, of human cytomegalovirus (HCMV) in transgenic tobacco plants. Viral glycoprotein was detectable in the protein extracts of mature tobacco seeds using neutralizing and non-neutralizing monoclonal antibodies specific for gB. Although several mammalian proteins have been expressed in tobacco, localization of these proteins in transgenic tobacco tissue has not been extensively examined. The objective of this study was to identify the site(s) of recombinant gB deposition in mature tobacco seeds. Using immunogold labelling and electron microscopy, we found specific labelling for gB in the endosperm of transgenic seeds, with gB localized almost exclusively in protein storage vesicles (PSV). This occurred in seeds that were freshly harvested and in seeds that had been stored for several months. These data indicate that gB behaves like a plant storage protein when expressed in tobacco seeds, and provide further support for the suitability of plants for producing recombinant proteins of potential clinical relevance.     

Pharming vaccines for hepatitis and cytomegalovirus: Towards the development of multivalent and subunit vaccines for oral delivery of antigens

A plant based high fidelity vaccine production system is being developed with emphasis on producing antigens capable of being orally delivered in multivalent or subunit plant packets. Plant-based edible vaccines may provide an attractive, safe and inexpensive alternative to conventional vaccine production. Edible plant tissues are not normally antigenic in nature. However, foreign antigens from common infectious organisms like hepatitis-B virus (HBV) can be produced along with naturally occurring storage proteins in DNA-transformed plants. Upon administration via the oral route, these transgenic plant tissues may mobilize the protective humoral and mucosal immune responses to challenge the natural infectious agent. When tobacco, carrot and rice plants were transformed with the truncated version of the HBV nucleocapsid gene expression construct, non-infective hepatitis B viral core particles were observed via electron microscopy. A second plant codon-optimised HBV expression construct was designed that included the extensin signal sequence for augmented HBV particle accumulation. Upon transformation of tobacco plants with the codon-optimised construct, over 4 times more transgenic plants with high levels of expression of the HBV nucleocapsid protein were generated in comparison with a similar vector containing the unmodified wild-type HBV gene codon sequence. Further analysis via Western blotting confirmed the presence of the viral antigen in the total protein extracts from transgenic tobacco leaves and seeds. Electron microscopy showed that the expressed protein self-assembled into viral-like particles of 25–30 nm in diameter. To develop an edible subunit vaccine in plant seeds, a third plant transformation construct was used for the synthesis of the human cytomegalovirus glycoprotein B (HCMV gB) subunit. The gB protein derived from tobacco seeds retained critical structural features including epitopes for neutralizing antibodies and was targeted to the protein storage vesicles of tobacco seed endosperm. Two different strains of mice were orally immunized with tobacco seeds containing low concentrations of HCMV gB, with varying dosages, but without adjuvant. No anti-gB response was detected in intestinal or serum samples. However, a systemic immune response to normal tobacco seed proteins was observed in both strains of mice. While higher expression levels of antigens in seeds must be achieved, seeds may provide an effective and immunostimulatory vehicle for delivering edible vaccines to the intestinal mucosa. One of the outstanding challenges includes defining optimum conditions of antigen presentation, dosage and immunization schedules that will induce strong mucosal and/or systemic immune responses in heterogeneous populations. Here we review the different strategies being employed to produce specific oral antigens in plant tissues.     

Increased yield of heterologous viral glycoprotein in the seeds of homozygous transgenic tobacco plants cultivated underground.

The use of transgenic plants in the production of recombinant proteins for human therapy, including subunit vaccines, is being investigated to evaluate the efficacy and safety of these emerging biopharmaceutical products. We have previously shown that synthesis of recombinant glycoprotein B (gB) of human cytomegalovirus can be targeted to seeds of transgenic tobacco when directed by the rice glutelin 3 promoter, with gB retaining critical features of immunological reactivity (E.S. Tackaberry et al. 1999. Vaccine, 17: 3020-3029). Here, we report development of second generation transgenic plant lines (T1) homozygous for the transgene. Twenty progeny plants from two lines (A23T(1)-2 and A24T(1)-3) were grown underground in an environmentally contained mine shaft. Based on yields of gB in their seeds, the A23T(1)-2 line was then selected for scale-up in the same facility. Analyses of mature seeds by ELISA showedthat gB specific activity in A23T(1)-2 seeds was over 30-fold greater than the best T0 plants from the same transformation series, representing 1.07% total seed protein. These data demonstrate stable inheritance, an absence of transgene inactivation, and enhanced levels of gB expression in a homozygous second generation plant line. They also provide evidence for the suitability of using this environmentally secure facility to grow transgenic plants producing therapeutic biopharmaceuticals.     

