Kajvan Saed Moocheshi; Ali Izadi Darbandi; Nima Namjoo; Namjoo Saed Moocheshi
Abstract
In most procedures that involve gene cloning, after the amplification of a target gene by PCR or by Real-time PCR, the purification of the trappedgene on agarose gel is a crucial stage. There are various methods for extracting genes from agarose gel by removing other contaminants. We isolated the amplified ...
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In most procedures that involve gene cloning, after the amplification of a target gene by PCR or by Real-time PCR, the purification of the trappedgene on agarose gel is a crucial stage. There are various methods for extracting genes from agarose gel by removing other contaminants. We isolated the amplified PqHMGR gene (derived from Ginseng (Panax quinquefolius)) from agarose gel by a quasi-electrophoresis device (similar to electro-elution technique). Moreover, the efficiency of this new approach was compared with that of the commercial kit ‘Silica Bead DNA Gel Extraction’ (Thermo Scientific American Company). Ligation to the PTG-19 plasmid and cloning in E. coli bacteria were also done. The results showed successful isolations of targeted DNA, along with a high efficiency in producing recombinant DNA and in concluding a successful cloning procedure through this new device. The invented method provided a better purification ability than the commercial kit, but because of using the TAE 1X buffer as the purified gene storage solution, the plasmid and bacterial transformation rates were slower than the commercial kit method. It was found that using the new method for the purification of nucleotide sequences by electrophoresis and electrophoresis buffer is feasible, and that these purified fragments can be applied in cloning and sequencing. Using the TAE 1X buffer instead of distilled water did not cause problems in gene binding to PTG-19 plasmid. It also allowed a successful transformation of E. coli bacteria by the modified plasmid. Nonetheless, using TAE 1X buffer reduced the modification rate of the PTG-19 plasmid and decreased the rate of E. coli transformation by the modified plasmid.Abbreviations:5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), Complementary DNA (cDNA), Diethyl Pyrocarbonate (DEPC), Escherichia coli (E. coli), Ethylenediaminetetraacetic acid (EDTA), Isopropyl ß-D-1-thiogalactopyranoside (IPTG), LB (Luria Broth), Optical density 260 (OD260), Optical density 280 (OD280), Panax quinquefolius HMGR (PqHMGR), Polymerase chain reaction (PCR), Reverse transcription polymerase chain reaction (RT-PCR), Tris/Borate/EDTA (TBE), Tris-acetate-EDTA. 1X (TAE).
Hossein Astaraki; Hossein Ramshini; Mahmoud Lotfi; Ali Izadi Darbandi
Abstract
Development of cultivars with high yield under normal conditions and maintaining their yield under abiotic stresses is the main purpose of plant breeding programs in arid and semi-arid areas. The present study aimed to evaluate the yield stability of a collection of commercial melon varieties under drought ...
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Development of cultivars with high yield under normal conditions and maintaining their yield under abiotic stresses is the main purpose of plant breeding programs in arid and semi-arid areas. The present study aimed to evaluate the yield stability of a collection of commercial melon varieties under drought stress. The trial was conducted in a field under normal conditions (plants were irrigated after 50 mm evaporation of a class A evaporation pan) and drought stress conditions (irrigation was carried out after 100 mm evaporation of a class A evaporation pan). In average, 3.32 kg fruit/plant and 2.76 kg fruit/plant were obtained under normal and drought stress conditions, respectively. The highest reduction in yield as the consequence of drought exposure was recorded for 'Mazandarani' (52%) and 'Samsoori' (48%). The most drought-tolerant genotypes were 'Mamaghani', 'Nahavandi', 'Shadegan', 'Crenshaw' and 'Suski-e-Sabz' as they had constant yield under both growing conditions. On the other hand, 'Samsoori' and 'Saveh' were the most sensitive genotypes to drought. For most of the measured traits, the values of broad-sense heritability were over 0.50 i.e. there was a large genetic diversity among melon genotypes. This variation can be utilized for selecting high potential fruit yield and drought-tolerant genotypes. Total soluble solids (TSS) (ºBrix) was 15.2% for 'Honey-Dew'. TSS (ºBrix) was obtained 10.7, 10.09, and 9.2% for Iranian genotypes of 'Khatooni', 'Samsoori', and 'Saveh', respectively. In conclusion, although some Iranian melon genotypes were recognized as drought tolerant, they need to be improved for TSS (ºBrix).
