This study was carried out to build a database system for amylose and protein contents of rice germplasm based on NIRS (Near-Infrared Reflectance Spectroscopy) analysis data. The average waxy type amylose contents was 8.7% in landrace, variety and weed type, whereas 10.3% in breeding line. In common rice, the average amylose contents was 22.3% for landrace, 22.7% for variety, 23.6% for weed type and 24.2% for breeding line. Waxy type resources comprised of 5% of the total germplasm collections, whereas low, intermediate and high amylose content resources share 5.5%, 20.5% and 69.0% of total germplasm collections, respectively. The average percent of protein contents was 8.2 for landrace, 8.0 for variety, and 7.9 for weed type and breeding line. The average Variability Index Value was 0.62 in waxy rice, 0.80 in common rice, and 0.51 in protein contents. The accession ratio in arbitrary ranges of landrace was 0.45 in amylose contents ranging from 6.4 to 8.7%, and 0.26 in protein ranging from 7.3 to 8.2%. In the variety, it was 0.32 in amylose ranging from 20.1 to 22.7%, and 0.51 in protein ranging from 6.1 to 8.3%. And also, weed type was 0.67 in amylose ranging from 6.6 to 9.7%, and 0.33 in protein ranging from 7.0 to 7.9%, whereas, in breeding line it was 0.47 in amylose ranging from 10.0 to 12.0%, and 0.26 in protein ranging from 7.0 to 7.9%. These results could be helpful to build database programming system for germplasm management.

A statistical analysis for 3651 genetic resources collected from China (1,542), Japan (1,409), Korea (413), and India (287) was conducted using normal distribution, variability index value (VIV), analysis of variation (ANOVA) and Ducan’s multiple range test (DMRT) based on a data obtained from NIRS analysis. In normal distribution, the average protein content was 8.0%, whereas waxy type amylose and common rice amylose were found to be 8.7% and 22.7%, respectively. The protein contents ranged from 5.4 to 10.6% at the level of 95%. The waxy amylose and common rice amylose ranged from 5.9 to 11.5%, and from 16.9 to 28.5% at 95% confidence level, respectively. The VIV was 0.59 for protein, 0.64 for low amylose, and 0.81 for high amylose contents. The average amylose contents were 18.85% in Japanese, 19.99% in Korean, 20.27% in Chinese, and 25.46% in Indian resources, while the average protein contents were found to be 7.23% in Korean, 7.73% in Japanese, 8.01% in Chinese, and 8.17% in Indian resources. The ANOVA of amylose and protein content showed significant differences at the level of 0.01. The F-test for amylose content was 158.34, and for protein content 53.95 compared to critical value 3.78. The DMRT of amylose and protein content showed significant difference (p<0.01) between resources of different countries. Japanese resources had the lowest level of amylose contents, whereas, the lowest level of protein content was found in Korean resources compared to other origins. Indian resources showed the highest level of amylose and protein contents. It is recommended these results should be helpful to future breeding experiments.

This study was conducted to characterize the amylose and protein contents of 4,948 rice landrace germplasm using the NIRS model developed in the previous study. The average amylose content of the germplasm was 20.39% and ranged between 3.97 and 37.13%. The amylose contents in the standard rice were 4.99, 18.63 and 20.55% in Sinseonchal, Chucheong and Goami, respectively. The average protein content was 8.17% and ranged from 5.20 to 17.45%. Protein contents in Sinseonchal, Chucheong and Goami were 6.824, 6.869 and 7.839%, respectively. A total of 62% germplasm were distributed between 20.06% and 27.02% in amylose content. Germplasm of 81.60% represented protein content of 6.78-9.75%. The distinguishable ranges of amylose contents according to origin were 16.58-20.06% in Korea, 20.06-23.25% in Japan, 23.25-27.02% in North Korea, and 27.02-37.13% in China. In the protein content, approximately 30% of Chinese resources ranged from 9.75 to 17.45%, whereas less than 10% were detected in other origin accessions. Fifty resources were selected with low and high amylose ranging from 3.97-6.66% and 30.41-37.13%, respectively. Similarly, fifty resources were selected with low and high protein ranging from 5.20-6.09% and 13.21-17.45%, respectively. Landraces with higher protein could be adapted to practical utilization of food sources.

