Investigation of the expression of Glb-1 and GluA2 promoters in indica and japonica rice in different tissues via GUS staining

Author: Antonio Pupulin

Pupulin, Antonio, 2023 Investigation of the expression of Glb-1 and GluA2 promoters in indica and japonica rice in different tissues via GUS staining, Flinders University, College of Medicine and Public Health

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This study investigated the gene expression patterns of the promoters governing two seed storage proteins in rice, the globulin 1 (Glb-1) and glutelin A2 (GluA2) promoters. Previous research in which these japonica rice-derived promoters controlled the expression of a barley sucrose transporter (HvSUT1) and a rice nicotianamine synthase (OsNAS2) in transgenic indica rice varieties has cast doubt on the long-held notion that their expression is confined to the endosperm. These transgenic plants showed increased or decreased vigour, depending on the transgene combination, compared to their wild-type counterparts, suggesting the leaky activity of these promoters. This research investigated this hypothesis by using the GUS reporter gene fused to the promoter elements. Previous research had generated transgenic lines with the japonica-derived actin and globulin-1 promoters (jActp and jGlb-1p) driving GUS expression in japonica rice, and the jGlu2A promoter (jGluA2p) driving GUS expression in indica rice. To complete this study with appropriate controls, novel transgenic lines with the indica Glb-1 promoter (iGlb-1p) fused to the GUS gene (iGlb-1p::GUS), and the jGlu2Ap::GUS fusion construct were transformed into japonica rice via callus infection with Agrobacterium. The characterisation of the transgenic plants was performed by end-point PCR followed by gel electrophoresis to confirm the inheritance or integration of the GUS-containing constructs. GUS staining was used to visualise any promoter-driven activity in seed, leaf, crown, and root tissue to shed light on the role of these two promoters in the physiology of those transgenic lines expressing the sucrose transporter and nicotianamine synthase genes. However, preliminary results showed no evidence of ectopic expression of Glb-1p or GluA2p under the conditions of this experiment. Further research on these transgenic lines is warranted, but it is hoped that by exploring the expression patterns of these two promoters this study could encourage further investigation of their role and function to inform potential applications in plant biotechnology.

Keywords: rice, promoters, GUS, GUS staining, Glb-1, globulin-1, globulin, glutelin, GluA2, glutelin A2, japonica, indica, Oryza sativa, ectopic, ectopic expression, SSP, seed storage protein, storage protein,

Subject: Medical Biotechnology thesis

Thesis type: Masters
Completed: 2023
School: College of Medicine and Public Health
Supervisor: Peter Anderson