Genetic transformation with untranslatable coat protein gene of <Emphasis Type="Italic">sugarcane yellow leaf virus</Emphasis> reduces virus titers in sugarcane

Sugarcane yellow leaf syndrome, characterized by a yellowing of the leaf midrib followed by leaf necrosis and growth suppression, is caused by sugarcane yellow leaf virus (SCYLV). We produced SCYLV-resistant transgenic sugarcane from a susceptible cultivar (H62-4671) and determined the amount of virus present following inoculation. The transgenic plants were produced through biolistic bombardment of cell cultures with an untranslatable coat protein gene. Presence of the transgene in regenerated plants was confirmed using PCR and Southern blot analysis. The transgenic lines were inoculated by viruliferous aphids and the level of SCYLV in the plants was determined. Six out of nine transgenic lines had at least 10³-fold lower virus titer than the non-transformed, susceptible parent line. This resistance level, as measured by virus titer and symptom development, was similar to that of a resistant cultivar (H78-4153). The selected SCYLV-resistant transgenic sugarcane lines will be available for integration of the resistance gene into other commercial cultivars and for quantification of viral effects on yield.     

Expression of immunogenic VP2 protein of infectious bursal disease virus in Arabidopsis thaliana

VP2 protein is the major host-protective immunogen of infectious bursal disease virus (IBDV) of chickens. Transgenic lines of Arabidopsis thaliana expressing recombinant VP2 were developed. The VP2 gene of an IBDV antigenic variant E strain was isolated, amplified by RT-PCR and introduced into a plant expression vector, pE1857, having a strong promoter for plant expression. A resulting construct with a Bar gene cassette for bialaphos selection in plant (rpE-VP2) was introduced into Agrobacterium tumefaciens by electroporation. Agrobacterium containing the rpE-VP2 construct was used to transform Ar. thaliana and transgenic plants were selected using bialaphos. The presence of VP2 transgene in plants was confirmed by PCR and Southern blot analysis and its expression was confirmed by RT-PCR. Western blot analysis and antigen-capture ELISA assay using monoclonal anti-VP2 were used to determine the expression of VP2 protein in transgenic plants. The level of VP2 protein in the leaf extracts of selected transgenic plants varied from 0.5% to 4.8% of the total soluble protein. Recombinant VP2 protein produced in plants induced antibody response against IBDV in orally-fed chickens.     

Expression of a synthetic cry1AcF gene in transgenic Pigeon pea confers resistance to Helicoverpa armigera

Pigeon pea is an important legume. Yield losses due to insect pests are enormous in the cultivation of this crop. Expression of cry proteins has led to increased resistance to pests in several crops. We report in this paper, expression of a chimeric cry1AcF (encoding cry1Ac and cry1F domains) gene in transgenic pigeon pea and its resistance towards Helicoverpa armigera. PCR, Southern hybridization, RT‐PCR and Western analysis confirmed stable integration and expression of the cry1AcF gene in pigeon pea transgenics. When screened for efficacy of the transformants for resistance against H. armigera, the transgenics showed not only high mortality of the larva but could also resist the damage caused by the larvae. Analysis for the stable integration, expression and efficacy of the transgenics resulted in the identification of four T3 plants arising from two T1 backgrounds as highly promising. The results demonstrate potentiality of the chimeric cry1AcF gene in developing H. armigera‐resistant pigeon pea.     

Synthesis and accumulation of pea plastocyanin in transgenic tobacco plants

The pea plastocyanin gene in a 3.5 kbp Eco RI fragment of pea nuclear DNA was introduced into tobacco by Agrobacterium-mediated transformation. Regenerated plants contained pea plastocyanin located within the chloroplast thylakoid membrane system. Analysis of seedlings from a self-pollinated transgenic plant containing a single copy of the pea plastocyanin gene indicated that seedlings homozygous for the pea gene contained almost twice as much pea plastocyanin as seedlings hemizygous for the pea gene. Homozygous seedlings contained approximately equal amounts of pea and tobacco plastocyanins. The amount of tobacco plastocyanin in leaves of transgenic plants was unaffected by the expression of the pea plastocyanin gene. The mRNA from the pea gene in tobacco was indistinguishable by northern blotting and S1 nuclease protection from the mRNA found in pea. In both pea and transgenic tobacco, expression of the pea plastocyanin gene was induced by light in leaves but was suppressed in roots. Pea plastocyanin free of contaminating tobacco plastocyanin was purified from transgenic tobacco plants and shown to be indistinguishable from natural pea plastocyanin by N-terminal protein sequencing and ¹H NMR spectroscopy.     

