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Modification of Recombination-based Gatewaytm BINAry Destination Vector with Novel Promoter for Agrobacterium-mediated Transformation of Rice

Md. Rakibul Islam
Rice Research and Biotechnology Laboratory, Department of Biochemistry and Molecular Biology, University of Dhaka

Richard Malo
Rice Research and Biotechnology Laboratory, Department of Biochemistry and Molecular Biology, University of Dhaka

Rumana S. Tammi
Rice Research and Biotechnology Laboratory, Department of Biochemistry and Molecular Biology, University of Dhaka

Sharmin Jahan
Rice Research and Biotechnology Laboratory, Department of Biochemistry and Molecular Biology, University of Dhaka

Lisa Parvin
Rice Research and Biotechnology Laboratory, Department of Biochemistry and Molecular Biology, University of Dhaka

Zeba I. Seraj
Rice Research and Biotechnology Laboratory, Department of Biochemistry and Molecular Biology, University of Dhaka

Abstract/ Executive Summary:

The GATEWAY^TM Binary Destination Vector pH7WG2 is available for easy insertion of genes for transformation into plants. The gene of interest integrates downstream of the Cauliflower Mosaic Virus Promoter CaMV 35S by recombination. The CaMV 35S promoter is however not suitable for transformation and expression of genes in monocots like rice. We isolated and cloned a ~1100 bp upstream region from two rice (Pokkali and IR64) Na/H antiporter genes into the GATEWAY^TM promoter cloning vector pHGWFS7. The Pokkali promoter expressed the β-glucuronidase or GUS gene ~25-fold more efficiently than the CaMV 35S promoter in rice calli, while that of IR64 was 7-fold more. The IR64 promoter however showed efficient expression in transgenic rice leaves. The promoter from Pokkali Na/H antiporter was used to replace the CaMV 35S sequence in pH7WG2. The CaMV 35S region was cut out and the linear vector fragment blunted and T-tailed. After amplification of the promoter from Pokkali rice DNA, it was A-tailed and ligated to the modified T-vector. The resultant vector, named pH7WG3, following the nomenclature at the gateway site, www.plantgenetics.rug.ac.be/gatewayT, can now be used for recombination of any genes for efficient rice transformation.

Keywords: Recombination, Binary vector, Promoter, Transformation, Rice

Location:

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Rice

Objectives:

This work was undertaken to identify a promoter showing efficient expression in rice and replace the CaMV 35S promoter in the Gateway destination expression vector, pH7GW2, with the rice promoter.

Methodology:

A versatile set of vectors are available for plant gene transformation and analysis (Karimi et al. 2002). For LR recombination reactions, Entry vectors contain the site specific sequence attL1-attL4, while the destination vectors contain attR1-attR4. Genes or promoters of interest are cloned into the Entry vector by ligase-free topoisomerase reactions, (Invitrogen Topo-cloning). Thereafter, recombina-tion between the two vectors using LR recombinase allows recombination of thegene of interest or promoter into the appropriate location on the destination vector. Destination vectors: Two types of commercially available destination vectors were used for transformation in this work.(a) pHGWFS7 for promoter-gus constructs. This vector with binary pPZP200 backbone (Hajdukiewicz et al. 1994) contains CmR-ccdB selection flanked by attR1 and attR2 recombination sites and followed by gfp-gus sequence with t35s termination sequence. The vector can be grown in the presence of chloramphenicol (Cm). To ensure that only recombinants grow, the cytotoxic ccdB gene gets replaced after recombination as well as the chloramphenicol resistance gene (CmR). Recombinants can also be selected by growth on streptomycin and/or spectinomycin. The recombination sites allow transfer of a promoter that drives the gfp-gus gene hence allowing its characterization and testing(b) pH7WG2 for gene transfer. This vector has the same pPZP200 backbone but contains CaMV35S promoter followed by CmR-ccdB selectionflanked by attR1 and attR2 recombination sites. The recombinations sites allow transfer the gene of interest downstream of the CaMV 35S promoter

Source of Information: Plant Tissue Cult. & Biotech. 17(1): 47-58, 2007 (June)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The TA ligation is not directional and so the insert may ligate in a flipped orientation which will result no activity of the promoter. To pick the ligation product with the correct orientation, a new forward primer was designed from the vector sequence, where the reverse primer was the same as for the amplicon. Only the clones with correct orientation gave PCR amplification . The new vector was named pH7WG3 and we will be testing its efficacy for gene expression by recombining GOIs downstream of the promoter PkN. This vector should prove to be useful for expression of GOIs in monocots like rice. Substituting CaMV 35S by PkN was difficult by restriction digestion-ligation method because the restriction enzyme used for cutting out of the CaMV 35S sequence digested PkN. Therefore we decided to blunt the restriction enzyme cut ends. However blunt end ligation is not efficient, therefore the vector was T-tailed and the insert was A-tailed. The process described in this work is therefore useful in transferring a DNA sequence into any site of interest.

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