The objective of this research was to develop Near-Infrared Reflectance Spectroscopy (NIRS) model for amylose and protein contents analysis of large accessions of rice germplasm. A total of 511 accessions of rice germplasm were obtained from National Agrobiodiversity Center to make calibration equation. The accessions were measured by NIRS for both brown and milled brown rice which was additionally assayed by iodine and Kjeldahl method for amylose and crude protein contents. The range of amylose and protein content in milled brown rice were 6.15-32.25% and 4.72-14.81%, respectively. The correlation coefficient (R 2 ), standard error of calibration (SEC) and slope of brown rice were 0.906, 1.741, 0.995 in amylose and 0.941, 0.276, 1.011 in protein, respectively, whereas R 2 , SEC and slope of milled brown rice values were 0.956, 1.159, 1.001 in amylose and 0.982, 0.164, 1.003 in protein, respectively. Validation results of this NIRS equation showed a high coefficient determination in prediction for amylose (0.962) and protein (0.986), and also low standard error in prediction (SEP) for amylose (2.349) and protein (0.415). These results suggest that NIRS equation model should be practically applied for determination of amylose and crude protein contents in large accessions of rice germplasm.

In this study, we analyzed seed storage proteins in order to investigate the main factors related to the eating quality of japonica and tongil-type rice varieties. Sensory evaluation was performed by a trained panel to assess the appearance (color and glossiness), flavor, taste, stickiness, texture, and overall score of nine japonica and three tongil-type rice cultivars. Moreover, the pattern of variation in rice storage proteins was examined by electrophoresis of protein extracts. The electrophoretic pattern of rice proteins showed 16.4 kDa albumin, 26.4 kDa globulin, 34-39 kDa and 21-22 kDa glutelin, and 14.3 kDa prolamin. In terms of storage protein, the varietal differences between japonica and tongil-type rice were found in albumin, globulin, and the α-1, and α-2 sub-units of acidic glutelin. Furthermore, the overall sensory evaluation score was observed to be positively correlated with albumin (0.495 ** ) and globulin (0.567 ** ), and negatively correlated with α-1 glutelin (-0.612 ** ). Therefore, the results indicated that albumin, globulin, and α-1 glutelin can affect the eating quality of japonica and tongil-type rice varieties, with the latter having lower eating quality than the former.

Background : Alzheimer’s disease (AD) is a neurodegenerative disease characterized by progressive memory loss, cognitive impairment and personality defects accompanied by diffuse structural abnormalities in the brain. The major pathological hallmarks of AD include beta amyloid (Aß) protein deposition, presence of neurofibrillary tangles and neurodegeneration of cholinergic neurons. Aß, a 39-43 amino acid proteolytic fragment of amyloid precursor protein, is the major constituent of the senile plaques. Rice bran, the major byproduct of the rice milling industry, is the source of a high quality vegetable oil. Rice bran oil (RBO) has attracted much medicinal attention for its strong hypocholesterolemic properties because of its balanced fatty acid composition and high levels of antioxidant phytochemicals such as oryzanols, tocopherols and tocotrienols. The present study aims to investigate the protective effect of RBO against Aß (25-35)-induced neurotoxicity in in vitro and in vivo. Methods and Results : Memory impairment was produced by intracerebroventricular (i.c.v) microinjection of 15 nmol Aß (25-35) and measured by passive avoidance test in ICR mice. Glutathione concentration, lipid peroxidation rate and acetylcholine esterase activity were measured in mice brain. The expression levels of phosphorylated mitogen activated proteins kinases (MAPKs), inflammatory factors, and anti-apoptotic and pro-apoptotic proteins in mice brains were detected by Western blot. Cerebral cortical neuronal cells were cultured from 15-days-old fetus. Cortical neurons were incubated with 10 μM Aß (25-35) for 36 h. Cell viability was measured by MTT assay. Chronic treatments of RBO (0.1-1 ml/kg, 8 days, p.o.) protected against memory impairment induced by Aß (25-35). Depletion of glutathione level, lipid peroxidation and increased acetylcholine esterase activity by the treatment with Aß (25-35) were inhibited by administration of RBO. The increase of phosphorylated MAPKs, inflammatory factors, and proapoptotic proteins and the decrease of antiapoptotic protein in Aß (25-35)-administered mice brain were significantly inhibited by treatment with RBO. RBO (0.1-5ul/ml) inhibited 10μM Aß (25-35)-induced neuronal cell death in cultured cortical neurons. Conclusion : The present study suggests the role of RBO as a promising therapeutic for neurodegenerative diseases like AD and stroke.

