Optimisation of expression of a rice (Oryza sativa L.cv Nipponbare) plant natriuretic peptide (OsPNP-B) and its functional characterisation
- Authors: Affun, Ogheneochuko Janet
- Date: 2012
- Subjects: Rice , Plant hormones , Water-electrolyte imbalances
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/24354 , vital:62644
- Description: Maintenance of water and solute homeostasis is a key requirement for living systems, and in vertebrates, homeostasis is in part achieved by natriuretic peptides (NP), a family of peptide hormones. A related family of peptide hormones have also been found in the plant kingdom. Plant natriuretic peptides (PNP) are a novel class of plant proteins with two closely related homologous genes (AtPNP-A and AtPNP-B) identified in the model plant Arabidopsis thaliana. AtPNP-A has been extensively studied and evidence obtained points to a role in plant water homeostasis. No research has been conducted on the function of PNP-B proteins. In this study, we focus on the bioinformatic analysis of the PNP-B gene in various plants, as well investigating whether PNP-B plays a role in water homeostasis in rice plants exposed to drought stress. Basic local alignment search tool (BLAST) queries of the ―The Gene Index‖, EST and available plant genome databases revealed the presence of the PNP-B mRNA in rice, oil seed rape, oak, leafy spurge, poplar, sugarcane, pepper, cotton, apple and maize. All the identified genome sequences contained a predicted intron/insert, which was not present in the related mRNA sequences. However, RT-PCR analysis revealed the presence of the 101 bp insert in the 976 bp amplified rice PNP-B (OsPNP-B) cDNA and therefore expression was optimized for the protein encoded by only the exon2 sequence as this contained the predicted active site region of PNP-B. OsPNP-B was shown to be translated to a protein of 14 kDa with a sequence similarity to AtPNP-B (54percent), AtPNP-A (37percent) and CjBAp12 (55percent), suggesting two possible functions for PNP-B viz water homeostasis and/or pathogenesis defence. To determine whether PNP-B is involved in water homeostasis, total protein extracted from 4 weeks old (4 leaves stage) rice plants subjected to drought treatment for a period of 24, 48, 72, 120, 168 and 240hrs were resolved by 17percent SDS-PAGE and analysed by western blot analysis. The PNP-B protein was found to be down-regulated during drought stress, implying that PNP-B may play a role in water homeostasis through the release of water from cells rather than the up-take of water as seen At-PNP-A. PNP-B could therefore also be involved in plant defence mechanisms to pathogens where plants induce desiccation of infected leaves, thereby ridding the plant of the relevant pathogen. , Thesis (MSc) -- Faculty of Science and Agriculture, 2012
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- Date Issued: 2012
Evolutionary development and functional role of plant natriuretic peptide (PNP)-B
- Authors: Hove, Runyararo Memory
- Date: 2009
- Subjects: Plant hormones , Peptides , Plant gene expression , Peptide hormones , Peptides -- Separation
- Language: English
- Type: Thesis , Masters , MSc (Biochemistry)
- Identifier: vital:11251 , http://hdl.handle.net/10353/155 , Plant hormones , Peptides , Plant gene expression , Peptide hormones , Peptides -- Separation
- Description: Plant natriuretic peptides (PNP) are novel peptides which, like in vertebrates, have been shown to have a function associated with water and salt homeostasis. Two PNP-encoding genes have been identified and isolated from Arabidopsis thaliana, namely; AtPNP-A and AtPNP-B. In this study, the focus was on PNP-B, which has not been extensively studied. Bioinformatic analysis was done on the AtPNP-B gene. This included the bioinformatic study of its primary structure, secondary structure, tertiary structure, transcription factor binding sites (TFBS) and its relation to other known proteins. The AtPNP-B gene was shown to be a 510 bp long, including a predicted 138 bp intron. AtPNP-B was also shown to have some sequence similarity with AtPNP-A and CjBAp12. The TFBS for AtPNP-B and OsJPNP-B were compared and they comprised of TFBS that are related to water homeostasis and pathogenesis. This suggested two possible functions; water stress and homeostasis and a pathogenesis related function for PNP-B. Following bioinformatic analysis, the heterologous expression of the AtPNP-B was attempted to investigate whether the AtPNP-B gene encoded a functional protein and to determine the functional role of PNP-B. However, expression was unsuccessful. An evolutionary study was then carried out which revealed that there were some plants without the intron such as, rice, leafy spurge, oilseed rape, onion, poplar, sugar cane, sunflower and tobacco. These plants would therefore be used for expression and functional studies in the future. The evolutionary studies also revealed that PNP-B had a relationship with expansins and the endoglucanase family 45. Other PNP-B related molecules were also obtained from other plant genomes and therefore used in the construction of a phylogenetic tree. The phylogenetic tree revealed that AtPNP-B clustered in the same group as CjBAp12 while AtPNP-A had its own cluster group. There were also other PNP-B like molecules that clustered in the same group as expansins (α- and β-). Thus, we postulate that, like PNP-A, PNP-B also has a possible function in water and salt homeostasis. However, due to the clustering iii of AtPNP-B into the same group as CjBAp12, a possible role of PNP-B in pathogenesis-related response is also postulated.
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- Date Issued: 2009