Knock down Os1bglu1 β-glucosidase in rice by Agrobacterium-mediated transformation

This research attempted to study the function of Os1bglu1 by RNAi technique. The suppression of Os1bglu1genewas done using the 3’UTR region. The target gene fragment was cloned into the pHELLSGATE8 vector. The high percentagesof effective callus induction of 93% were obtained when the seeds were cultured on N6D medium for 4-6 weeks at 28°C. Thesuitable transformation conditions were to incubate the calli with Agrobacterium (OD 600 = 0.02) and blot dry to remove excessbacteria cells, then transferred to co-cultivation medium (pH 5.2) with 200 M acetosyringone and incubate for three days at25°C. The 20% transformation efficiency was obtained from the transformed calli with control plasmid, while transformationefficiency of only 15% was obtained from pHELLSGATE8 Os1bglu1 constructs. The transformed calli with control constructshowed higher growth rate than the transformed calli with pHELLSGATE8 Os1bglu1construct. The expression of Os1bglu1mRNA was not found in the transformed calli and siRNAs were found in the transformed calli. However no siRNAs weredetected in the control transformed calli. The regeneration efficiencies of 6% were obtained from only the calli transformedwith the control construct. The calli transformed with the knock down Os1bglu1 constructs were not able to regenerate. This may indicated that Os1bglu1 is involved in regeneration of rice from callus tissue

รายงานการวิจัยการบ่งชี้และศึกษาการทำงานของจิบเบอริลินเบตากูลโคไซเดสจากข้าว

Funded by Suranaree University of Technology budget year 2012-201

Genomic and expression analysis of glycosyl hydrolase family 35 genes from rice (Oryza sativa L.)

<p>Abstract</p> <p>Background</p> <p>Many plant β-galactosidases (Bgals) have been well characterized and their deduced biological functions mainly involve degradation of structural pectins, xyloglucans or arabinogalactoproteins in plant cell walls. However, gene multiplicity in glycosyl hydrolase family 35 (GH35), to which these proteins belong, implies diverse functions. In this study, the gene multiplicity, apparent evolutionary relationships and transcript expression of rice Bgal genes were examined, in order to predict their biological functions.</p> <p>Results</p> <p>Fifteen rice Bgal genes were identified in the plant genome, one of which encodes a protein similar to animal Bgals (<it>OsBgal9</it>), and the remaining 14 fall in a nearly plant-specific subfamily of Bgals. The presence of both classes of Bgals in bryophytes, as well as vascular plants, suggests both gene lineages were present early in plant evolution. All 15 proteins were predicted to contain secretory signal sequences, suggesting they have secretory pathway or external roles. RT-PCR and database analysis found two distinct lineages to be expressed nearly exclusively in reproductive tissues and to be closely related to <it>Arabidopsis </it>Bgals expressed most highly in flower and pollen. On the other hand, <it>OsBgal6 </it>is expressed primarily in young vegetative tissues, and alternative splicing in panicle prevents its production of full-length protein in this reproductive tissue. <it>OsBgal11 </it>also showed alternative splicing to produce different length proteins. OsBgal13 produced by recombinant expression in <it>Escherichia coli </it>hydrolyzed α-L-arabinoside in addition to β-D-galactoside and β-(1→3)-, β-(1→4)- and β-(1→6)- linked galacto-oligosaccharides.</p> <p>Conclusion</p> <p>Rice <it>GH35 </it>contains fifteen genes with a diversity of protein sequences, predicted locations and expression and splicing patterns that suggest that OsBgals enzymes may play a variety of roles in metabolism of cell wall polysaccharides, glycoproteins and glycolipids.</p

Analysis of rice glycosyl hydrolase family 1 and expression of Os4bglu12 β-glucosidase