Fibroin silk proteins from spider or silkworm are attractive biomaterials that are of particular biotechnological interest for industrial and medical purposes because of their unique physical and mechanical properties. In this study, we generated and characterized the transgenic rice plant expressing a spider silk protein. Spider silks have great potential as biomaterials with extraordinary properties. Here, we report the cloning and characterization of the major ampullate silk protein gene from the spider Araneus ventricosus. A cDNA encoding the partial major ampullate silk protein (AvMaSp) was cloned from A. ventricosus. An analysis of the cDNA sequence shows that AvMaSp consists of a 240 amino acid repetitive region and a 99 amino acid C-terminal non-repetitive domain. The peptide motifs that were found in the spider major ampullate silk proteins, (A)n, (GA)n, and (GGX)n, were conserved in the repetitive region of AvMaSp. Phylogenetic analysis further confirmed that AvMaSp belongs to the spider major ampullate spidroin family of proteins. Recombinant AvMaSp-R was degraded abruptly by trypsin. However, AvMaSp-R was stable at 100 °C for at least 30 min. Additionally, the AvMaSp-R was stable at pH values from 2 to 12 for at least 1 h. Taken together, our findings describe the molecular structure and biochemical properties of the A. ventricosus major ampullate silk protein and demonstrate its potential as a biomaterial.

As sessile organisms, plants have evolved mechanisms that allow them to adapt and survive periods of various environmental stresses including high salinity and drought. The ubiquitin-proteasome system (UPS) is an integral player in plant response and adaptation to various abiotic stresses. Understanding UPS function has centered mainly on defining the role of E3 ubiquitin ligases, which are the substrate-recruiting component of the ubiquitination pathway. Here, we report on Ring finger E3 ligase, Oryza sativa salt- and drought-induced RING finger protein1 gene (OsSDRFP1) in defense responses to osmotic stresses. Results of qRT-PCR and In vitro ubiquitination assay demonstrated that OsSDRFP1 act as an E3 ligase in response to salt and drought stresses. in this study, Subcellular localizations showed that the OsSDRFP1 was observed in cytosol (66%) and nucleus (34%) under non-treated conditions. However, the florescence signals of rice protoplasts after salt treatments detected in nucleus (60%) higher than in cytosol (30%). The Arabidopsis plants overexpressing OsSDRFP1 clearly exhibited hypersensitive responses to salt stress. whereas, OsSDRFP1-overexpressing plants were more tolerant to both drought- and ABA-stresses than the wild-type plants. These results might suggest that OsSDRFP1 has a dual function as a regulator of high salt- and drought-stresses.

Rice flour is used in many food products. However, dough made from rice lacks extensibility and elasticity, whereas that of wheat is suitable for many food products including breads. We have produced marker-free transgenic rice plants containing a wheat TaGlu-Ax1 gene encoding the HMG-GS from the Korean wheat cultivar ‘Jokyeong’ using the Agrobacteriummediated co-transformation method. The TaGlu-Bx7-own promoter was inserted into a binary vector for seed-specific expression of the TaGlu-Ax1 gene. Two expression cassettes comprised of separate DNA fragments containing only TaGlu-Ax1 and hygromycin phosphotransferase II (HPTII) resistance genes were introduced separately to the Agrobacterium tumefaciens EHA105 strain for co-infection. Each EHA105 strain harboring TaGlu-Ax1 or HPTII was infected to rice calli at a 3:1 ratio of TaGlu-Ax1 and HPTII, respectively. Then, among 210 hygromycin-resistant T0 plants, we obtained 20 transgenic lines with both TaGlu-Ax1 and HPTII genes inserted into the rice genome. We reconfirmed integration of the TaGlu-Ax1 gene into the rice genome by Southern blot analysis. Transcripts and proteins of the wheat TaGlu-Ax1 were stably expressed in the rice T1 seeds. Finally, the marker-free plants harboring only the TaGlu-Ax1 gene were successfully screened at the T1 generation.

The aim of the study was to assess the safety of methionine sulfoxide reductase B2(CaMsrB2) protein as toxicity, allergenecity and identity of inserted gene product that transformed rice. Through bioinfomatical research of CaMsrB2, amino acid sequence of CaMsrB2 did not share overall homology with any known or suspected to be allergen or toxin protein. For the biochemical research, CaMsrB2 protein was expressed and purified. Using purified protein, we made a specific antibody. Purified protein was sequenced by Edman degradation methods and confirmed sequence identify.. The amino acid sequences of purified protein were the same as deduced amino acid sequences exclude N-terminal Histidine. And for the internal sequences of CaMsrB2, we performed MALDI-TOF Mass. The results of MALDI-TOF MS was compared Mascot Database and confirmed the sequence coverage was 56%. These results mean bacterially produced CaMsrB2 was the same with inserted gene product. With these purified and identified CaMsrB2 protein, we performed acute toxicity test. Following the OECD guideline 423, 2,000mg/Kg body weight protein were injected as oral administration. After 2 weeks, there did not shown any death and special symptoms.

