Transformation of rice plants by plant reovirus genes

Rice dwarf phytoreovirus (RDV), and rice ragged stunt oryzairus (RRSV) genes were introduced into rice protoplasts by using the cauliflower mosaic virus 35S promoter, tissue culture techniques and electroporation. The translation products of cDNA to RDV segment 8 were detected in transformed rice. Plants transgenic for RRSV S9 also expressed an mRNA of appropriate size but the protein was not apparently expressed. These latter plants did not show any resistance when inoculated with RRSV; on the contrary, symptom expression was intensified. Since most plant reoviruses are phloem-limited, an alternative promoter could be that of rice tungro bacilliform virus (RTBV), which is itself phloem-limited. When the β-glucuronidase (GUS) gene was coupled to this promoter and introduced into rice, GUS activity was successfully expressed only in the phloem, so the system could be of interest in the reovirus context.     

利用转hpRNA基因水稻抗水稻矮缩病毒

具有发夹结构的双链RNA(hairpin RNA,hpRNA)能高效诱导转录后基因沉默的发生.以水稻(Oryza sativaL.)矮缩病毒(RDV)基因组中第八片段编码区128～754 bp的序列为臂构建hpRNA,并克隆到植物表达载体pROK-2上.通过农杆菌介导的方法转化水稻"中花11".Southern blot分析表明,共获得12株阳性转化体.用带有RDV的叶蝉(Nephotettix cincticeps)接种Tl代转hpRNA水稻,结果表明转基因水稻对RDV具有高抗性或表现为症状延迟.而相同序列的有义链的转基因水稻和空载体的转基因水稻表现为典型的RDV侵染症状.HpRNA在转基因水稻中对RDV高抗性发挥重要作用.     

表达反义核酶RNA的转基因水稻对矮缩病毒复制和症状的抑制作用

用基因枪法将含有ＲＤＶ第五片段反义核酶序列基因的植物表达载体ｐＲＯＫＩＩ转化水稻幼胚,在Ｇ４１８存在的条件下,约２～３个月可筛选出抗性愈伤,转入分化培养基中培养可分化出幼苗。经Ｓｏｕｔｈｅｒｎ杂交法检测为阳性的水稻幼苗进行抗病性测定显示,转ＲＤＶ反义核酶基因的水稻植株对ＲＤＶ的复制和症状有显著抑制作用。转基因植株发病较轻,并能部分结实,而对照植株则明显矮化且大多不能抽穗。     

Constitutive and tissue-specific differential expression of the cryIA(b) gene in transgenic rice plants conferring resistance to rice insect pest

 The truncated chimeric Bt gene, cryIA(b) of Bacillus thuringiensis, driven by two constitutive promoters, 35S from CaMV and Actin-1 from rice, and two tissue-specific promoters, pith tissue and pepcarboxylase (PEPC) for green tissue from maize, was introduced into several varieties of rice (indica and japonica) by microprojectile bombardment and protoplast systems. A total of 1800 putative transgenic Bt rice plants could be produced. Southern analysis revealed that more than 100 independently transformed plants could be confirmed for integration of the cryIA(b) gene. High levels of CryIA(b) proteins were obtained in the green tissue (leaves and stem) of many plants using the PEPC promoter. There was little difference in Bt protein level in leaves and stems from transgenic plants with the 35 S or Actin-1 promoter. Out of 800 Southern-positive plants that were bioassayed, 81 transgenic plants showed 100% mortality of insect larvae of the yellow stem borer (Scirpophaga incertulas). The transgene, cryIA(b), driven by different promoters showed a wide range of expression (low to high) of Bt proteins stably inherited in a number of rice varieties with enhanced yellow stem borer resistance. This first report of transgenic indica Bt rice plants with the PEPC or pith promoter either alone or in combination should provide a better strategy for providing rice plants with protection against insect pest resistance, minimizing the expression of the CryIA(b) protein in seeds and other tissues. Received: 12 November 1997 / Accepted: 25 November 1997     

