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Pollen-Specific Expression of Oryza sativa Indica Pollen Allergen Gene (OSIPA) Promoter in Rice and Arabidopsis Transgenic Systems

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Abstract

Earlier, a pollen-specific Oryza sativa indica pollen allergen gene (OSIPA), coding for expansins/pollen allergens, was isolated from rice, and its promoter—upon expression in tobacco and Arabidopsis—was found active during the late stages of pollen development. In this investigation, to analyze the effects of different putative regulatory motifs of OSIPA promoter, a series of 5′ deletions were fused to β-glucuronidase gene (GUS) which were stably introduced into rice and Arabidopsis. Histochemical GUS analysis of the transgenic plants revealed that a 1631 bp promoter fragment mediates maximum GUS expression at different stages of anther/pollen development. Promoter deletions to −1272, −966, −617, and −199 bp did not change the expression profile of the pollen specificity. However, the activity of promoter was reduced as the length of promoter decreased. The region between −1567 and −199 bp was found adequate to confer pollen-specific expression in both rice and Arabidopsis systems. An approximate 4-fold increase in the GUS activity was observed in the pollen of rice when compared to that of Arabidopsis. As such, the OSIPA promoter seems promising for generation of stable male-sterile lines required for the production of hybrids in rice and other crop plants.

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Acknowledgments

This work was supported by a grant from Department of Biotechnology, Government of India. RK and SVK are recipients of JRF and SRF given by Council of Scientific & Industrial Research. We would like to thank Drs. Sanjay Kapoor and Vikrant Gupta for their critical inputs.

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  1. A. K. Tyagi

    Present address: National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, 110067, India

Authors and Affiliations

  1. Centre for Plant Molecular Biology, Osmania University, Hyderabad, 500007, Andhra Pradesh, India

    L. Swapna, S. Vijaya Kumar & K. V. Rao

  2. Interdisciplinary Centre For Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, 110021, India

    R. Khurana & A. K. Tyagi

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Fig.1S

A OSIPA transformants of rice showing tolerance to herbicide-Basta; (UC: Control without Basta, C: Control with Basta treatment, 1-12: Transformants with various constructs of OSIPA gene promoter). B OSIPA transformants of Arabidopsis transformants showing kanamycin resistance (PAΔ1) (TIFF 386 kb)

Fig. 2S

Confirmation of the transgenic nature of rice plants carrying OSIPA promoter constructs by using (A) bar forward and nos reverse primers used to amplify 750 bp fragment of bar coding region or (B) OSIPA forward and GUS reverse primers used to amplify 600 bp fragment of OSIPA region. M: marker, P: plasmid, C: untransformed control, 1-15: different transformants (TIFF 59 kb)

Fig. 3S

Confirmation of the transgenic nature of Arabidopsis thaliana plants carrying PAΔ1 A, PAΔ2 B, PAΔ3 C, PAΔ4 D and PAΔ5 E constructs. GUS gene-specific primers was used to amplify ~2 kb fragment. M: Mass ruler, +: positive control, -: negative control (TIFF 105 kb)

Fig. 4S

Quantitative measurement of GUS activity in different tissues of transgenic Arabidopsis thaliana plants harboring different PAΔ1 A, PAΔ2 B, PAΔ3 C, PAΔ4 D and PAΔ5 E constructs. GUS activity was determined using protein extracts from roots, stem, leaves, sepal, petal, anther, gynoecium and silique of transgenic plants as well as wild-type plants. Each column represents the mean GUS activity from 3 plants of 5 independent transgenic lines. The value was obtained after subtracting any background specific GUS activity observed in root, stem, leaf, sepal, petal, anther gynoecium and silique of wild type from the activity observed in respective organs of transgenic Arabidopsis. Standard error bars are shown (TIFF 106 kb)

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Swapna, L., Khurana, R., Vijaya Kumar, S. et al. Pollen-Specific Expression of Oryza sativa Indica Pollen Allergen Gene (OSIPA) Promoter in Rice and Arabidopsis Transgenic Systems. Mol Biotechnol 48, 49–59 (2011). https://doi.org/10.1007/s12033-010-9347-5

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