Production of two Highly Active Bacterial Phytases with Broad pH Optima in Germinated Transgenic Rice Seeds

Phytate is the main storage form of phosphorus in many plant seeds, but phosphate bound in this form is not available to monogastric animals. Phytase, an enzyme that hydrolyzes phosphate from phytate, has the potential to enhance phosphorus availability in animal diets when engineered in rice seeds as a feed additive. Two genes, derived from a ruminal bacterium Selenomonas ruminantium (SrPf6) and Escherichia coli (appA), encoding highly active phytases were expressed in germinated transgenic rice seeds. Phytase expression was controlled by a germination inducible alpha-amylase gene (alphaAmy8) promoter, and extracellular phytase secretion directed by an betaAmy8 signal peptide sequence. The two phytases were expressed in germinated transgenic rice seeds transiently and in a temporally controlled and tissue-specific manner. No adverse effect on plant development or seed formation was observed. Up to 0.6 and 1.4 U of phytase activity per mg of total extracted cellular proteins were obtained in germinated transgenic rice seeds expressing appA and SrPf6 phytases, respectively, which represent 46-60 times of phytase activities compared to the non-transformant. The appA and SrPf6 phytases produced in germinated transgenic rice seeds had high activity over broad pH ranges of 3.0-5.5 and 2.0-6.0, respectively. Phytase levels and inheritance of transgenes in one highly expressing plant were stable over four generations. Germinated transgenic rice seeds, which produce a highly active recombinant phytase and are rich in hydrolytic enzymes, nutrients and minerals, could potentially be an ideal feed additive for improving the phytate-phosphorus digestibility in monogastric animals.     

Oral immunization with transgenic rice seeds expressing VP2 protein of infectious bursal disease virus induces protective immune responses in chickens

The expression of infectious bursal disease virus (IBDV) host-protective immunogen VP2 protein in rice seeds, its immunogenicity and protective capability in chickens were investigated. The VP2 cDNA of IBDV strain ZJ2000 was cloned downstream of the Gt1 promoter of the rice glutelin GluA-2 gene in the binary expression vector, pCambia1301-Gt1. Agrobacterium tumefaciens containing the recombinant vector was used to transform rice embryogenic calli, and 121 transgenic lines were obtained and grown to maturity in a greenhouse. The expression level of VP2 protein in transgenic rice seeds varied from 0.678% to 4.521% µg/mg of the total soluble seed protein. Specific pathogen-free chickens orally vaccinated with transgenic rice seeds expressing VP2 protein produced neutralizing antibodies against IBDV and were protected when challenged with a highly virulent IBDV strain, BC6/85. These results demonstrate that transgenic rice seeds expressing IBDV VP2 can be used as an effective, safe and inexpensive vaccine against IBDV.     

新城疫病毒融合蛋白在转基因水稻叶片中的表达及其免疫原性

利用转基因植物作为生物反应器表达抗原蛋白具有广阔的应用前景。以新城疫病毒融合蛋白（NDVF）基因1.7kb全长编码区序列为外源基因与组成型表达的玉米泛素蛋白基因（Ubi）启动子和农杆菌胭脂碱合成酶基因（nos）终止子组成嵌合基因,构建了适宜于农杆菌介导转化水稻的转化载体pUNDV,经根癌农杆菌介导的遗传转化方法将由Ubi动子驱动的NDVF嵌合基因导入水稻细胞中,经潮霉素抗性筛选,共再生获得了6个独立的转基因株系。PCR分析结果表明NDVF基因已整合到水稻基因组中。ELISA和Western blot分析结果证实NDVF蛋白在部分转基因水稻叶片组织中获得表达,其中植株F5叶片组织中具有较高的表达水平。将F5叶片可溶性总蛋白皮下注射免疫BALB／c小鼠,结果表明能够诱导小鼠产生一定水平的NDVF蛋白特异抗体。     

