Cloning of Three Antiporter Genes from Arabidopsis and Rice for Over-Expressing Them in Farmer Popular Tomato Varieties of Bangladesh

Razzaque, Samsad; Chakraborty, Debashis; Tammi, Rumana S; Elias, Sabrina M.; Seraj, Zeba I.; Islam, Aparna

doi:10.4236/ajps.2014.526414

Cited by 2 publications

(3 citation statements)

References 23 publications

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“…antiporter increased upon treatment with NaCl and showed more increase plant tolerance to NaCl in transformants plants compared to wild types (Yu et al 2007;DoraniUliaie et al 2012). Also, overexpression of AtNHX1 in tomato resulted in transgenic plants that were able to grow, flower and set fruits at higher salt concentrations (Zhang and Blumwald 2001;Razzaque et al 2014).…”

Section: Introductionmentioning

confidence: 99%

Molecular cloning and expression of a vacuolar Na+/H+ antiporter gene (AgNHX1) in fig (Ficus carica L.) under salt stress

Metwali

Soliman

Fuller

et al. 2015

Plant Cell Tiss Organ Cult

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Soil salinity can be a major limiting factor for productivity in agriculture and forestry and in order to fully utilize saline lands productively in plantation forestry for fig production, the genetic modification of tree species for salt tolerance may be required. Na ? /H ? antiporters have been suggested to play important roles in salt tolerance in plants.Here, we isolated AgNHX1 a vacuolar Na ? /H ? antiporter from a halophytic species Atriplex gmelini and introduced it into fig (Ficus carica L.) cv. Black Mission via Agrobacterium-mediated transformation. Leaf discs explants of fig were co-cultivated for 2 days with Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pBI121 containing the AgNHX1 gene and the hpt selectable marker gene which encodes hygromycin phosphotransferase. Explants were cultured on MS medium containing 30 mg L -1 hygromycin, 3 % sucrose, 0.2 mg L -1 kinetin and 2.0 mg L -1 2,4-dichlorophenoxyacetic acid solidified with 2.5 g L -1 phytagel in darkness for callus formation. The calli were cultured on MS medium containing 2.0 mg L -1 zeatin riboside in combination with 0.4 mg L -1 indole acetic acid in the light for plant regeneration. Putative regenerated transformant shoots were confirmed by polymerase chain reaction (PCR) and Southern hybridization for the AgNHX1 gene. Reverse transcriptase polymerase chain reaction analysis indicated that the gene was highly expressed in transgenic plants, but the degree of this expression varied among transformants. Overexpression of the AgNHX1 gene conferred high tolerance to salt stress and transgenic fig plants overexpressing AgNHX1 developed normally under salinity conditions compared to those of non-transgenic plants. Salt treated transgenic plants contained high proline and K ? but less Na ? compared to non-transgenic control plants.

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Section: Introductionmentioning

confidence: 99%

Molecular cloning and expression of a vacuolar Na+/H+ antiporter gene (AgNHX1) in fig (Ficus carica L.) under salt stress

Metwali

Soliman

Fuller

et al. 2015

Plant Cell Tiss Organ Cult

View full text Add to dashboard Cite

show abstract

“…Then, for RE analysis [28,29], a 2-3 white/light blue colonies were inoculated into 5mL selective LB broth and shaken at 150 rpm, 37 o C overnight. The recombinant vector was isolated from the overnight cultured using Invitrogen Pure Link TM Quick Plasmid Miniprep kit as described by the manufacturer.…”

Section: Screening Of Positive Transformantsmentioning

confidence: 99%

“…Consequently, transformed cells containing the recombinant vector without the functional β-galactosidase may be formed as white or light blue on the plate. The compound is cleaved by β-galactosidase to form 5-bromo-4-chloro-indoxyl, that spontaneously oxidizes and dimerizes to form a bright blue insoluble pigment 5,5'-dibromo-4,4'-dichloro-indigo [29,60,61]. The blue colonies characteristic, hence, showed that the cells contain an uninterrupted lacZ gene or fully functional β-galactosidase enzyme, whereas the white/light blue colonies (X-gal is not hydrolyzed) indicated the presence recombinant vector in the bacterial cells [62].…”

Section: Construction Of Rft1 Gene and Transformation Analysismentioning

confidence: 99%

Quality RNA Isolation and Efficient Construction of Rft1 (Peasy:Rft1) Gene

Mohammed,

Bello Mahmoud,

Sa’idu

et al. 2021

J Biotechnol Bioeng

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Introduction-High-quality RNA isolation from a polysaccharide-rich plant such as rice for downstream application is challenging. Equally, successful gene cloning for molecular analysis is tricky as it produces negative transformants.Objective-Here we reported on qualitative and quantitative RNA, RT-PCR amplification of RFT1 gene from matured leaves of Malaysian upland rice, then construction of the gene. Methodology-The RNA was isolated from Wai cultivar leaves (indica sub-species) using three different methods, synthesized cDNA and RT-PCR amplification of RFT1 gene. A T-A cloning of the RFT1 gene into p-EASY T3 vector (pEASY:RFT1), chemical transformation into E. coliTrans1-T1 and screening of the transformed cells on selective Laix-plate, by colony PCR and RE analysis were observed.Results-The RNA integrity indicated that using guanidine isothiocyanate based method, precisely Trizol was the superlative (yield = 36.50±0.59 and purity = 2.04±0.09) for isolating quality RNA from upland rice leaves. The RT-PCR and bioinformatics analysis resulted in full-length RFT1 gene amplification; responsible for promoting flowering under LD condition. Both recombinant DNA construction and transformation were efficient with more than 50% white colonies due to deactivation of the lacZ reporter gene. Also, the gel analysis of the colony-PCR indicated an absolute RFT1 gene amplification with a singleband and double-bands (vector and insert) after RE analysis all at expected positions. Conclusion-These have shown the suitability of the RNA isolation protocols observed in producing qualityRNA for downstream applications, first ever reported on full-length RFT1 gene isolation from Malaysian upland rice and successful gene construction (pEASY:RFT1).

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Cloning of Three Antiporter Genes from Arabidopsis and Rice for Over-Expressing Them in Farmer Popular Tomato Varieties of Bangladesh

Cited by 2 publications

References 23 publications

Molecular cloning and expression of a vacuolar Na+/H+ antiporter gene (AgNHX1) in fig (Ficus carica L.) under salt stress

Molecular cloning and expression of a vacuolar Na+/H+ antiporter gene (AgNHX1) in fig (Ficus carica L.) under salt stress

Quality RNA Isolation and Efficient Construction of Rft1 (Peasy:Rft1) Gene

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