Expression of a synthetic cholera toxin B subunit in tobacco using ubiquitin promoter and bar gene as a selectable marker

A protocol has been developed to produce a cholera toxin B subunit (CTB) in tobacco tolerant to the herbicide phosphinothricin (PPT) by means of in vitro selection. The synthetic CTB subunit gene was altered to modify the codon usage to that of tobacco plant genes. The gene was then cloned into a plant expression vector and was under the control of the ubiquitin promoter and transformed into tobacco plants by Agrobacterium-mediated transformation. Transgenic plantlets were selected in a medium supplemented with 5 mg/L PPT. Polymerase chain reaction analysis confirmed stable integration of the synthetic CTB gene into a chromosomal DNA. A high level of CTB (1.8% of total soluble protein) was expressed in transgenic plants, which was 18-fold higher than that under the control of the expressed CaMV 35S promoter with native gene. The transgenic plants when transferred to a greenhouse proved to be resistant to 2% PPT.     

Expression of a cholera toxin B subunit in transgenic lettuce (Lactuca sativa L.) using Agrobacterium-mediated transformation system

To increase expression level of cholera toxin B subunit (CTB) in lettuce plants, synthetic CTB (sCTB) gene based on the optimized codon usage was fused with an endoplasmic reticulum retention signal, KDEL. The sCTB gene was introduced into a plant expression vector and transformed to lettuce plants using Agrobacterium-mediated transformation system. As a selection marker, a bialaphos resistance (bar) gene that encodes phosphinothricin acetyltransferase (PAT), conferring tolerance to the herbicide phosphinothricin (PPT), was used. PCR amplification of genomic DNA confirmed the presence of the sCTB gene in the transgenic lettuce plants. Expressions of mRNA and protein of sCTB were observed by Northern and Western blot analyses, respectively. The sCTB synthesized in the transgenic lettuce showed strong affinity for GM₁-ganglioside suggesting that the sCTB conserved the antigenic sites for binding and proper folding of pentameric CTB structure. The expression level of CTB was relatively high, reaching total soluble protein (TSP) levels of 0.24% in transgenic lettuce.     

Highly expressed cholera toxin B subunit in the fruit of a transgenic tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> L.)

The cholera toxin B subunit (CTB), a nontoxic molecule with potent biological properties, is a powerful mucosal and parenteral adjuvant that induces a strong immune response against co-administered or coupled antigens. A gene encoding CTB, which was modified based on the optimized codon usage in the plant, was synthesized and fused to the endoplasmic reticulum retention signal KDEL to enhance its expression level in plants. The synthetic CTB (sCTB) gene was introduced into a plant expression vector adjacent to the CaMV 35S promoter, and was transformed into tomato using an Agrobacterium-mediated transformation method. The integration of the sCTB gene into the genomic DNA of transgenic plants was confirmed by genomic DNA PCR amplification. The synthesis and assembly of CTB protein in transgenic plants was demonstrated through immunoblot analysis and G_M1-ELISA. The highest amount of CTB protein produced in transgenic tomatoes was approximately 0.9% of total soluble fruit protein which was 10-fold greater than the previously 0.081%. G_M1-ELISA indicated that plant-synthesized CTB protein bound specifically to G_M1-gangliosides, suggesting that the CTB subunits formed active pentamers.     

Modification of the cholera toxin B subunit coding sequence to enhance expression in plants

The cholera toxin B subunit (CTB) contains five identical polypeptides and targets glycosphingolipid receptors on eukaryotic cell surfaces. Increased expression of CTB in plants is critical for the development of edible vaccines. In this study, the coding sequence of the CTB gene was optimized, based on the modification of codon usage to that of tobacco plant genes and the removal of mRNA-destabilizing sequences. The synthetic CTB gene was cloned into a plant expression vector and expressed in tobacco plants under the control of the CaMV 35S promoter. The recombinant CTB protein constituted approximately 1.5% of the total soluble protein in transgenic tobacco leaves. This level of CTB production was approximately 15-fold higher than that in tobacco plants that were transformed with the bacterial CTB gene. The recombinant CTB produced by tobacco plants demonstrated strong affinity for GM1-ganglioside, which indicates that the sites required for binding and proper folding of the pentameric CTB structure were conserved. This is the first report on the optimization of the CTB-coding sequence to give a dramatic increase in CTB expression in plants.     

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.     

OsRRM, a Spen-like rice gene expressed specifically in the endosperm

We used the promoter trap technique to identify a rice plant, named 107^#, in which the β-glucuronidase （GUS） reporter gene was expressed specifically in the endosperm. A single copy of the T-DNA was inserted into the plant genome, and a candidate gene OsRRM was identified by the insertion. The OsRRM promoter directed GUS expression specifically in rice endosperm, analogous to the GUS expression pattern observed in 107^#. OsRRMis a single-copy gene in rice and encodes a nuclear protein containing 1 005 amino-acid residues with two RNA recognition motifs and one Spen paralog and ortholog C-terminal domain. Westem blot analysis confirmed that the OsRRM protein was specifically expressed in rice endosperm. Ectopic expression of OsRRM in transgenic plants led to abnormalities, such as short stature, retarded growth and low fructification rates. Our data, in conjunction with the reported function of Spen genes, implicated OsRRM in the regulation of cell development in rice endosperm.     