Expression and Inheritance of Nine Transgenes in Rice

A total of 66 transgenic rice cell lines were produced by simultaneously transforming rice callus with nine different plasmids/genes. PCR analysis indicated that the co-transformation frequency of each gene was about 70%. All the cell lines carried at least three genes and 11 cell lines carried all nine genes. Thirty-two fertile transgenic plants (R₀) were generated from the transgenic cell lines and seeds of 32 transgenic R₁ lines and 5 R₂ lines were harvested and analyzed for gene inheritance and protein expression. Progeny segregation analysis indicated that the multiple transgenes were integrated into the same locus of the rice genome, resulting in a 3:1 segregation ratio of the transgenes. Expression analysis of all nine transgenes revealed that the transgenes were expressed in all generations (R₀, R₁, and R₂) and about half of the transgenes from each line were expressed. The expression of one transgene appears to have no effect on the expression of another transgene. Among the 66 cell lines, six lines (9.1%) expressed seven or eight transgenes out of the nine transformed genes. All together, our results showed that multiple genes could be delivered into rice cells simultaneously and cell lines expressing multiple genes could be generated. The results and procedures reported here should be useful in designing multi-plasmid transformation experiments such as those required for plant metabolic engineering.     

Constitutive Expression of Soybean Ferritin cDNA Intransgenic Wheat and Rice Results in Increased Iron Levels in Vegetative Tissues but not in Seeds

We used particle bombardment to produce transgenic wheat and rice plants expressing recombinant soybean ferritin, a protein that can store large amounts of iron. The cDNA sequence was isolated from soybean by RT-PCR and expressed using the constitutive maize ubiquitin-1 promoter. The presence of ferritin mRNA and protein was confirmed in the vegetative tissues and seeds of transgenic wheat and rice plants by northern and western blot analysis, respectively. The levels of ferritin mRNA were similar in the vegetative tissues of both species, but ferritin protein levels were higher in rice. Both ferritin mRNA and protein levels were lower in wheat and rice seeds. ICAP spectrometry showed that iron levels increased only in vegetative tissues of transgenic plants, and not in the seeds. These data indicate that recombinant ferritin expression under the control of the maize ubiquitin promoter significantly increases iron levels invegetative tissues, but that the levels of recombinant ferritin in seeds are not sufficient to increase iron levels significantly over those in the seeds of non-transgenic plants.     

Production of human growth hormone in transgenic rice seeds: co-introduction of RNA interference cassette for suppressing the gene expression of endogenous storage proteins

Rice seeds are potentially useful hosts for the production of pharmaceutical proteins. However, low yields of recombinant proteins have been observed in many cases because recombinant proteins compete with endogenous storage proteins. Therefore, we attempt to suppress endogenous seed storage proteins by RNA interference (RNAi) to develop rice seeds as a more efficient protein expression system. In this study, human growth hormone (hGH) was expressed in transgenic rice seeds using an endosperm-specific promoter from a 10 kDa rice prolamin gene. In addition, an RNAi cassette for reduction of endogenous storage protein expressions was inserted into the hGH expression construct. Using this system, the expression levels of 13 kDa prolamin and glutelin were effectively suppressed and hGH polypeptides accumulated to 470 μg/g dry weight at the maximum level in transgenic rice seeds. These results suggest that the suppression of endogenous protein gene expression by RNAi could be of great utility for increasing transgene products.     

