Molecular Biotechnology

, Volume 48, Issue 1, pp 49–59

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

  • L. Swapna
  • R. Khurana
  • S. Vijaya Kumar
  • A. K. Tyagi
  • K. V. Rao
Research

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.

Keywords

Arabidopsis Cis-regulatory element GUS Pollen-specific promoter Rice Transgenics 

Supplementary material

12033_2010_9347_MOESM1_ESM.ppt (696 kb)
Supplementary material 1 (PPT 695 kb)
12033_2010_9347_MOESM2_ESM.tif (386 kb)
Fig.1SA 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)
12033_2010_9347_MOESM3_ESM.tif (59 kb)
Fig. 2SConfirmation 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)
12033_2010_9347_MOESM4_ESM.tif (105 kb)
Fig. 3SConfirmation 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)
12033_2010_9347_MOESM5_ESM.tif (107 kb)
Fig. 4SQuantitative 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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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • L. Swapna
    • 1
  • R. Khurana
    • 2
  • S. Vijaya Kumar
    • 1
  • A. K. Tyagi
    • 2
    • 3
  • K. V. Rao
    • 1
  1. 1.Centre for Plant Molecular BiologyOsmania UniversityHyderabadIndia
  2. 2.Interdisciplinary Centre For Plant Genomics and Department of Plant Molecular BiologyUniversity of Delhi South CampusNew DelhiIndia
  3. 3.National Institute of Plant Genome ResearchNew DelhiIndia

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