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Cloning of the Plasma Membrane Sodium/hydrogen Antiporter Sos1 for Its Over Expression in Rice

Samsad Razzaque
Plant Biotechnology Lab., Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka

Sabrina M. Elias
Plant Biotechnology Lab., Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka

Sudip Biswas
Plant Biotechnology Lab., Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka

Taslima Haque
Plant Biotechnology Lab., Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka

Zeba I. Seraj
Plant Biotechnology Lab., Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka

Abstract/ Executive Summary:

Soil salinity adversely affects plant growth, development and disturbs intracellular ion homeostasis which results in cellular toxicity. The salt overly sensitive 1 (SOS1) gene is a critical component of salt tolerance in many species and encodes a plasma membrane Na+/H+ antiporter that plays an important role in germination and growth in saline environments. In the current study, the coding sequence of SOS1 gene (3447 bp) was amplified from a Bangladeshi rice landrace, Pokkali, by applying the fusion PCR strategy. SOS1 PCR amplicons were firstly cloned into pENTR/D‐TOPO and then recombined with the binary vector, pH7WG2, through LR reaction. Positive colonies were validated by PCR, restriction digestion and sequencing. Finally, the constructed vector was transformed into Agrobacterium tumeficiens, LBA4404 strain, to initiate Agrobacterium‐mediated transformation with the provision to transfer the cloned SOS1 into farmer popular rice varieties.

Keywords: Antiporter, SOS1, Cloning, Plasma membrane, Overexpression

Location:

Start Date:

End Date:

Program Area: Knowledge Management

Commodity: Rice

Objectives:

In the current study, SOS1 has been cloned from a salt tolerant rice landrace Pokkali, with the aim to transform it into farmer popular BRRI varieties for enhancing the salinity tolerance of the latter

Methodology:

Rice landrace Pokkali was grown for 15 days and 150 mM salt stress was applied to extract total RNA using TRIZOL (Invitrogen) method. The extracted RNA was quantified using Nanodrop® spectrophotometer ND‐1000 (Thermo Fisher Scientific inc.), and the cDNA was synthesized from isolated RNA by using SuperScript™ first‐strand synthesis system (Invitrogen). The amplification of SOS1 coding region was accomplished applying fusion PCR/overlap PCR strategy. Primers were designed accordingly to attain the task. The first fragment was amplified by PCR with the SOS1 forward primer (SOS1_F) and SOS1 overlap reverse primer (SOS1_OL_R) . PCR reaction program for amplifying the first fragment was optimized as follows. Initial denaturation at 95°C for 5 min and 35 cycles of denaturation at 95°C for 1 min, annealing at 61.5°C for 1 min, extension at 72°C for 2.10 min followed by a final extension at 72°C for 10 min. A final concentration of 2.3 mM MgCl₂, 0.1 mM dNTPs, 0.3 μM of each primer and 1 unit of recombinant Taq polymerase (Invitrogen, Carlsbad, CA, USA) was used. The forward primer was designed adding CACC overhang to ensure compatibility with pENTR/D‐TOPO vector Successful pENTR/D‐TOPO cloning allowed recombining the desired sequence of SOS1 into a destination vector by using the Gateway® LR recombination reaction (invitrogen). The LR reaction was carried out following the manufacturer’s protocol. Positive colonies were screened out by gene specific primers and restriction digestions. Restriction endonuclease NdeI (NEBr inc.) was used and confirmation was carried out by comparing the transformed plasmid with non‐transformed plasmid. Finally, Agrobacterium tumefaciens (LBA4404) was electroporated with the constructed pH7WG2_ SOS1 using standard protocols (Sambrook et al. 1989). Positive colonies were authenticated by PCR reactions with gene specific primers

Source of Information: Plant Tissue Cult. & Biotech. 23(2): 263-273, 2013 (December)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The challenge in cloning the SOS1 CDS was its sequence length. Amplification of the full 3.4 kb with high fidelity Taq polymerases specific for larger fragments was not considered since it amplifies the insert with a mixture of both blunt and A ends (Platinum® Taq DNA polymerase high fidelity protocol, Invitrogen). pENTR/D‐TOPO cloning kit only allows insertion of the Blunt end products, but high fidelity Taq polymerase adds a single deoxyadenosine (A) to the 3ʹ ends of PCR products which make the amplified product incompatible to clone into pENTR/D‐TOPO vector. Hence the overlapping fusion PCR strategy needed to be adopted which could successfully accomplish the task. Moreover the gene length was larger than the insert size recommended for cloning in pENTR‐D‐TOPO vector (Invitrogen). Modification in the incubation time and volume of the reaction during cloning could successfully overcome the insert size restriction; though the efficiency rate was drastically reduced as observed by the low number of positive colonies. Successful cloning was accomplished by initiating some major changes in the manufacturer’s protocol. The incubation period was extended to an overnight period and reaction volume was maintained to 10 instead of 6 microlitre. Combination of the strategies adapted in the experiment can be beneficial in other studies where large sequence length of the gene is a limiting factor in cloning.

Knowledge Management: Journal