Ectopic expression of the spike protein of Rice Ragged Stunt Oryzavirus in transgenic rice plants inhibits transmission of the virus to insects

Rice ragged stunt oryzavirus (RRSV) replicates in both its insect vector, Nilaparvata lugens, and its plant host, rice, and has a complex multi-component particle bearing spikes on its outer surface. Transgenic rice lines expressing the 39 kDa spike protein showed good resistance to infection by RRSV. Furthermore, N. lugens fed on these plants prior to feeding on RRSV-infected plants were significantly protected against RRSV infection. The viral titre in insects initially fed on transgenic plants and then on RRSV-infected plants was inversely proportional to the levels of the 39 kDa protein expressed in the transgenic plants. This suggests that the 39 kDa protein interferes with the interaction between the intact virus particles and insect cell receptors and that the spike protein of RRSV contributes to vector specificity. This approach would probably be a more environment-friendly and sustainable method of virus control than by actual eradication of insect vectors.     

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.     

根癌农杆菌介导的转新城疫病毒融合蛋白基因水稻植株的获得

利用转基因植物作为生物反应器可以表达重组蛋白、生产外源蛋白质,也可以成为动物疫苗的廉价生产系统。以编码新城疫病毒融合蛋白(NDV-F)的基因为外源基因,以玉米泛素蛋白(Ubi)启动子为启动子,以潮霉素磷酸转移酶(HPT)基因作为选择标记基因,β-半乳糖苷酸酶(GUS)基因作为报告基因构建了适宜于农杆菌介导转化水稻的表达质粒pUNDV,并通过农杆菌介导转化水稻,获得了多株转基因植株。通过PCR分析和GUS活性检测,证实含有NDV-F基因的T-DNA已整合到水稻核基因组中,为研制廉价安全的转基因水稻新城疫基因工程疫苗奠定了基础。     

Expression of a human lactoferrin cDNA in tobacco cells produces antibacterial protein(s).

A suspension tobacco (Nicotiana tabacum L.) cell line was transformed to express human lactoferrin, an iron-binding glycoprotein. The transgenic calli produced a protein that was significantly smaller than the full-length lactoferrin protein. Total protein extracts made from transgenic tobacco callus exhibited much higher antibacterial activity than commercially available purified lactoferrin as determined by the decrease of colony-forming units when tested with four phytopathogenic species of bacteria. Introduction of the lactoferrin gene in crop plants may provide resistance against phytopathogenic bacteria.     

RNAi-directed down-regulation of RSV results in increased resistance in rice (Oryza sativa L.)

Rice stripe disease (RSD), caused by rice stripe virus (RSV), is a serious disease in temperate rice-growing areas. We have created an RNAi construct containing coat protein gene (CP) and disease specific protein gene (SP) sequences from RSV. The RNAi construct was transformed into two susceptible japonica varieties, Suyunuo and Guanglingxiangjing, to develop resistance against RSD. The homozygous progeny of rice plants in the T(5) and T(7) generations containing RNAi constructs, after self-fertilization were strongly resistant to viral infection. RT-PCR indicated that viral replication of SP and CP in the transgenic plants was significantly inhibited. There were no obvious morphological or developmental differences between the transgenic and wild-type plants from seedling stage to maturity. The excellent agronomic traits of these two varieties, such as high yield and good quality were maintained. Suppression of virus genes using RNAi is therefore a practical and effective strategy for controlling viral infection in crops.     

Molecular breeding of transgenic rice plants expressing a bacterial chlorocatechol dioxygenase gene

The cbnA gene encoding the chlorocatechol dioxygenase gene from Ralstonia eutropha NH9 was introduced into rice plants. The cbnA gene was expressed in transgenic rice plants under the control of a modified cauliflower mosaic virus 35S promoter. Western blot analysis using anti-CbnA protein indicated that the cbnA gene was expressed in leaf tissue, roots, culms, and seeds. Transgenic rice calluses expressing the cbnA gene converted 3-chlorocatechol to 2-chloromucote efficiently. Growth and morphology of the transgenic rice plants expressing the cbnA gene were not distinguished from those of control rice plants harboring only a Ti binary vector. It is thus possible to breed transgenic plants that degrade chloroaromatic compounds in soil and surface water.     

猪心脏脂肪酸结合蛋白基因PCR-RFLP分子标记研究

利用PCR-RFLP分子标记技术,检测了杜洛克、长白、大白、内江、荣昌、汉江黑、汉白、八眉和野猪共计265头猪心脏脂肪酸结合蛋白基因5'上游区和第二内含子区的遗传变异。结果表明,在HinfI-RFLP位点上,上述猪种和野猪均存在多态性,等位基因H的频率分别为0.7500,0.7188,0.9167,0.3333,0.1250,0.6909,0.1167,0.8500和0.9375;除汉江黑猪(P<0.05)和野猪(P<0.01)外,其余的猪种基因频率和基因型频率都处于Hardy-Weinderg平衡状态(P>0.05);大白、八眉、汉江黑、汉白和野猪表现为低度多态(PIC<0.25),杜洛克、长白、内江和荣昌猪为中度多态性(0.25相似文献   