In this study, we generated and characterized the transgenic rice plant expressing a spider silk protein. Spider silks have great potential as biomaterials with extraordinary properties. We report the cloning and characterization of the major ampullate silk protein gene from the spider Araneus ventricosus. A cDNA encoding the partial major ampullate silk protein (AvMaSp) was cloned from A. ventricosus. An analysis of the cDNA sequence shows that AvMaSp consists of a 240 amino acid repetitive region and a 99 amino acid C-terminal non-repetitive domain. The peptide motifs that were found in the spider major ampullate silk proteins, (A)n, (GA)n, and (GGX)n, were conserved in the repetitive region of AvMaSp. Phylogenetic analysis further confirmed that AvMaSp belongs to the spider major ampullate spidroin family of proteins. The AvMaSp-R cDNA, which encodes the 240 amino acid repetitive domain, was expressed as a soluble 22 kDa polypeptide in baculovirus-infected insect cells. To produce transgenic rice plant with high contents of glycine and alanine, the prolamin promoter-driven AvDrag was introduced into rice plant via agrobacterium tumefaciens-mediated gene transformation. The introduction and copy number of the AvDrag gene in transgenic rice plants were determined by PCR and Southern blot analysis. AvDrag expression in transgenic rice seeds was examined by Northern blot and Western blot analysis. Immunofluorescence staining with the AvDrag antiserum revealed that the recombinant AvDrag protein were localized in transgenic rice seed. Furthermore, the amino acid content analysis showed that transgenic rice seeds were greatly increased in glycine and alanine as compared to controls

Since global climate changes drastically, pre-harvest sprouting (PHS) is expected to pose serious problems in rice production. CBL-interacting serine/threonine protein kinases (CIPKs) have been implicated to play important role in regulating various abiotic stresses such as cold, salinity and drought. In this study, to understand the function of this gene under pre-harvest sprouting in rice, a cDNA clone encoding CBL-interacting protein kinase 15 (CIPK15) was isolated from rice flowers. This gene is 2,818 bp long with 1,332 bp coding region that encodes a polypeptide of 443 amino acids. We constructed a recombinant vector carrying the OsCIPK15 under the control of the CaMV 35S promoter and Tnos terminator and transformed into rice using Agrobacterium tumefaciens. Insertion of the gene was verified in transformants using HPT resistance test and genomic PCR. Transcriptional profiling using tissues of wild type, Gopum, revealed expression of the gene in whole plant tissues with level of expression highest in the seeds suggesting possible role in dormancy. Comparative expression analysis of the gene in transgenic and wild type through semi-quantitative RT-PCR and real-time PCR showed higher expression in transgenic rice lines. Moreover, screening in the mist chamber showed overexpression lines that were resistant to the PHS. This result suggests the involvement of OsCIPK15 in the regulation of pre-harvest sprouting.

The proteins from functional rice cultivars (Nogwonchalbyeo, Giant embryonic, Arhyangchalbyeo, and Goamibyeo) and general white rice were extracted and separated using two-dimensional (2D) gel electrophoresis. A wide variation in the molecular weight (MW) and pH range of the expressed proteins in rice samples were observed. The green-kerneled rice (Nogwonchalbyeo) exhibited proteins with MW of 9-57 kDa and appeared at a pH range of 4-7. The Giant embryonic contained proteins with MW of 31-63 kDa and a pH range of 5-6. The aromatic glutinous rice (Arhyangchalbyeo) showed proteins with MW of 24-28 and pH of 5.8-6.8. The high-amylose rice (Goamibyeo) exhibited proteins with MW of 3-63 and pH of 5.2-5.6. The identified proteins uniquely found and highly expressed in each cultivar may have a significant role on rice functionality. The results illustrate that the 2D gel electrophoresis is a valuable method in the determination of the protein expression profiles in functional rice grains and may be useful in the identification of specific marker proteins associated with the functional property of rice.

We used a microarray dataset that is deposited in the public database to evaluate plant responses to heat stress and selected two genes, OsSHSP1 (Os03g16030) and OsSHSP2 (Os01g04380), that are highly expressed under heat stress in rice. OsSHSP1 and OsSHSP2 gene transcripts were highly induced in response to salt and drought. In addition, OsSHSP1 and OsSHSP2 gene transcripts were induced under ABA and SA. Subcellular localization of proteins of 35S::OsSHSP1 were associated with the cytosol, whereas those of and 35S::OsSHSP2 were associated with the cytosol and nucleus. Heterogeneous overexpression of both genes exhibited higher germination rates than those of wild-type plants under the salt treatment, but not under heat or drought stress. The network of both genes harboring 9 sHSPs as well as at least 13 other chaperone genes might support the idea of a role for sHSPs in the chaperone network. Our findings might provide clues to shed light on the molecular functions of OsSHSP1 and OsSHSP2 in response to abiotic stresses, especially heat stress.