Enhancement of salt tolerance in transgenic rice expressing an Escherichia coli catalase gene, katE

Rice (Oryza sativa) is sensitive to salt stresses and cannot survive under low salt conditions, such as 50 mM NaCl. In an attempt to improve salt tolerance of rice, we introduced katE, a catalase gene of Escherichia coli, into japonica rice cultivar, Nipponbare. The resultant transgenic rice plants constitutively expressing katE were able to grow for more than 14 days in the presence of 250 mM NaCl, and were able to form flower and produce seeds in the presence of 100 mM NaCl. Catalase activity in the transgenic rice plants was 1.5- to 2.5-fold higher than non-transgenic rice plants. Our results clearly indicate that simple genetic modification of rice to express E. coli-derived catalase can efficiently increase its tolerance against salt stresses. The transformant presented here is one of the most salt-tolerant rice plants created by molecular breeding so far.     

Characterization of rice tryptophan decarboxylases and their direct involvement in serotonin biosynthesis in transgenic rice

l-Tryptophan decarboxylase (TDC) and l-tyrosine decarboxylase (TYDC) belong to a family of aromatic l-amino acid decarboxylases and catalyze the conversion of tryptophan and tyrosine into tryptamine and tyramine, respectively. The rice genome has been shown to contain seven TDC or TYDC-like genes. Three of these genes for which cDNA clones were available were characterized to assign their functions using heterologous expression in Escherichia coli and rice (Oryza sativa cv. Dongjin). The purified products of two of the genes were expressed in E. coli and exhibited TDC activity, whereas the remaining gene could not be expressed in E. coli. The recombinant TDC protein with the greatest TDC activity showed a K _m of 0.69 mM for tryptophan, and its activity was not inhibited by phenylalanine or tyrosine, indicating a high level of substrate specificity toward tryptophan. The ectopic expression of the three cDNA clones in rice led to the abundant production of the products of the encoded enzymes, tyramine and tryptamine. The overproduction of TYDC resulted in stunted growth and a lack of seed production due to tyramine accumulation, which increased as the plant aged. In contrast, transgenic plants that produced TDC showed a normal phenotype and contained 25-fold and 11-fold higher serotonin in the leaves and seeds, respectively, than the wild-type plants. The overproduction of either tyramine or serotonin was not strongly related to the enhanced synthesis of tyramine or serotonin derivatives, such as feruloyltyramine and feruloylserotonin, which are secondary metabolites that act as phytoalexins in plants.     

Expression of rice gall dwarf virus outer coat protein gene (<Emphasis Type="Italic">S8</Emphasis>) in insect cells

To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity, its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system. The S8 gene was subcloned into the pFastBac™1 vector, to produce the recombinant baculovirus transfer vector pFB-S8. After transformation, pFB-S8 was introduced into the competent cells (E. coli DH10Bac) containing a shuttle vector, Bacmid, generating the recombinant bacmid rbpFB-S8. After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection, Sf9 cells were collected at different times and analyzed by SDS-PAGE, Western blotting and immunofluorescence microscopy. The expression level of the P8 protein was highest between 48–72 h after transfection of Sf9 cells. Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.     

Transfer of a grapevine stilbene synthase gene to rice (Oryza sativa L.)

