Expression of granule-bound starch synthase in developing rice grain

The expression of granule-bound starch synthase (GBSS) in rice (Oryza sativa) genotype Tainung 67 (200 g kg−1 grain amylose content) and its two NaN3-induced mutants SA419 and SA418 were examined. G/T polymorphism analysis indicated that SA418 (300 g kg−1 grain amylose content) carried Wxa allele. The insertion of 23 base pairs sequence was found only in SA419 (80 g kg−1 grain amylose content), suggesting that it was a waxy mutant. Microsatellite polymorphisms (CT)n were also detected on the Wx gene encoding GBSS in the tested genotypes. The activities of several key enzymes involving starch biosynthesis in developing grains of field-grown rice plants were also compared during grain filling period. Significant genotypic differences were only found in the expression of GBSS. The content of amylose in SA418 grain was higher than Tainung 67 and SA419 grains throughout the entire grain filling period, possibly due to its superiority to synthesize amylose through GBSS. The lowest amylose content of SA 419 grain was attributable to its extremely low activity of GBSS in comparison with the two other genotypes. The mutation effects on the expression of GBSS were confirmed by two-dimensional electrophoresis and mass spectrometry. Copyright © 2007 Society of Chemical Industr

Nucleotide polymorphisms in the waxy gene of NaN(3)-induced waxy rice mutants

Spontaneous and induced waxy phenotype, associated with endosperm containing little or no amylose, has been recognized in rice (Oryza sativa L.). Mutation of a dominant gene Wx into a recessive gene wx, which causes the inactivation or absence of granule bound starch synthase, is believed to be responsible for the change in endosperm starch leading to the waxy grain. In the present study, the nucleotide polymorphism in the Wx gene of rice genotype Tainung 67 (wild type) and its 35 NaN3-induced wx mutants were examined. Iodine staining confirmed that all the mutants had waxy grain trait. The G-to-T single base substitution analysis indicated that the wild type genotype Tainung 67 and its waxy mutants carried Wxb allele. Moreover, 23-bp duplication in exon 2 was detected in all the waxy mutants. Microsatellite polymorphism (CT)n was also detectable on the Wx gene of the tested genotypes and mutants, with at least 5 classes of (CT)n microsatellites identified at the Wx locus. Electrophoretic analyses also confirmed the observed nucleotide polymorphsim. Thus, nucleotide polymorphsim exist among NaN3-induced waxy mutants in rice. However, only the 23-bp duplication in exon 2 may be used as a molecular marker to characterize waxy grain trait in rice genotypes

OIP30, a RuvB-Like DNA Helicase 2, is a Potential Substrate for the Pollen-Predominant OsCPK25/26 in Rice

Calcium ions are a well-known essential component for pollen germination and tube elongation. Several calcium-dependent protein kinases (CDPKs) are expressed predominantly in mature pollen grains and play a critical role in pollen. However, none of their interacting proteins or downstream substrates has been identified. Using yeast two-hybrid screening, we isolated OsCPK25/26-interacting protein 30 (OIP30), which is also predominantly expressed in pollen. OIP30 encodes a RuvB-like DNA helicase 2 (RuvBL2) that is well conserved in eukaryotic species from yeast to human. Yeast and Drosophila defective in RuvBL2 are non-viable. The interaction between OsCPK26 and OIP30 was confirmed by far-Western blot and pull-down experiments. OIP30 was phosphorylated in a calcium-dependent manner by OsCPK26 but not OsCPK2, which is highly similar to OsCPK26 in sequence and expression profile. OIP30 unwound partial duplex DNA with a 3′ to 5′ directionality by ATP hydrolysis. Concurrently, the ATPase activity of OIP30 depended on single-stranded DNA. OsCPK26 phosphorylated OIP30 and enhanced both its helicase and ATPase activity about 3-fold. OIP30 may be the potential downstream substrate for OsCPK25/26 in pollen. This report characterizes a RuvBL in plants and links its activities with its upstream regulator