Co-transformation of gene expression cassettes via particle bombardment to generate safe transgenic plant without any unwanted DNA

The presence of resistant selectable marker genes and other added DNAs such as the vector backbone sequence in transgenic plant might be an unpredictable hazard to the ecosystem as well as to human health, which have affected the safe assessment of transgenic plants seriously. Using minimal gene expression cassette (containing the promoter, coding region, and terminator) without vector backbone sequence for particle bombardment is the new trend of plant genetic transformation. In the present paper, we co-transformed the selectable marker bar gene cassette and non-selected cecropinB gene cassette into rice (Oryza sativa L.) by particle bombardment, then eliminated the selectable marker bar gene in R₁ generation applying the hereditary segregation strategy and attained two safe transgenic plants only harboring cecropinB gene cassettes without any superfluous DNA. This is the fist report indicating that the combination of minimal gene cassettes transformation with the co-transformation and segregation strategy can generate selectable marker-free transgenic plants, which will promote the advancement in plant genetic engineering greatly.     

Expression of CaMV35S-GUS gene in transgenic rice plants

Summary To understand the properties of the cauliflower mosaic virus (CaMV) 35S promoter in a monocotyledonous plant, rice (Oryza sativa L.), a transgenic plant and its progeny expressing the CaMV35S-GUS gene were examined by histochemical and fluorometric assays. The histochemical study showed that -glucuronidase (GUS) activity was primarily localized at or around the vascular tissue in leaf, root and flower organs. The activity was also detected in the embryo and endosperm of dormant and germinating seeds. The fluorometric assay of various organs showed that GUS activity in transgenic rice plants was comparable to the reported GUS activity in transgenic tobacco plants expressing the CaMV35S-GUS gene. The results indicate that the level of expression of the CaMV 35S promoter in rice is similar to that in tobacco, a dicotyledonous plant, suggesting that it is useful for expression of a variety of foreign genes in rice plants.     

Isolation of the endosperm-specific LPAAT gene promoter from coconut (Cocos nucifera L.) and its functional analysis in transgenic rice plants

As one of the key tropical crops, coconut (Cocos nucifera L.) is a member of the monocotyledonous family Aracaceae (Palmaceae). In this study, we amplified the upstream region of an endosperm-specific expression gene, Lysophosphatidyl acyltransferase (LPAAT), from the coconut genomic DNA by chromosome walking. In this sequence, we found several types of promoter-related elements including TATA-box, CAAT-box and Skn1-motif. In order to further examine its function, three different 5′-deletion fragments were inserted into pBI101.3, a plant expression vector harboring the LPAAT upstream sequence, leading to pBI101.3-L1, pBI101.3-L2 and pBI101.3-L3, respectively. We obtained transgenic plants of rice by Agrobacterium-mediated callus transformation and plant regeneration and detected the expression of gus gene by histochemical staining and fluorometric determination. We found that gus gene driven by the three deletion fragments was specifically expressed in the endosperm of rice seeds, but not in the empty vector of pBI101.3 and other tissues. The highest expression level of GUS was at 15 DAF in pBI101.3-L3 and pBI101.3-L2 transgenic lines, while the same level was detected at 10 DAF in pBI101.3-L1. The expression driven by the whole fragment was up to 1.76- and 2.8-fold higher than those driven by the −817 bp and −453 bp upstream fragments, and 10.7-fold higher than that driven by the vector without the promoter. Taken together, our results strongly suggest that these promoter fragments from coconut have a significant potential in genetically improving endosperm in main crops.     

Enhanced resistance to the rice blast fungus Magnaporthe grisea conferred by expression of a cecropin A gene in transgenic rice

Cecropins are a family of antimicrobial peptides, which constitute an important key component of the immune response in insects. Here, we demonstrate that transgenic rice (Oryza sativa L.) plants expressing the cecropin A gene from the giant silk moth Hyalophora cecropia show enhanced resistance to Magnaporthe grisea, the causal agent of the rice blast disease. Two plant codon-optimized synthetic cecropin A genes, which were designed either to retain the cecropin A peptide in the endoplasmic reticulum, the ER-CecA gene, or to secrete cecropin A to the extracellular space, the Ap-CecA gene, were prepared. Both cecropin A genes were efficiently expressed in transgenic rice. The inhibitory activity of protein extracts prepared from leaves of cecropin A-expressing plants on the in vitro growth of M. grisea indicated that the cecropin A protein produced by the transgenic rice plants was biologically active. Whereas no effect on plant phenotype was observed in ER-CecA plants, most of the rice lines expressing the Ap-CecA gene were non-fertile. Cecropin A rice plants exhibited resistance to rice blast at various levels. Transgene expression of cecropin A genes was not accompanied by an induction of pathogenesis-related (PR) gene expression supporting that the transgene product itself is directly active against the pathogen. Taken together, the results presented in this study suggest that the cecropin A gene, when designed for retention of cecropin A into the endoplasmic reticulum, could be a useful candidate for protection of rice plants against the rice blast fungus M. grisea.     