BACKGROUND: Glycosyl hydrolase family 1 (GH1) β-glucosidases have been implicated in physiologically important processes in plants, such as response to biotic and abiotic stresses, defense against herbivores, activation of phytohormones, lignification, and cell wall remodeling. Plant GH1 β-glucosidases are encoded by a multigene family, so we predicted the structures of the genes and the properties of their protein products, and characterized their phylogenetic relationship to other plant GH1 members, their expression and the activity of one of them, to begin to decipher their roles in rice. RESULTS: Forty GH1 genes could be identified in rice databases, including 2 possible endophyte genes, 2 likely pseudogenes, 2 gene fragments, and 34 apparently competent rice glycosidase genes. Phylogenetic analysis revealed that GH1 members with closely related sequences have similar gene structures and are often clustered together on the same chromosome. Most of the genes appear to have been derived from duplications that occurred after the divergence of rice and Arabidopsis thaliana lineages from their common ancestor, and the two plants share only 8 common gene lineages. At least 31 GH1 genes are expressed in a range of organs and stages of rice, based on the cDNA and EST sequences in public databases. The cDNA of the Os4bglu12 gene, which encodes a protein identical at 40 of 44 amino acid residues with the N-terminal sequence of a cell wall-bound enzyme previously purified from germinating rice, was isolated by RT-PCR from rice seedlings. A thioredoxin-Os4bglu12 fusion protein expressed in Escherichia coli efficiently hydrolyzed β-(1,4)-linked oligosaccharides of 3–6 glucose residues and laminaribiose. CONCLUSION: Careful analysis of the database sequences produced more reliable rice GH1 gene structure and protein product predictions. Since most of these genes diverged after the divergence of the ancestors of rice and Arabidopsis thaliana, only a few of their functions could be implied from those of GH1 enzymes from Arabidopsis and other dicots. This implies that analysis of GH1 enzymes in monocots is necessary to understand their function in the major grain crops. To begin this analysis, Os4bglu12 β-glucosidase was characterized and found to have high exoglucanase activity, consistent with a role in cell wall metabolism

The performance of a resazurin chromogenic agar plate with a combined disc method for rapid screening of extended-spectrum-β-lactamases, AmpC β-lactamases and co-β-lactamases in Enterobacteriaceae.

A resazurin chromogenic agar (RCA) along with combined disc method has been developed as a promising method for rapid screening of extended-spectrum-β-lactamase (ESBL), AmpC β-lactamase, and co-production of ESBL and AmpC. Cefpodoxime (CPD) discs supplemented with and without clavulanic acid (CA), cloxacillin (CX), or CA+CX were evaluated against 86-molecularly confirmed β-lactamase-producing Enterobacteriaceae, including 15 ESBLs, 32 AmpCs, 9 co-producers of ESBL and AmpC, and 30 carbapenemase producers. The CA and CX synergy test successfully detected all ESBL producers (100% sensitivity and 98.6% specificity) and all AmpC producers (100% sensitivity and 96.36% specificity). This assay also exhibited a good performance in the screening for the co-existence of ESBL and AmpC (88.89% sensitivity and 100% specificity). The RCA assay is a simple and inexpensive method that allows observation of results within 7 h. It can be applicable in any microbiological laboratory, especially in the endemic areas of ESBL, AmpC, or co-β-lactamase-producing Enterobacteriaceae

Bacterial β-Glucosidase Reveals the Structural and Functional Basis of Genetic Defects in Human Glucocerebrosidase 2 (GBA2)

Human glucosylcerebrosidase 2 (GBA2) of the CAZy family GH116 is responsible for the breakdown of glycosphingolipids on the cytoplasmic face of the endoplasmic reticulum and Golgi apparatus. Genetic defects in GBA2 result in spastic paraplegia and cerebellar ataxia, while cross-talk between GBA2 and GBA1 glucosylceramidases may affect Gaucher disease. Here, we report the first three-dimensional structure for any GH116 enzyme, Thermoanaerobacterium xylanolyticum TxGH116 β-glucosidase, alone and in complex with diverse ligands. These structures allow identification of the glucoside binding and active site residues, which are shown to be conserved with GBA2. Mutagenic analysis of TxGH116 and structural modeling of GBA2 provide a detailed structural and functional rationale for pathogenic missense mutations of GBA2