藉由共轉殖表現促轉基因以增進穠桿菌媒介之植物轉殖效率的方法

本發明係關於一種促進穠桿菌所媒介之植物轉殖效率的方法，特別係指其T－DNA轉殖效率的改進，方法係利用共轉殖雙T-DNA進入植物細胞，其中一個T-DNA包含至少一促轉基因(enhance transformation gene，簡稱ET gene)，可大量表現促轉蛋白，以提高另一個T-DNA區域內之基因於胞內之傳送、入核、或插入染色體之機率，最終提昇獲得成功轉殖植物的效率

Production of two Highly Active Bacterial Phytases with Broad pH Optima in Germinated Transgenic Rice Seeds

Phytate is the main storage form of phosphorus in many plant seeds, but phosphate bound in this form is not available to monogastric animals. Phytase, an enzyme that hydrolyzes phosphate from phytate, has the potential to enhance phosphorus availability in animal diets when engineered in rice seeds as a feed additive. Two genes, derived from a ruminal bacterium Selenomonas ruminantium (SrPf6) and Escherichia coli (appA), encoding highly active phytases were expressed in germinated transgenic rice seeds. Phytase expression was controlled by a germination inducible -amylase gene (Amy8) promoter, and extracellular phytase secretion directed by an Amy8 signal peptide sequence. The two phytases were expressed in germinated transgenic rice seeds transiently and in a temporally controlled and tissue-specific manner. No adverse effect on plant development or seed formation was observed. Up to 0.6 and 1.4 U of phytase activity per mg of total extracted cellular proteins were obtained in germinated transgenic rice seeds expressing appA and SrPf6 phytases, respectively, which represent 46–60 times of phytase activities compared to the non-transformant. The appA and SrPf6 phytases produced in germinated transgenic rice seeds had high activity over broad pH ranges of 3.0–5.5 and 2.0–6.0, respectively. Phytase levels and inheritance of transgenes in one highly expressing plant were stable over four generations. Germinated transgenic rice seeds, which produce a highly active recombinant phytase and are rich in hydrolytic enzymes, nutrients and minerals, could potentially be an ideal feed additive for improving the phytate-phosphorus digestibility in monogastric animals

Different effects on triacylglycerol packaging to oil bodies in transgenic rice seeds by specifically eliminating one of their two oleosin isoforms

Expression of OLE16 and OLE18, two oleosin isoforms in oil bodies of rice seeds, was suppressed by RNA interference. Electron microscopy revealed a few large, irregular oil clusters in 35S::ole16i transgenic seed cells, whereas accumulated oil bodies in 35S::ole18i transgenic seed cells were comparable to or slightly larger than those in wild-type seed cells. Large and irregular oil clusters were observed in cells of double mutant seeds. These unexpected differences observed in oil bodies of 35S::ole16i and 35S::ole18i transgenic seeds were further analyzed. In comparison to wild-type plants, OLE18 levels were reduced to approximately 40% when OLE16 was completely eliminated in 35S::ole16i transgenic plants. In contrast, OLE16 was reduced to only 80% of wild-type levels when OLE18 was completely eliminated in 35S::ole18i transgenic plants. While the triacylglycerol content of crude seed extracts of 35S::ole16i and 35S::ole18i transgenic seeds was reduced to approximately 60% and 80%, respectively, triacylglycerol in isolated oil bodies was respectively reduced to 45% and 80% in accordance with the reduction of their oleosin contents. Oil bodies isolated from both 35S::ole16i and 35S::ole18i transgenic seeds were found to be of comparable size and stability to those isolated from wild-type rice seeds, although they were merely sheltered by a single oleosin isoform. The drastic difference between the triacylglycerol contents of crude seed extracts and isolated oil bodies from 35S::ole16i transgenic plants could be attributed to the presence of large, unstable oil clusters that were sheltered by insufficient amounts of oleosin and therefore could not be isolated together with stable oil bodies

Down-Regulation of Cytokinin Oxidase 2 Expression Increases Tiller Number and Improves Rice Yield

