Identification of O-acetylserine(thiol)lyase (OASTL) genes in sorghum (Sorghum bicolor) and gene expression analysis under cadmium stress

Cysteine (Cys) is the first identified molecule in plant metabolism which includes both sulfur and nitrogen. It can be synthesized in three cellular compartments, containing chloroplast, cytoplasm and mitochondrion. The final step of cysteine biosynthesis is catalyzed by the O-acetylserine(thiol)lyase enzyme (OASTL, E.C. 4.2.99). In the present study, seven members of the OASTL gene family in the sorghum (Sorghum bicolor) genome were identified at a genome-wide scale and comparative bioinformatics analyses were performed between sorghum and Arabidopsis OASTLs. In all OASTL proteins, a pyridoxal-phosphate dependent domain structure (PALP, PF00291) was identified. The gene ontology annotations also revealed that all sorghum OASTL genes have KOG1252 (Cystathionine beta-synthase and related enzyme) and K01738 (cysteine synthase A) activities. In promotor sequences of OASTL genes, diverse cis-acting elements were found, including hormone and light responsiveness, abiotic stress responsiveness, and tissue-specific ones (meristem and endosperm). Sorghum OASTL genes demonstrated medium or high level expressions in anatomical parts and developmental stages based on the digital expression data. Expression of OASTL genes were also analyzed under cadmium (Cd) stress in sorghum by Real Time-quantitative PCR (RT-qPCR). The results exclusively showed that OASTL A1-2 gene was 1.12 fold up-regulated in roots, whereas cysteine synthase 26 was 2.25 fold down-regulated in leaves. The predicted 3D structure of OASTLs indicated some structural diversities as well as variations in the secondary structures.

     

Genome-wide identification and cadmium induced expression profiling of sulfate transporter (<Emphasis Type="Italic">SULTR</Emphasis>) genes in sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis> L.)

Sulfur is an essential element for all living organisms. Plants can convert inorganic sulfur into organic sulfur compounds by complex enzymatic steps. In this study, we conducted a genome-wide analysis of sulfate transporter genes (SULTRs) in the sorghum (Sorghum bicolor) genome and examined expression profiles of SbSULTR genes under 200 µM cadmium (Cd) exposure. As a result of sorghum genome analysis, 11 SULTR genes were identified, including SbSULTR1;1, SbSULTR1;2, SbSULTR1;3, SbSULTR2;1, SbSULTR2;2, SbSULTR3;1, SbSULTR3;2, SbSULTR3;3, SbSULTR3;4, SbSULTR3;5, and SbSULTR4. Given names are based on phylogeny and chromosomal locations. Except SbSULTR4, all SbSULTR proteins contained Sulfate_transp (PF00916), STAS (PF01740) domains and 12 trans-membrane domains. Phylogenetic analysis revealed that four major groups were identified such as SULTR1, 2, 3, and 4 groups and SULTR4 group was separated to other SULTR groups. In promotor sequences of SbSULTR genes, many diverse cis-acting elements were found mainly related with physiological processes such as light, stress and hormone responsiveness. The expression profiles of SbSULTR genes showed that SULTR1;2, 1;3, 3;3, and 3;5 genes up-regulated in root, while expression level of SULTR4 decreased under 200 µM Cd exposure. The predicted 3D structures of SULTR proteins showed some conformational changes, suggesting functional diversities of SbSULTRs. Finally, results of this study may contribute towards understanding SbSULTR genes and their regulations and roles in Cd stress in sorghum.     

Improved FLP Recombinase,FLPe, Efficiently Removes Marker Gene from Transgene Locus Developed by Cre–<Emphasis Type="Italic">lox</Emphasis> Mediated Site-Specific Gene Integration in Rice

Site-specific recombination systems, such as FLP–FRT and Cre–lox, carry out precise recombination reactions on their respective targets in plant cells. This has led to the development of two important applications in plant biotechnology: marker-gene deletion and site-specific gene integration. To draw benefits of both applications, it is necessary to implement them in a single transformation process. In order to develop this new process, the present study evaluated the efficiency of FLP–FRT system for excising marker gene from the transgene locus developed by Cre–lox mediated site-specific integration in rice. Two different FLP recombinases, the wild-type FLP (FLPwt) and its thermostable derivative, FLPe, were used for the excision of marker gene flanked by FLP recombination targets (FRT). While marker excision mediated by FLPwt was undetectable, use of FLPe resulted in efficient marker excision in a number of transgenic lines, with the relative efficiency reaching up to ~100%. Thus, thermo-stability of FLP recombinase in rice cells is critical for efficient site-specific recombination, and use of FLPe offers practical solutions to FLP–FRT-based biotechnology applications in plants.     

Mitochondrial iron transporter (MIT) gene in potato (Solanum tuberosum): comparative bioinformatics,physiological and expression analyses in response to drought and salinity

BioMetals - Mitochondrial iron transporter (MIT) genes are essential for mitochondrial acquisition/import of iron and vital to proper functioning of mitochondria. Unlike other organisms, research...     

Dosage-Dependent Gene Expression from Direct Repeat Locus in Rice Developed by Site-Specific Gene Integration

In the standard plant transformation practice, transgene copy number is often inversely correlated with transgene expression. As the integration locus generated by standard methods is mostly complex, consisting of both full-length and partial copies arranged in direct or inverted repeat configurations, it is difficult to parse the effect of copy number and locus structure. To clearly study the effect of transgene copy number on gene expression, it is important to control the locus structure and integrate full-length copies. In this study, the effect of transgene copy number on transgene expression in plant cells was determined using rice callus as a model. To generate full-length integrations, Cre-lox-mediated site-specific gene integration method was used. Transgenic rice lines consisting of one to three copies of β-glucuronidase or green fluorescent protein genes were developed. Site-specific integration lines were characterized and subjected to expression analysis. Lines containing two or three copies of either reporter genes displayed 2–4 times higher expression compared to the single-copy lines. Therefore, dosage-dependent transgene expression can be obtained by integrating full-length copies, and site-specific gene integration approach can serve as an efficient tool for generating precise multi-copy integrations.