Overexpression of a new rice vacuolar antiporter regulating protein OsARP improves salt tolerance in tobacco

We examined the function of the rice (Oryza sativa L.) antiporter-regulating protein OsARP by overexpressing it in tobacco (Nicotiana tabacum L.). In public databases, this protein was annotated as a putative Os02g0465900 protein of rice. The OsARP gene was introduced into tobacco under the control of the cauliflower mosaic virus 35S promoter. The transformants were selected for their ability to grow on medium containing kanamycin. Incorporation of the transgene in the genome of tobacco was confirmed by PCR, and its expression was confirmed by Western blot analysis. Transgenic plants had better growth and vigor than non-transgenic plants under salt stress in vitro. Overexpression of OsARP in transgenic tobacco plants resulted in salt tolerance, and the plants had a higher rate of photosynthesis and effective PSII photon yield when compared with the wild type. The OsARP protein was localized in the tonoplast of rice plants. Transgenic plants accumulated more Na+ in their leaf tissue than did wild-type plants. It is conceivable that the toxic effect of Na+ in the cytosol might be reduced by sequestration into vacuoles. The rate of water loss was higher in the wild type than in transgenic plants under salt stress. Increased vacuolar solute accumulation and water retention could confer salt tolerance in transgenic plants. Tonoplast vesicles isolated from OsARP transgenic plants showed Na+/H+ exchange rates 3-fold higher than those of wild-type plants. These results suggest that OsARP on the tonoplasts plays an important role in compartmentation of Na+ into vacuoles. We suggest that OsARP is a new type of protein participating in Na+ uptake in vacuoles.     

Engineering broad-spectrum resistance against RNA viruses in potato

RNA silencing technology has become the tool of choice for inducing resistance against viruses in plants. A significant discovery of this technology is that double-stranded RNA (dsRNA), which is diced into small interfering RNAs (siRNAs), is a potent trigger for RNA silencing. By exploiting this phenomenon in transgenic plants, it is possible to confer high level of virus resistance by specific targeting of cognate viral RNA. In order to maximize the efficiency and versatility of the vector-based siRNA approach, we have constructed a chimeric expression vector containing three partial gene sequences derived from the ORF2 gene of Potato virus X, Helper Component Protease gene of Potato virus Y and Coat protein gene of Potato leaf roll virus. Solanum tuberosum cv. Desiree and Kuroda were transformed with this chimeric gene cassette via Agrobacterium tumefaciens-mediated transformation and transgenic status was confirmed by PCR, Southern and double antibody sandwich ELISA detection. Due to simultaneous RNA silencing, as demonstrated by accumulation of specific siRNAs, the expression of partial triple-gene sequence cassette depicted 20% of the transgenic plants are immune against all three viruses. Thus, expression of a single transgene construct can effectively confer resistance to multiple viruses in transgenic plants.     

羽衣甘蓝胁迫应答基因BoRS1转化烟草的初步研究

将克隆于羽衣甘蓝的胁迫应答基因BoRS1连入中间载体p35S-2300：：gus：：noster相应位点,成功地构建了含BoRS1基因的植物双元表达载体p35S-2300：：BoRS1：：noster,并通过农杆菌介导法对烟草进行了遗传转化。PCR检测结果表明目的基因BoRS1已成功地导入并整合到烟草基因组中。RT-PCR分析显示,在不同的转基因烟草植株中BoRS1表达量存在差异。转BoRS1烟草的耐干性和甘露醇胁迫研究表明,BoRS1基因的表达对提高植物抗干旱胁迫能力有一定的作用。     

