Characterization of the spatial and temporal expression of the OsSSII-3 gene encoding a key soluble starch synthase in rice.

BACKGROUND: Starch, the major component of rice grain, consists of amylose and amylopectin. SSIIa, a key soluble starch synthase involved in the biosynthesis of rice amylopectin, is a major factor that controls the gelatinization temperature of rice grain. Extensive work has been done and impressive progress has been made in elaborating the function of the gene encoding SSIIa (OsSSII-3). However, the systematic expression analysis of OsSSII-3 is still rare. RESULTS: In the present study, we performed a comprehensive expression analysis of OsSSII-3 in both the developing seeds and other tissues of indica rice 9311 by using quantitative real-time PCR. The results showed that the gene was dominantly expressed in the developing seeds. In addition, the promoter sequence of OsSSII-3 was cloned and fused with the GUS reporter gene and its expression was carefully monitored in the transgenic rice. The data from both histochemical and fluorometric analyses showed that the OsSSII-3 promoter was capable of driving the target gene to have an endosperm-specific expression, which may be due to the existing of several endosperm-specific motifs in the promoter, including the -300 elements, AACA motifs and GCN4 motifs. This result was quite consistent with that of the endogenous transcription analysis of OsSSII-3. CONCLUSION: This study not only advanced our understanding of the spatial and temporal expression characteristics of OsSSII-3, but also provided a valuable promoter for future application in generating elite rice varieties with high nutritional or medicinal value.

Ubiquitin fusion expression and tissue-dependent targeting of hG-CSF in transgenic tobacco.

BACKGROUND: Human granulocyte colony-stimulating factor (hG-CSF) is an important human cytokine which has been widely used in oncology and infection protection. To satisfy clinical needs, expression of recombinant hG-CSF has been studied in several organisms, including rice cell suspension culture and transient expression in tobacco leaves, but there was no published report on its expression in stably transformed plants which can serve as a more economical expression platform with potential industrial application. RESULTS: In this study, hG-CSF expression was investigated in transgenic tobacco leaves and seeds in which the accumulation of hG-CSF could be enhanced through fusion with ubiquitin by up to 7 fold in leaves and 2 fold in seeds, leading to an accumulation level of 2.5 mg/g total soluble protein (TSP) in leaves and 1.3 mg/g TSP in seeds, relative to hG-CSF expressed without a fusion partner. Immunoblot analysis showed that ubiquitin was processed from the final protein product, and ubiquitination was up-regulated in all transgenic plants analyzed. Driven by CaMV 35S promoter and phaseolin signal peptide, hG-CSF was observed to be secreted into apoplast in leaves but deposited in protein storage vacuole (PSV) in seeds, indicating that targeting of the hG-CSF was tissue-dependent in transgenic tobacco. Bioactivity assay showed that hG-CSF expressed in both seeds and leaves was bioactive to support the proliferation of NFS-60 cells. CONCLUSIONS: In this study, the expression of bioactive hG-CSF in transgenic plants was improved through ubiquitin fusion strategy, demonstrating that protein expression can be enhanced in both plant leaves and seeds through fusion with ubiquitin and providing a typical case of tissue-dependent expression of recombinant protein in transgenic plants.

Glucose lowering effect of transgenic human insulin-like growth factor-I from rice: in vitro and in vivo studies.

BACKGROUND: Human insulin-like growth factor-I (hIGF-I) is a growth factor which is highly resemble to insulin. It is essential for cell proliferation and has been proposed for treatment of various endocrine-associated diseases including growth hormone insensitivity syndrome and diabetes mellitus. In the present study, an efficient plant expression system was developed to produce biologically active recombinant hIGF-I (rhIGF-I) in transgenic rice grains. RESULTS: The plant-codon-optimized hIGF-I was introduced into rice via Agrobacterium-mediated transformation. To enhance the stability and yield of rhIGF-I, the endoplasmic reticulum-retention signal and glutelin signal peptide were used to deliver rhIGF-I to endoplasmic reticulum for stable accumulation. We found that only glutelin signal peptide could lead to successful expression of hIGF-I and one gram of hIGF-I rice grain possessed the maximum activity level equivalent to 3.2 micro molar of commercial rhIGF-I. In vitro functional analysis showed that the rice-derived rhIGF-I was effective in inducing membrane ruffling and glucose uptake on rat skeletal muscle cells. Oral meal test with rice-containing rhIGF-I acutely reduced blood glucose levels in streptozotocin-induced and Zucker diabetic rats, whereas it had no effect in normal rats. CONCLUSION: Our findings provided an alternative expression system to produce large quantities of biologically active rhIGF-I. The provision of large quantity of recombinant proteins will promote further research on the therapeutic potential of rhIGF-I.