A gene derived from grapevine (Vitis vinifera) coding for stilbene synthase has been transferred into protoplasts of the commercially important japonica rice cultivar Nipponbare using PEG-mediated direct gene transfer. Transgenic plants were regenerated from calli selected on kanamycin. Southern blot analysis of genomic DNA isolated from regenerants and progeny plants demonstrated that the stilbene synthase gene is stably integrated in the genome of transgenic rice plants and inherited in the offspring. The transient formation of stilbene-synthase-specific mRNA shortly after inoculation with the fungus of the rice blast Pyricularia oryzae has demonstrated that the grapevine stilbene synthase promoter is also active in monocotyledonous plants. Preliminary results indicate an enhanced resistance of transgenic rice to P. oryzae. Received: 1 July 1996 / Revision received: 5 November 1996 / Accepted: 30 November 1996     

Effective selection and regeneration of transgenic rice plants with mannose as selective agent

A new method for the selection of transgenic rice plants without the use of antibiotics or herbicides has been developed. The phosphomannose isomerase (PMI) gene from Escherichia coli has been cloned and consitutively expressed in japonica rice variety TP 309. The PMI gene was transferred to immature rice embryos by Agrobacterium-mediated transformation, which allowed the selection of transgenic plants with mannose as selective agent. The integration and expression of the transgene was confirmed by Southern and northern blot analysis and the activity of PMI indirectly proved with the chlorophenol red assay. The results of genetic analysis showed that the transgenes were segregated in a Mendelian fashion in the T₁ generation. The establishment of this selection system in rice provides an efficient way for producing transgenic plants without using antibiotics or herbicides with a transformation frequency of up to 41%.     

Heterologous expression of artificial miRNAs from rice dwarf virus in transgenic rice

In contrast to hairpin RNAs, in which heterogeneous small RNAs are processed from double-stranded RNA to have potential off-target effects on endogenous other genes, artificial miRNAs (amiRNAs) have advantages of exquisite specificity and non-transitivity to thus target individual genes and groups of endogenous genes. Earlier studies showed that amiRNA engineering based on osa-miRNA528 precursor could efficiently trigger endogenous gene silencing and modulate agronomic traits in rice. However, both the expression efficiency of heterologous amiRNAs based on osa-miRNA528 precursor and the correlation of copy number with the relative expression level of amiRNAs remain unknown. In the present study, five amiRNAs (S9-1174, S9-1192, S11-864, S11-868 and S11-869) targeting different sites of S9 and S11 negative strands in rice dwarf virus (RDV) genome were constructed using endogenous osa-miRNA528 precursor as backbone. After identification by Northern blot, two amiRNAs (S9-1174 and S9-1192) targeting S9 negative strand in RDV genome were highly expressed, whereas in three tested amiRNAs targeting S11 negative strand in RDV genome, only two amiRNAs (S11-868 and S11-869) were processed efficiently. T0 generation transgenic rice containing amiRNAs (S9-1174, S9-1192, S11-868 and S11-869) exhibited different expression ratios of amiRNAs, accounting for 90.0, 90.0, 66.7 and 77.8 %, respectively. In addition, combination analysis with the relative amiRNA expression levels and its copy number revealed that the relative expression levels of amiRNAs had no relation to the copy number of T-DNA insert in transgenic rice.     

Arabitol dehydrogenase as a selectable marker for rice

Arabitol dehydrogenase has been adapted for use as a plant selectable marker. Arabitol is a five-carbon sugar alcohol that can be used by E. coli strain C, but not by the laboratory K12 strains. The enzyme converts the non-plant-metabolizable sugar arabitol into xylulose, which is metabolized by plant cells. Rice was transformed with a plant-expression-optimized synthetic gene using Biolistic-mediated transformation. Selection on 2.75% arabitol and 0.25% sucrose yielded a transformation efficiency (9.3%) equal to that obtained with hygromycin (9.2%). Molecular analyses showed that the atlD gene was integrated into the rice genome of selected plants and was inherited in a Mendelian manner. This study indicates that arabitol could serve as an effective means of plant selection.     