Development of transgenic finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.) resistant to leaf blast disease

Finger millet plants conferring resistance to leaf blast disease have been developed by inserting a rice chitinase (chi11) gene through Agrobacterium-mediated transformation. Plasmid pHyg-Chi.11 harbouring the rice chitinase gene under the control of maize ubiquitin promoter was introduced into finger millet using Agrobacterium strain LBA4404 (pSB1). Transformed plants were selected and regenerated on hygromycin-supplemented medium. Transient expression of transgene was confirmed by GUS histochemical staining. The incorporation of rice chitinase gene in R₀ and R₁ progenies was confirmed by PCR and Southern blot analyses. Expression of chitinase gene in finger millet was confirmed by Western blot analysis with a barley chitinase antibody. A leaf blast assay was also performed by challenging the transgenic plants with spores of Pyricularia grisea. The frequency of transient expression was 16.3% to 19.3%. Stable frequency was 3.5% to 3.9%. Southern blot analysis confirmed the integration of 3.1 kb chitinase gene. Western blot analysis detected the presence of 35 kDa chitinase enzyme. Chitinase activity ranged from 19.4 to 24.8. In segregation analysis, the transgenic R₁ lines produced three resistant and one sensitive for hygromycin, confirming the normal Mendelian pattern of transgene segregation. Transgenic plants showed high level of resistance to leaf blast disease compared to control plants. This is the first study reporting the introduction of rice chitinase gene into finger millet for leaf blast resistance.     

Coat Protein-mediated Resistance to Pea Enation Mosaic Virus in Transgenic Pisum Sativum L.

Pea (Pisum sativum L.) plants were transformed in planta by injection/electroporation of axillary meristems with a chimeric pea enation mosaic virus (PEMV) coat protein gene contruct. R1 progenies of these plants were shown to harbor the transgene by polymerase chain reaction (PCR) and genomic Southern analysis, while transgene expression was demonstrated by western blot analysis. Transgenic R2, R3 and R4 plants displayed delayed or transient PEMV multiplication and attenuated symptoms as compated to control inoculated individuals.     

The expression of the <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> HAL1 gene increases salt tolerance in transgenic watermelon [<Emphasis Type="Italic">Citrullus lanatus</Emphasis> (Thunb.) Matsun. &; Nakai.]

An optimised Agrobacterium-mediated gene transfer protocol was developed in order to obtain watermelon transgenic plants [Citrullus lanatus (Thunb.) Matsun. & Nakai.]. Transformation efficiencies ranged from 2.8% to 5.3%, depending on the cultivar. The method was applied to obtain genetically engineered watermelon plants expressing the Saccharomyces cerevisiae HAL1 gene related to salt tolerance. In order to enhance its constitutive expression in plants, the HAL1 gene was cloned in a pBiN19 plasmid under control of the 35S promoter with a double enhancer sequence from the cauliflower mosaic virus and the RNA4 leader sequence of the alfalfa mosaic virus. This vector was introduced into Agrobacterium tumefaciens strain LBA4404 for further inoculation of watermelon half-cotyledon explants. The introduction of both the neomycin phosphotransferase II and HAL1 genes was assessed in primary transformants (TG1) by polymerase chain reaction analysis and Southern hybridisation. The expression of the HAL1 gene was determined by Northern analysis, and the diploid level of transgenic plants was confirmed by flow cytometry. The presence of the selectable marker gene in the expected Mendelian ratios was demonstrated in TG2 progenies. The TG2 kanamycin-resistant plantlets elongated better and produced new roots and leaves in culture media supplemented with NaCl compared with the control. Salt tolerance was confirmed in a semi-hydroponic system (EC=6 dS m(-1)) on the basis of the higher growth performance of homozygous TG3 lines with respect to their respective azygous control lines without the transgene. The halotolerance observed confirmed the inheritance of the trait and supports the potential usefulness of the HAL1 gene of S. cerevisiae as a molecular tool for genetic engineering of salt-stress protection in other crop species.     

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。