In this study, we generated and characterized the transgenic rice plant expressing a spider silk protein. A cDNA coding for the C-terminus of spider silk protein (AvMaSp) was cloned from the spider Araneus ventricosus. Analysis of the cDNA sequence shows that the C-terminus of AvMaSp consists of 165 amino acids of are petitive region and 99 amino acids of a C-terminalnon-repetitive region. The peptide motifs found in spider silk proteins, GGX and An, were conserved in the repetitive region of AvDrag. The AvMaSp cDNA was expressed as a 28kDa polypeptide in baculovirus-infected insect cells. To produce transgenic rice plant with high contents of glycine and alanine, the prolamin promoter-driven AvMaSp was introduced into rice plant via Agrobacteriumtumefaciens-mediated gene transformation. Because of seeds pecific prolamin promoter, expression of AvMaSp protein has been achieved inriceseed. The introduction and copy number of the AvMaSp gene in transgenic rice plants were determined by PCR and Southern blot analysis. AvMaSp expression in transgenic rice seeds was examined by Northern blot and Western blot analysis. Immuno fluorescence staining with the AvMaSpantiserum revealed that the recombinant AvMaSp proteins were localized in transgenic rice seeds. Furthermore, the amino acid content analysis showed that transgenic rice seeds were greatly increased in glycine and alanine as compared to controls. The present study is the first to show the expression of spider silk protein in rice seed.

Development of transgenic plant increasing crop yield or disease resistance is good way to solve the world food shortage. However, the persistence of marker genes in crops leads to serious public concerns about the safety of transgenic crops. In the present study, we developed marker-free transgenic rice inserted high molecular-weight glutenin subunit (HMW-GS) gene (Dx5) from the Korean wheat cultivar ‘Jokyeong’ using Agrobacterium-mediated co-transformation method. The Dx5’s own promoter was used for protein expression. Two expression cassettes comprised of separate DNA fragments containing only the Dx5 and hygromycin resistance (HPTII) genes were introduced separately into Agrobacterium tumefaciens EHA105 strain for co-infection. Each EHA105 strain harboring Dx5 or HPTII was infected into rice calli at a 3: 1 ratio of EHA105 with Dx5 gene and EHA105 with HPTII gene expressing cassette. Then, among 270 hygromycin-resistant transformants, we obtained 27 transgenic lines inserted with both the Dx5 and HPTII genes into the rice genome. We reconfirmed integration of the Dx5 gene into the rice genome by Southern blot analysis. Wheat Dx5 transcripts in T1 rice seeds were examined with semi-quantitative RT-PCR. Protein expression of the Dx5 was analyzed with Western blot using polyclonal antibody recognising x-type of glutenin subunits in T1 seeds. It was suggested that the protein-processing system was conserved between rice and wheat. Finally, the marker-free plants containing only the Dx5 gene were successfully screened at the T1 generation.

Protein disulfide isomerase (PDI) is a chaperone protein that involves in oxidative protein folding by acting as catalysts and folding assistants in the endoplasmic reticulum (ER). Genome database showed that rice contains three PDI-like genes. But, their functions and subcellullar localization are not clearly identified. Here, we show possible functions of rice PDI (OsPDI) during seed development. Seeds of OsPDI T-DNA insertion mutants which were identified by genomic DNA PCR and western blot display chalky phenotype. Electron microscope analysis revealed that endosperms of the OsPDIL1-1Δ mutant show imperfect packing of round starch granules, causing floury-white color. Abnormal form of protein body I (PB-I) containing prolamin and thick aleurone layer were also observed in the OsPDIL1-1Δ mutants. Protein content per seed was significantly low in the OsPDIL1-1Δ mutant. However, free sugar content was high in the OsPDIL1-1Δ mutant seed. Northern and western blot analyses showed that during seed development, OsPDI protein is steadily accumulated in the seed until maturation while its transcript level was highest at 10 days after flowering and rapidly decreased to basal level. In addition, OsPDI strongly interacts with cysteine protease OsCP1 and chaperone BiP protein accumulates in OsPDIL1-1Δ mutant. Besides, proteomic analysis of the OsPDIL1-1Δ mutant seed showed that OsPDI is post-translationally regulated and its loss causes accumulation of many types of seed proteins. Our results indicate that OsPDI plays a critical role in seed development through its regulatory activity for various proteins.