Background: Cytokinins are plant-specific hormones that affect plant growth and development. The endogenous level of cytokinins in plant cells is regulated in part by irreversible degradation via cytokinin oxidase/dehydrogenase (CKX). Among the 11 rice CKXs, CKX2 has been implicated in regulation of rice grain yield. Results: To specifically down-regulate OsCKX2 expression, we have chosen two conserved glycosylation regions of OsCKX2 for designing artificial short hairpin RNA interference genes (shRNA-CX3 and -CX5, representing the 5' and 3' glycosylation region sequences, respectively) for transformation by the Agrobacterium-mediated method. For each construct, 5 independent transgenic lines were obtained for detailed analysis. Southern blot analysis confirmed the integration of the shRNA genes into the rice genome, and quantitative real time RT-PCR and northern blot analyses showed reduced OsCKX2 expression in the young stem of transgenic rice at varying degrees. However, the expression of other rice CKX genes, such as CKX1 and CKX3, in these transgenic lines was not altered. Transgenic rice plants grown in the greenhouse were greener and more vigorous with delayed senescence, compared to the wild type. In field experiments, both CX3 and CX5 transgenic rice plants produced more tillers (27-81 %) and grains (24-67 %) per plant and had a heavier 1000 grain weight (5-15 %) than the wild type. The increases in grain yield were highly correlated with increased tiller numbers. Consistently, insertional activation of OsCKX2 led to increased expression of CKX2 and reduced tiller number and growth in a gene-dosage dependant manner. Conclusions: Taken together, these results demonstrate that specific suppression of OsCKX2 expression through shRNA-mediated gene silencing leads to enhanced growth and productivity in rice by increasing tiller number and grain weight

Cloning and Characterization of Pigment Transportation Gene －Glutathione S-transferase (GST) from Soybean Seed Coat

利用大豆EST基因庫中穀胱苷肽轉移酶(glutathione-S-transferase)(GST2，Y10820)基因序列設計特異核酸引子組，進行逆轉錄酶鏈合反應(RT-PCR)，獲得GST2之基因片段GST420 殖系。經序列分析及基因庫比對確認後，用以篩選黑色(iRT 基因型)大豆青仁烏豆(CRWD)種皮之cDNA 基因庫，共得到二個完整的 GST cDNA 殖系 GST11-1(AY382831, 926 bp)與 GST70-1(AY382832,930 bp)，分別編碼具有 219 個及 216 個胺基酸的蛋白質。由蛋白質結構分析顯示，二殖系均具有 GST 共同之 N 端與 C 端domain 及與 glutathione 結合之 G-site 等三個保留區，依特性分類應屬於第三群之GST基因。以七個不同I, R, T 基因型組合Clark近同源品系進行南方雜交(Southern hybridization)結果顯示，在大豆基因組中GST基因至多為二套，且未發現多型性。由北方分析(Northern hybrid-dization)結果發現，此二個GST基因在種皮的全生育時期及葉部均有表現，但在花及豆莢組織中並未偵測到基因的表現或是表現量極低。由於在大豆EST基因庫中仍有其他GST基因相關殖系存在，因此在花瓣組織中是否另有專一性表現之GST基因，扮演花青素轉移之角色仍有待研究。 Primers specific for GST gene (GST2, Y10820) were synthesized according to the results of soybean EST database search. Expected cDNA fragment GST420 was amplified from total RNA extracted from soybean seed coats of CRWD variety with iRT genotype for experiment. Sequence analysis of GST cDNA fragment confirmed that a partial GST gene fragment was cloned. Two complete cDNA clones, GST11-1 (926 bp, AY382831) and GST70-1 (930 bp, AY382832) of type III GST were obtained by screening the cDNA library with GST420 fragment. The deduced amino acid sequences showed 41-100% identity to ten registered GST proteins by Blast analysis of NCBI. Southern blot analysis indicated that two copies of GST were presented in the genome of Clark isogenic lines with no polymorphism. Northern hybridization showed that these two GST genes expressed in the seed coats and leaves, but not expressed in flowers. Further experiment will be conducted to study the function of GST genes in these tissues