DNA methylation is one of the epigenetic changes, which plays a major role in regulating gene expression and, thus, many biological processes and diseases. There are several methods for determining the methylation of DNA samples. However, selecting the most appropriate method for answering biological questions appears to be a challenging task. The primary methods in DNA methylation focused on identifying the state of methylation of the examined genes and determining the total amount of 5-methyl cytosine. The study of DNA methylation at a large scale of genomic levels became possible following the use of microarray hybridization technology. The new generation of sequencing platforms now allows the preparation of genomic maps of DNA methylation at the single-open level. This review includes the majority of methods available to date, introducing the most widely used methods, the bisulfite treatment, biological identification, and chemical cutting along with their advantages and disadvantages. The techniques are then scrutinized according to their robustness, high throughput capabilities, and cost.
We studied the correlation of synonymous codon usage (SCU) on gene expression levels under drought stress in rice. Sixty genes related to drought stress (with high, intermediate and low expression) were selected from rice meta-analysis data and various codon usage indices such as the effective number of codon usage (ENC), codon adaptation index (CAI) and relative synonymous codon usage (RSCU) were calculated. We found that in genes highly expressing under drought 1) GC content was higher, 2) ENC value was lower, 3) the preferred codons of some amino acids changed and 4) the RSCU ratio of GC-end codons relative to AT-end codons for 18 amino acids increased significantly compared with those in other genes. We introduce ARSCU as the Average ratio of RSCUs of GC-end codons to AT-end codons in each gene that could significantly separate high-expression genes under drought from low-expression genes. ARSCU is calculated using the program ARSCU-Calculator developed by our group to help predicting expression level of rice genes under drought. An index above ARSCU threshold is expected to indicate that the gene under study may belong to the "high expression group under drought". This information may be applied for codon optimization of genes for rice genetic engineering. To validate these findings, we further used 60 other genes (randomly selected subset of 43233 genes studied for their response to drought stress). ARSCU value was able to predict the level of expression at 88.33% of the cases. Using third set of 60 genes selected amongst high expressing genes not related to drought, only 31.65% of the genes showed ARSCU value of higher than the set threshold. This indicates that the phenomenon we described in this report may be unique for drought related genes. To justify the observed correlation between CUB and high expressing genes under drought, possible role of tRNA post transcriptional modification and tRFs was hypothesized as possible underlying biological mechanism.
BackgroundKochia scoparia is a dicotyledonous annual herb and belongs to the Amaranthaceae family. Genetic diversity and resistance to drought stress of this plant has made it widely scattered in different regions which contains highly genetic diversity and great potential as fodder and can grow on salty, drought affected areas. Since the soil salinity has become widely spread, environmental concern has sparked so many debates. An important limiting factor in agricultural production worldwide is the sensitivity of most of the crop to salinity caused by high concentration of salts soil. Plants use three different strategies to prevent and adapt to high Na+ concentrations. Antiporters are important category of genes that play a pivotal role in ion homeostasis in plants. Na+/H+ antiporters (NHX1 and SOS1) are located in tonoplasts and reduce cytosolic Na+ concentration by pumping in the vacuole whereas SOS1 is localized at the plasma membrane and extrudes Na+ in apoplasts.ResultsCoding sequence of plasma membrane Na+/H+ antiporter (SOS1) and vacuole membrane Na+/H+ antiporter (NHX) in Kochia scoparia were isolated using conserved sequences of SOS1 and NHX. Also, expression profile under salinity stress was studied in this study. The amino acid sequences (aa) of the isolated region of K.SSOS1 and K.SNHX showed the maximum identity up to 84% and 90% to its orthologous in salicornia brachiate and suede maritime, respectively. The results of semi-quantitative RT-PCR revealed that salinization has affected positively on SOS1 transcription level. The expression of K.SSOS1 and K.SNHX in leaves and roots of Kochia scoparia were progressively increased under all salinity levels compared to control.ConclusionThe results suggest that K.SSOS1 and K.SNHX play an essential role in salt tolerance of K.scoparia and they can be useful to improve salt tolerance in other crops.
Background Flower color is one of the main characteristics of ornamental plants. Aurones are light yellow flavonoids produced in the petals of a limited number of plant species including snapdragon (Antirrhinum majus). As a commercially-recognized species, African violet can be found in various colors except yellow. This research, aiming at changing the petals’ color of African violet from white to yellow, was conducted using the simultaneous expressions of chalcone 4’-O-glucosyltransferase (4’CGT) and aureusidin synthase (AS1) genes without the need for silencing anthocyanin biosynthesis pathway genes via both transient and stable transfer methods. Results The transient gene transfer among transgenic plants led to a clear change of petals’ color from white to light yellow. This occurs while no change was observed in non-transgenic (Wild type) petals. In total, 15 positive transgenic plants, produced via stable gene transfer, were detected. Moreover, since their flower color was yellow, both genes were present. Meanwhile, the corresponding transformation yield was determined 20-30%. The transformation, expression and integration of genes among T0 transgenic plants were verified using the PCR, qRT-PCR and Southern blotting techniques, respectively. Furthermore, the probable color change of petals’ cross-section and existence of Aureusidin 6-O-glucoside (AOG) compound were determined using a light microscope and HPLC-DAD-MSn analysis, correspondingly. Conclusions Generally, the creation of aurones biosynthesis pathway is only viable through the simultaneous expression of genes which leads to color change of African violet’s petal from white to yellow. This conclusion can lead to an effective strategy to produce yellow color in ornamental plant species.
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