水稻谷蛋白GluA-2基因5′上游序列控制下的UidA基因在转基因水稻胚乳中的表达模式

为了研究谷蛋白胚乳特异性表达启动子在我国栽培稻品种中的表达模式,将UidA基因分别置于水稻谷蛋白GluA—2基因750bp和2．3kb上游序列下游,利用农杆菌转化法导人栽培稻品种中花8号并获得转基因植株。Southern blot检测表明,UidA基因已经整合到水稻基因组当中并以单拷贝存在。Northern blot检测表明,开花后13～15d和11～13d,UidA基因和水稻内源的GluA—2基因的表达量分别达到最高,随后逐渐降低。对转基因植株种子的GUS染色表明,UidA基因仅在胚乳中表达,在糊粉层中GUS表达量最高。测定了2．3kb和750bp转基因植株种子的GUS活性,结果表明前者的GUS活性是后者的2～3倍。序列分析表明,位于GluA—2基因转录启始位点上游2170bD的G-box可能是一个与表达量相关的顺式调控元件。     

Strategies for precise quantification of transgene expression levels over several generations in rice

Variation in transgene expression levels can result from uncontrolled differences in experimental protocols. Studies conducted over generations could, by their design, generate additional unwanted variation. To study sources of spurious variation, transgene expression levels were quantified over five homozygous generations in two independent transgenic rice lines created by particle bombardment. Both lines contained the same gus expression unit and had been shown to exhibit stable inheritance of transgene structure and expression. All plants were cultured and sampled using previously developed standardized protocols. Plants representative of each generation (T2, T3, T4, T5, T6) were grown either all together or across several different growth periods. GUS activity in plants from different generations was quantified either in the same assay or over multiple independent assays. Strategies in which plants were grown and phenotyped independently, significantly increased (up to 3-fold) extraneous variation in transgene expression level quantification, thus reducing the precision of molecular genetic studies and generating artefactual results in transgenic studies conducted over generations. Identification of sources of unwanted variation and quantification of their effect allowed the development of new strategies designed to control spurious variation. Growth and phenotyping of all plants from all generations together, using standard operating procedures (SOP), led to a reduction in extraneous variation associated with transgene expression level quantification. Adoption of such strategies is key to improving the reproducibility of transgenic studies conducted over generations.     

High accumulation of bioactive peptide in transgenic rice seeds by expression of introduced multiple genes

We have developed a simple binary vector construction system for the simultaneous expression of multiple genes in plants. Up to three independent gene cassettes can be easily integrated into one binary vector using the MultiSite Gateway System. Using this system, we produced transgenic rice plants that accumulated high levels of the hypocholesterolaemic peptide lactostatin (IIAEK) in endosperm. Binary vectors were constructed that could accommodate up to three independent modified glutelin gene cassettes encoding multimer lactostatin in the variable regions. Eight construct permutations were used for rice transformation. We measured the accumulation of lactostatin expressed as a glutelin fusion protein in the mature seeds of 105 independent transgenic rice lines. A general correlation was observed between accumulation level and gene number in the vector constructs, indicating that a higher accumulation of lactostatin was obtained from transgenic rice plants containing the maximum number of gene inserts. These results indicate that this strategy is applicable for the selection of transgenic lines containing large amounts of bioactive peptides in rice seeds.     

Induced tyramine overproduction in transgenic rice plants expressing a rice tyrosine decarboxylase under the control of methanol-inducible rice tryptophan decarboxylase promoter

Tyramine, one of the various biogenic amines found in plants, is derived from the aromatic L-amino acid tyrosine through the catalytic reaction of tyrosine decarboxylase (TYDC). Tyramine overproduction by constitutive expression of TYDC in rice plants leads to stunted growth, but an increased number of tillers. To regulate tyramine production in rice plants, we expressed TYDC under the control of a methanol-inducible plant tryptophan decarboxylase (TDC) promoter and generated transgenic T(2) homozygous rice plants. The transgenic rice plants showed normal growth phenotypes with slightly increased levels of tyramine in seeds relative to wild type. Upon treatment with 1% methanol, the transgenic rice leaves produced large amounts of tyramine, whereas no increase in tyramine production was observed in wild-type plants. The methanol-induced accumulation of tyramine in the transgenic rice leaves was inversely correlated with the tyrosine level. These data indicate that tyramine production in rice plants can be artificially controlled using the methanol-inducible TDC promoter, suggesting that this promoter could be used to selectively induce the expression of other proteins or metabolites in rice plants.     