Enhancing disease resistances of Super Hybrid Rice with four antifungal genes

A plant expression vector harboring four antifungal genes was delivered into the embryogenic calli of ‘9311’, an indica restorer line of Super Hybrid Rice, via modified biolistic particle bombardment. Southern blot analysis indicated that in the regenerated hygromycin-resistant plants, all the four antifungal genes, including RCH10, RAC22, β-Glu and B-RIP, were integrated into the genome of ‘9311’, co-transmitted altogether with the marker gene hpt in a Mendelian pattern. Some transgenic R₁ and R₂ progenies, with all transgenes displaying a normal expression level in the Northern blot analysis, showed high resistance to Magnaporthe grisea when tested in the typical blast nurseries located in Yanxi and Sanya respectively. Furthermore, transgenic F₁ plants, resulting from a cross of R₂ homozygous lines with high resistance to rice blast with the non-transgenic male sterile line Peiai 64S, showed not only high resistance to M. grisea but also enhanced resistance to rice false smut (a disease caused by Ustilaginoidea virens) and rice kernel smut (another disease caused by Tilletia barclayana).     

Recovery of transgenic rice plants expressing the rice dwarf virus outer coat protein gene (S8)

 The coding region of the eighth largest segment (S8) of the rice dwarf virus (RDV) was obtained from a RDV Fujian isolate. It was then cloned into pTrcHisA for expression in E. coli and into vector pE3 for plant transformation. By using callus derived from mature rice embryos as the target tissue, we obtained regenerated rice plants after bombardment of the former with plasmid pE3R8 containing the RDV S8 gene and the marker gene neomycin phosphotransferase (NPT II). Southern blotting confirmed the integration of the RDV S8 gene into the rice genome. The expression of the outer coat protein in both E. coli and rice plants was confirmed by western blotting. The recovery of transgenic rice plants expressing S8 gene is an important step towards studying the function of the RDV genes and obtaining RDV-resistant rice plants. Received: 1 March 1996 / Accepted: 2 August 1996     

Excision of a selective marker in transgenic rice using a novel Cre/<Emphasis Type="Italic">loxP</Emphasis> system controlled by a floral specific promoter

Based on the Cre/loxP system, we have developed a novel marker-free system mediating a direct auto-excision of loxP-flanked marker genes from T₁ transgenic rice without any treatment or further offspring crossing. To achieve this, the floral-specific promoter OsMADS45 was isolated from rice and the expression patterns of OsMADS45 promoter was characterised by using the pOs45:GUS transgenic plants. Furthermore, the binary vector with Cre recombinase under the control of OsMADS45 promoter was constructed and introduced into rice by Agrobacterium-mediated transformation and transgenic rice plants were generated. Southern blot analysis showed that auto-excision of the selective markers occurred in some T₁ progeny of the transgenic plants, suggesting that a high auto-excision frequency can be achieved with our Cre/loxP system. This auto-excision strategy provides an efficient way of removing the selectable marker gene from transgenic rice. Xianquan Bai and Qiuyun Wang contributed equally to the work.     

Transgenic tobacco plants containing <Emphasis Type="Italic">Bt</Emphasis> and <Emphasis Type="Italic">GNA</Emphasis> genes

A new plant expression vector (pBSbtCry1Ac-GNA) containing two insect resistant genes, a synthetic chimeric gene SbtCry1Ac encoding the insecticidal protein CrylAc and a gene GNA encoding snowdrop lectin (Galanthus nivalis agglutinin) was constructed. Transgenic tobacco plants containing these two genes were obtained through Agrobacterium-mediated transformation of tobacco leaf discs. Results from PCR detection and genomic DNA Southern blot analysis indicated that both SbtCrylAc gene and GNA gene were integrated into the genome of these plants. Results of Western blot analysis indicated that these two proteins were expressed in the analyzed plants. Bioassays of Myzus persicae and Helicoverpa assulta on detached leaves of transformed tobacco plants were carried out. The average aphid inhibition rate of these plants tested at 12 d post-infestation was 71.9 %. The average H. assulta mortality of these plants tested at 6 d post-infestation was up to 89.8 %. The kanamycin resistance of the T₁ progeny of these transgenic plants was analyzed and a typical 3:1 segregation was observed.     

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。     

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.