Biofortification of rice with the essential amino acid lysine: molecular characterization, nutritional evaluation, and field performance.

Rice (Oryza sativa L.), a major staple crop worldwide, has limited levels of the essential amino acid lysine. We previously produced engineered rice with increased lysine content by expressing bacterial aspartate kinase and dihydrodipicolinate synthase and inhibiting rice lysine ketoglutarate reductase/saccharopine dehydrogenase activity. However, the grain quality, field performance, and integration patterns of the transgenes in these lysine-enriched lines remain unclear. In the present study, we selected several elite transgenic lines with endosperm-specific or constitutive regulation of the above key enzymes but lacking the selectable marker gene. All target transgenes were integrated into the intragenic region in the rice genome. Two pyramid transgenic lines (High Free Lysine; HFL1 and HFL2) with free lysine levels in seeds up to 25-fold that of wild type were obtained via a combination of the above two transgenic events. We observed a dramatic increase in total free amino acids and a slight increase in total protein content in both pyramid lines. Moreover, the general physicochemical properties were improved in pyramid transgenic rice, but the starch composition was not affected. Field trials indicated that the growth of HFL transgenic rice was normal, except for a slight difference in plant height and grain colour. Taken together, these findings will be useful for the potential commercialization of high-lysine transgenic rice.

The WRKY transcription factor OsWRKY78 regulates stem elongation and seed development in rice.

WRKY proteins are a large super family of transcriptional regulators primarily involved in various plant physiological programs. In present study, the expression profile and putative function of the WRKY transcriptional factor, WRKY78, in rice were identified. Real-time RT-PCR analysis showed that OsWRKY78 transcript was most abundant in elongating stems though its expression was detected in all the tested organs. The expression profiles were further confirmed by using promoter-GUS analysis in transgenic rice. OsWRKY78::GFP fusion gene transient expression analysis demonstrated that OsWRKY78 targeted to the nuclei of onion epidermal cell. Furthermore, OsWRKY78 RNAi and overexpression transgenic rice lines were generated. Transgenic plants with OsWRKY78 overexpression exhibited a phenotype identical to the wild type, whereas inhibition of OsWRKY78 expression resulted in a semi-dwarf and small kernel phenotype due to reduced cell length in transgenic plants. In addition, a T-DNA insertion mutant line oswrky78 was identified and a phenotype similar to that of RNAi plants was also observed. Grain quality analysis data showed no significant differences, with the exception of minor changes in endosperm starch crystal structure in RNAi plants. Taken together, these results suggest that OsWRKY78 may acts as a stem elongation and seed development regulator in rice.

Down-Regulation of SSSII-2 Gene Expression Results in Novel Low-Amylose Rice with Soft, Transparent Grains.

Although soft rice, with low amylose content (AC), has high eating and cooking quality (ECQ), its appearance is poor due to the opaque endosperm. Here, a novel soft rice with low AC but a transparent appearance was generated by knocking-down the expression of SSSII-2, a gene encoding one isoform of soluble starch synthase (SSS). The physicochemical properties of the SSSII-2 RNAi rice are quite different from the control but more like the popular soft rice "Nanjing 46". The taste value assay further demonstrated that the ECQ of SSSII-2 RNAi rice was as high as "Nanjing 46", but only SSSII-2 RNAi rice retained the transparent endosperm under low moisture conditions. Further examination showed that the different morphologies and fine structures of the starch granules may contribute to the specific properties of SSSII-2 RNAi rice. Therefore, SSSII-2 has potential application in future high quality rice breeding programs.

[Expression of the GUS fusion gene controlled by the tomato rbcS3A promoter in transgenic rice].

To use different types of promoters in transgenic rice research, the 1.1 kb 5'-upstream regulation region of one of the tomato (Solanum tuberosum L.) Rubisco small subunit gene, rbcS3A, was cloned and its sequences were confirmed by comparison with the known sequences in GenBank. The cloned rbcS3A promoter was fused to the 5'-upstream of GUS (beta-glucuronidase) coding region in a binary vector, and introduced into an elite japonica rice variety by Agrogacterium-mediated transformation. The integration of the GUS fusion gene into the genome of transgenic rice was confirmed by both PCR and Southern blot analysis. The results of both histochemical staining and quantitative analysis of GUS activity showed that the expression level of GUS fusion gene was significantly stronger in stem, leaf blade and sheath than in other organs of transgenic rice plants, and showed highest in the stem, which implies that the tomato rbcS3A promoter can make tissue-specific, in particular in the stem, expression of foreign genes in transgenic rice. The results present here also demonstrate that light induction had no effect on the expression of the foreign gene when regulated by the tomato rbcS3A promoter in transgenic rice. Our results show that the cloned tomato rbcS3A promoter might be very useful for the expression of target genes in transgenic rice, with particularly high efficiency in stem tissues.

[Expression and immunization testing of fusion protein of Newcastle disease virus in leaf tissue of transgenic rice].