Ectopic expression of MgSM1, a Cerato-platanin family protein from Magnaporthe grisea, confers broad-spectrum disease resistance in Arabidopsis

Proteins belonging to the newly identified Cerato-platanin (CP) family have been shown to have elicitor activity in inducing disease resistance responses in various plants. In this study, we characterized a gene, MgSM1 , from Magnaporthe grisea , encoding a putative small protein belonging to the CP family. MgSM1 was constitutively expressed not only in different fungal growth stages but also during its infection process in rice plants. Agrobacterium-mediated transient expression of MgSM1 in Arabidopsis resulted in hypersensitive response in the infiltrated local leaves and enhanced disease resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato ( Pst ) DC3000 in upper leaves of plants, accompanyed by up-regulated expression of defense genes ( PR-1 , PR-5 and PDF1.2 ). Transgenic Arabidopsis plants expressing MgSM1 under control of a dexamethasone (DEX)-inducible promoter were generated. Expression of MgSM1 in transgenic plants was induced by exogenous application of DEX. MgSM1- expressing plants showed normal growth with application of <10 μ m DEX. After DEX induction, the MgSM1 -expressing plants showed enhanced disease resistance against B. cinerea , Alternaria brassicicola and Psto DC3000 as well as up-regulated expression of some of defense genes. Moreover, accumulation of reactive oxygen species was observed in MgSM1 -expressing plants. These results collectively suggest that ectopic expression of MgSM1 in transgenic plants confers broad-spectrum resistance against different types of pathogens. Our study also provides a novel strategy to generate environment-friendly crops with enhanced broad-spectrum resistance through ectopic expression of microbe-derived disease resistance-inducing proteins.     

Transgenic indica rice expressing Mirabilis jalapa antimicrobial protein (Mj-AMP2) shows enhanced resistance to the rice blast fungus Magnaporthe oryzae

Mj-AMP2, a knottin-type antimicrobial peptide, in vitro inhibits the growth of several plant pathogenic fungi including Magnaporthe oryzae. We demonstrate that transgenic rice (Oryza sativa L.) plants expressing the Mj-AMP2 gene show enhanced resistance to M. grisea, the causal agent of the rice blast disease. Mj-AMP2 was efficiently expressed and the level of Mj-AMP2 ranged from 0.32% to 0.38% of the total protein in the transgenic rice plants. In vitro inhibitory activity assays with the crude protein extract from transgenic rice indicated that the Mj-AMP2 protein produced was biologically active. Constitutive expression of Mj-AMP2 in transgenic rice reduces the growth of M. grisea by 63% with respect to untransformed control plant, and no effect on plant phenotype was observed. Transgene expression of Mj-AMP2 gene was not accompanied by an induction of pathogenesis-related (PR) gene expression indicating that the transgene product itself is directly active against the pathogen. The results presented in this study suggest that the Mj-AMP2 gene could be a useful candidate for protection of rice plants against the rice blast fungus M. grisea.     

Constitutive expression of a cowpea trypsin inhibitor gene,CpTi, in transgenic rice plants confers resistance to two major rice insect pests

The gene encoding a cowpea trypsin inhibitor (CpTI), which confers insect resistance in trangenic tobacco, was introduced into rice. Expression of the CpTi gene driven by the constitutively active promoter of the rice actin 1 gene (Act1) leads to high-level accumulation of the CpTI protein in transgenic rice plants. Protein extracts from transgenic rice plants exhibit a strong inhibitory activity against bovine trypsin, suggesting that the proteinase inhibitor produced in transgenic rice is functionally active. Small-scale field tests showed that the transgenic rice plants expressing the CpTi gene had significantly increased resistance to two species of rice stem borers, which are major rice insect pests. Our results suggest that the cowpea trypsin inhibitor may be useful for the control of rice insect pests.     

Expression of Cry1Ab and Cry2Ab by a Polycistronic Transgene with a Self-Cleavage Peptide in Rice

Insect resistance to Bacillus thuringiensis (Bt) crystal protein is a major threat to the long-term use of transgenic Bt crops. Gene stacking is a readily deployable strategy to delay the development of insect resistance while it may also broaden insecticidal spectrum. Here, we report the creation of transgenic rice expressing discrete Cry1Ab and Cry2Ab simultaneously from a single expression cassette using 2A self-cleaving peptides, which are autonomous elements from virus guiding the polycistronic viral gene expression in eukaryotes. The synthetic coding sequences of Cry1Ab and Cry2Ab, linked by the coding sequence of a 2A peptide from either foot and mouth disease virus or porcine teschovirus-1, regardless of order, were all expressed as discrete Cry1Ab and Cry2Ab at high levels in the transgenic rice. Insect bioassays demonstrated that the transgenic plants were highly resistant to lepidopteran pests. This study suggested that 2A peptide can be utilized to express multiple Bt genes at high levels in transgenic crops.     