Oral immunogenicity and protective efficacy in mice of transgenic rice plants producing a vaccine candidate antigen (As16) of Ascaris suum fused with cholera toxin B subunit

Cereal crops such as maize and rice are considered attractive for vaccine production and oral delivery. Here, we evaluated the rice Oryza sativa for production of As16—an antigen protective against the roundworm Ascaris suum. The antigen was produced as a chimeric protein fused with cholera toxin B subunit (CTB), and its expression level in the endosperm reached 50 μg/g seed. Feeding the transgenic (Tg) rice seeds to mice elicited an As16-specific serum antibody response when administered in combination with cholera toxin (CT) as the mucosal adjuvant. Although omitting the adjuvant from the vaccine formulation resulted in failure to develop the specific immune response, subcutaneous booster immunization with bacterially expressed As16 induced the antibody response, indicating priming capability of the Tg rice. Tg rice/CT-fed mice orally administered A. suum eggs had a lower lung worm burden than control mice. This suggests that the rice-delivered antigen functions as a prophylactic edible vaccine for controlling parasitic infection in animals.     

玉米pepc基因在杂交转育的转基因水稻后代中的传递和表达特征

在合肥、海南两地以高光效转玉米pepc基因水稻为父本,与培矮64S、2302S、2304S、2306S、5129、02428、皖粳97和双九A等8个受体杂交,转育转pepc基因水稻新种质材料。至2002年已转育成一批转pepc基因水稻品系,对这些材料世代跟踪研究显示：(1)玉米pepc基因以一个显性基因稳定传递给后代,符合孟德尔分离规律,自交F2呈3：1分离,BC1为1：1分离;(2)玉米pepc基因在杂交转育的转基因水稻中高水平表达,与受体亲本相比,PEPC活性提高3.7～17．4倍,表达水平与受体亲本和转基因拷贝数有关,来源相同的世代间有相似的表达水平,但同一世代不同个体间表达水平有差异,这可能与其位置效应、拷贝数和环境条件有关;(3)杂交后代结实率不高,容易产生异交结实导致转基因材料混杂分离。采取抗生素催芽初筛、PCR分析抽检、PEPC活性测定和田间表型观测的转玉米pepc基因水稻选育筛选体系,辅之以受体为轮回亲本适当回交可有效地控制。利用该途径已育成3个稳定的高表达的转玉米pepc基因水稻品系H1596、H1597和Y1470,说明通过常规杂交转育的方法,可以培育实用的稳定高表达的转玉米pepc基因水稻品种。     

Functional analyses of TaHMA2, a P1B‐type ATPase in wheat

Currently, there are few studies concerning the function of heavy metal ATPase 2 (HMA2), particularly in monocotyledons, and the potential application of this protein in biofortification and phytoremediation. Thus, we isolated and characterized the TaHMA2 gene from wheat (Triticum aestivum L.). Our results indicate that TaHMA2 is localized to the plasma membrane and stably expressed, except in the nodes, which showed relatively high expression. Zinc/cadmium (Zn/Cd) resistance was observed in TaHMA2‐transformed yeast. The over‐expression of TaHMA2 increased the elongation and decreased the seed‐setting rate in rice (Oryza sativa L.), but not Arabidopsis thaliana, tobacco (Nicotiana tabacum L.) or wheat. TaHMA2 over‐expression also improved root‐shoot Zn/Cd translocation, especially in rice. The seeds of transgenic rice and wheat, not tobacco, showed decreased Zn concentrations. The Zn concentration was decreased in all parts of the transgenic rice seeds, but was decreased only in the ventral endosperm of wheat, which showed an increased Zn concentration in the embryo and aleurone. The over‐expression of TaHMA2 improved plant tolerance under moderate Zn stress and Zn deficiency, but Zn and Cd resistance decreased under high levels of Zn and Cd stress, respectively. The Cd concentration in transgenic rice seedlings was dramatically increased under Zn deficiency. Thus, over‐expression of TaHMA2 showed a more obvious phenotype in monocotyledons than in dicotyledons. These findings provide important information for TaHMA2, and more efforts should be made in the future to characterize the reduced Zn concentration in TaHMA2 transgenic grains and the diversity of TaHMA2 substrate specificity.     

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.