Transgenic plant is an attractive bioreactor to produce recombinant protein, such as safe and economic vaccine. In present study, a plant expression binary vector pUNDV, containing the 1.7 kb fusion protein gene of Newcastle Disease Virus (NDV F) under the control of maize ubiquitin (Ubi) promoter and nos terminator was constructed, in which, the Hygromycin phosphotransferase (HPT) gene was used as the selectable marker. The expression cassette was introduced into a japonica rice variety by Agrobacterium-mediated transformation, and 6 independent transgenic rice lines were regenerated and verified by hygromycin resistance selection. The integration of target gene in transgenic plants was confirmed by PCR analysis. The results from ELISA and Western blot analyses revealed that NDV F could be expressed in the leaf of several transgenic plants. The total salt soluble proteins were extracted from leaf of transgenic plant F5, which contained the highest expressing level of NDV F protein, and used in immunization of BALB/c mice. The specific antibodies could be elicited in mice immunized with NDV F protein expressed in transgenic rice.

[Specific expression of the foreign gene regulated by the rice rbcS promoter in transgenic rice].

To use different types of promoters in transgenic rice research, the 5'-upstream regulation region of rice Rubisco small subunit gene (rbcS) was cloned from a Chinese cultivar Wuyunjing 8, and its sequences were confirmed by comparison with the known genome sequences of both japonica and indica rice. The cloned rbcS promoter was fused to the 5'-upstream of GUS (beta-glucuronidase) coding region in a binary vector, and introduced into rice by Agrogacterium-mediated transformation. The integration of the rbcS-GUS fusion gene in transgenic rice was confirmed by PCR analysis. The results of both histochemical staining and quantitative analysis of GUS activity showed that the expression level of GUS fusion gene was significantly stronger in leaf blade and sheath than in other organs of transgenic rice plants, and the GUS activity was restricted to the mesophyll cells of leaf tissue, which showed that the rice rbcS promoter could control not only the tissue- but also the cell-specific expression of foreign genes in transgenic rice. The present results also demonstrated that light induction had a significant effect on the enhancement of transgene's expression when regulated by the rice rbcS promoter in transgenic rice. Our results showed that the rice rbcS promoter might be very useful for the expression of target genes in transgenic rice, with particularly high efficiency in leaf tissues.

[Agrobacterium-mediated transformation of rice mature embryos and regeneration of transgenic plants with Metr gene].

The mature embryos of a Japonica rice, Guanglingxiangnuo, were used for the study on optimization of Agrobacterium-mediated transformation. Several factors affecting the transformation were investigated and a suitable transformation system was set up. For this transformation system, the HRM medium, based on the MS medium, was suitable for inducing callus from rice mature embryos. The suitable time span of initial culture in this medium was 7-8 days before co-culturing with Agrobacterium and suitable medium for selection was CC medium. Using this transformation system, the Metr gene was introduced into Guanglingxiangnuo, and many transgenic plants were obtained. Most of these transgenic rice plants were confirmed by PCR technique and basta resistance, indicating the T-DNA had been integrated into the genome of transgenic rice plants.

Rice OsGL1-1 is involved in leaf cuticular wax and cuticle membrane.

Cuticular wax forms a hydrophobic barrier on aerial plant organs; it plays an important role in protecting a plant from damage caused by many forms of environmental stress. In the present study, we characterized a rice leaf wax-deficient mutant osgl1-1 derived from a spontaneous mutation, which exhibited a wax-deficient and highly hydrophilic leaf phenotype. We cloned the OsGL1-1 gene by the map-based cloning method and performed a complementation test to confirm the function of the candidate gene. Molecular studies revealed that OsGL1-1 was a member of the OsGL1 family, and contained regions that were homologous to some regions in sterol desaturases and short-chain dehydrogenases/reductases. Compared to the wild-type, the osgl1-1 mutant showed decreased cuticular wax deposition, thinner cuticular membrane, decreased chlorophyll leaching, increased rate of water loss, and enhanced sensitivity to drought. OsGL1-1 is expressed ubiquitously in rice. The transient expression of OsGL1-1-green fluorescent protein fusion protein indicated that OsGL1-1 is localized in the cytoplasm, plasma membrane, and nucleus.

Characterization of expression of the OsPUL gene encoding a pullulanase-type debranching enzyme during seed development and germination in rice.

Starch-debranching enzymes (DBEs) are key enzymes involved in starch metabolism in cereals, having a dual function, in both starch synthesis and degradation. However, their precise roles in this pathway, particularly their expression profiles, remain unclear. In the present study, we performed a quantitative real-time PCR (Q-PCR) analysis of the expression pattern of the OsPUL gene encoding a pullulanase-type DBE in different tissues as well as in seeds at different developmental stages. The results showed that this gene was expressed only in seeds. In addition, the 1177-bp OsPUL promoter sequence was cloned, and some endosperm-specific motifs such as the GCN4 and AACA motifs were observed to exist in this region. The promoter was then fused with the GUS reporter gene and its expression was carefully investigated in transgenic rice. The data from both histochemical and fluorometric analyses showed that the OsPUL promoter was capable of driving the target gene to have a high level of endosperm-specific expression. The OsPUL gene maintained a relatively high expression level during the entire period of seed development, and peaked in the middle and late stages. This observation was very consistent with that of the endogenous transcription analysis by Q-PCR. Furthermore, the seed germination experiment showed that the OsPUL promoter actively functions in the late stage of seed germination. The expression of the OsPUL gene was maintained at a significant level during the entire grain filling period and in the late stage of seed germination, which coincided with its involvement in starch anabolism and catabolism.