Expression and inheritance of hypersensitive resistance to rice hoja blanca virus mediated by the viral nucleocapsid protein gene in transgenic rice

Rice hoja blanca virus (RHBV) is a major virus disease of economic importance affecting rice in northern South America, Central America and the Caribbean. This is the first report of transgenic resistance to RHBV and the transformation of an indica rice variety from Latin America. Rice transformed with the RHBV nucleocapsid protein ( N) gene had a significant reduction in disease development. Several reactions were observed that ranged from susceptible to completely resistant plants (immunity). The resistant reactions were characterized by the production of local lesions like a hypersensitive reaction or a recovery phenotype with the emergence of symptom-less new leaves. These transgenic RHBV-resistant rice lines expressed the N gene RNA at low levels that were below the detection limit by Northern blots and only resolved by RT-PCR. The nucleocapsid protein could not be detected in any of the transgenic plants either by Western or ELISA tests. These results suggest that the resistance encoded by the N gene in these plants appears to be mediated by RNA. When challenged with RHBV, the resistant transgenic lines showed a significant increased performance for important agronomic traits including the number of tillers, the number of grains per plant and the yield as compared to the susceptible control. Furthermore, upon inoculation some of the most-resistant transgenic lines showed agronomic traits similar to the uninoculated non-transgenic Cica 8 control. Using both agronomic traits and disease severity as criteria, several of the most-resistant lines were followed through the R(4) generation and demonstrated that the N gene and RHBV resistance was inherited in a stable manner. These transgenic rice lines could become a new genetic resource in developing RHBV-resistant cultivars.     

Transgenic tobacco plants expressing a coat protein gene of tobacco mosaic virus are resistant to some other tobamoviruses

Transgenic tobacco plants expressing the coat protein (CP) gene of tobacco mosaic virus were tested for resistance against infection by five other tobamoviruses sharing 45-82% homology in CP amino acid sequence with the CP of tobacco mosaic virus. The transgenic plants (CP+) showed significant delays in systemic disease development after inoculation with tomato mosaic virus or tobacco mild green mosaic virus compared to the control (CP-) plants, but showed no resistance against infection by ribgrass mosaic virus. On a transgenic local lesion host, the CP+ plants showed greatly reduced numbers of necrotic lesions compared to the CP- plants after inoculation with tomato mosaic virus, pepper mild mottle virus, tobacco mild green mosaic virus, and Odontoglossum ringspot virus but not ribgrass mosaic virus. The implications of these results are discussed in relation to the possible mechanism(s) of CP-mediated protection.     

The hrpZ gene of Pseudomonas syringae pv. phaseolicola enhances resistance to rhizomania disease in transgenic Nicotiana benthamiana and sugar beet

To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confirmed that the protein HrpZ(Psph) was produced and the signal peptide was properly processed. Neither T0 plants nor selfed progeny (T1) showed macroscopically visible necrosis or any other macroscopic phenotypes. However, plants expressing the SP/HrpZ(Psph) showed increased vigor and grew faster in comparison with non-transgenic control plants. Transgenic resistance was assessed after challenge inoculation with BNYVV on T1 progeny by scoring of disease symptoms and by DAS-ELISA at 20 and 30 dpi. Transgenic and control lines showed significant differences in terms of the number of plants that became infected, the timing of infection and the disease symptoms displayed. Plants expressing the SP/HrpZ(Psph) developed localized leaf necrosis in the infection area and had enhanced resistance upon challenge with BNYVV. In order to evaluate the SP/HrpZ-based resistance in the sugar beet host, A. rhizogenes-mediated root transformation was exploited as a transgene expression platform. Upon BNYVV inoculation, transgenic sugar beet hairy roots showed high level of BNYVV resistance. In contrast, the aerial non-transgenic parts of the same seedlings had virus titers that were comparable to those of the seedlings that were untransformed or transformed with wild type R1000 cells. These findings indicate that the transgenically expressed SP/HrpZ protein results in enhanced rhizomania resistance both in a model plant and sugar beet, the natural host of BNYVV. Possible molecular mechanisms underlying the enhanced resistance and plant growth phenotypes observed in SP/HrpZ transgenic plants are discussed.     

Expression of the PVYO coat protein (CP) under the control of the PVX CP gene leader sequence: protection under greenhouse and field conditions against PVYO and PVYN infection in three potato cultivars

Coat protein-mediated resistance (CPMR), resistance conferred as a result of the expression of viral coat proteins in transgenic plants, has been illustrated to be an effective way of protecting plants against several plant viruses. Nonetheless, consistent protection has not been achieved for transgenic plants expressing the coat protein of potato virus Y (PVY), the type member of the potyvirus family. In this report, three different potato cultivars were transformed with a chimeric construct consisting of the capsid protein (CP) coding sequences of PVY flanked by the AUG codon and the translational enhancer from the coat protein gene of potato virus X (PVX). These cultivars were shown to express high levels of PVY CP and confer a high degree of protection against PVY^o and PVY^N under both greenhouse and field conditions. In addition, transgenic plants infected with potato virus A (PVA), a related potyvirus, exhibited a delay in virus accumulation, which could be easily overcome with increasing virus concentrations. Received: 26 October 1995 / Accepted: 14 June 1996