Expression of rice dwarf phytoreovirus Pns6 and the specificity analysis of its monoclonal antibodies

The genome of rice dwarf phytoreovirus (RDV) is composed of 12 double-stranded RNA segments, of which segment S6 encodes a non-structural protein Pns6 identified as the movement protein. In this report, Pns6 with a 6-histidine tag at the N-terminal was expressed in E. coli after induction under low temperature (18℃) and low concentration (0.4 mmol/L and 0.2 mmol/L) of IPTG, and then purified by Ni-chelated affinity chromatography. Stability analysis indicated that the expressed HisPns6 protein was stable at 37℃ after 24 h treatment. This recombinant protein was then used to make monoclonal antibody. Total 18 hybridoma clones were obtained. The specificity of antibodies was tested by Western blot using native Pns6 extracted from RDV-infected rice leaves, and 15 positive clones were confirmed. Mapping of the antigenic sites of Pns6 using antibodies showed that the most sensitive antigen determinant is located in the C-terminal region (the 296th—509th amino acids) of Pns6, which is confirms bioinformatics analysis.     

FPF1 transgene leads to altered flowering time and root development in rice

AtFPF1 (FLOWERING PROMOTING FACTOR 1) is a gene that promotes flowering in Arabidopsis. An expression vector containing AtFPF1 driven by a Ubi-1 promoter was constructed. The gene was introduced into rice callus by Agrobacterium-mediated transformation and fertile plants were obtained. The presence of AtFPF1 in rice plants was confirmed by PCR, Southern and Northern blot analyses, as well as by -glucuronidase assay. The results showed that, as in Arabidopsis, AtFPF1 reduced flowering time in rice. Furthermore, introduction of AtFPF1 enhanced adventitious root formation but inhibited root growth in rice during the seedling stage. The results suggest that AtFPF1 promotes flowering time in both dicots and monocots, and plays a role in the initiation of adventitious roots in rice.Ming-Li Xu and Jia-Fu Jiang contributed equally to this work     

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.     

Enhanced resistance to blast (Magnaporthe grisea) in transgenic Japonica rice by constitutive expression of rice chitinase

Rice blast is the most devastating plant disease in Japan. Our goal is to create new rice varieties which show enhanced resistance against blast, regardless of the race of blast. By an Agrobacterium-mediated transformation method, we reintroduced a rice class-I chitinase gene, Cht-2 or Cht-3, under the control of the enhanced CaMV 35S promoter and a hygromycin phosphotransferase gene, as a selection marker into the Japonica rice varieties Nipponbare and Koshihikari, which have retained the best popularity over a long period in Japan. In regenerated plants (R₀), the Cht-2 product was found to accumulate intracellularly whereas the Cht-3 product was found to be targeted extracellularly. The transgenic rice plants which constitutively expressed either chitinase gene showed significantly higher resistance against the rice blast pathogen Magnaporthe grisea races 007.0 and 333. Both high-level expression of the chitinase and blast-resistance were stably inherited by the next generation in several lines. Received: 16 November 1998 / Accepted: 30 January 1999     

Efficient microprojectile bombardment-mediated transformation of rice using gene cassettes

This study was aimed at determining whether gene cassettes (promoter-coding sequence-terminator) can be efficiently used in microprojectile acceleration-mediated co-transformation of rice in the place of whole plasmids, and to what extent their use influences the integration and expression of the co-transferred gene of interest. Two non-linked marker genes (yfp and hph) were co-introduced by microprojectile bombardment into cells of embryogenic calli in three separate experiments. Three different DNA structures were compared for their ability to transiently and stably transform rice cells: supercoiled or linearized whole-plasmid DNA, gene cassette DNA and single-stranded gene cassette DNA coated with Escherichia coli single-stranded binding (SSB) proteins. Our results demonstrate that microprojectile bombardment-mediated transformation of rice using gene cassettes is possible without significantly reducing transformation efficiency in comparison to the use of whole-plasmid DNA. Furthermore, no obvious difference in transgene integration pattern and inheritance was observed among plants transformed with gene cassettes compared to those transformed with the whole plasmid, except that concatemerization of molecules prior to integration was rarely observed in gene cassette transformants. Received: 4 April 2001 / Accepted: 13 August 2001