Expression analysis in self-compatible Petunia hybrida and promoter analysis of S-locus F-box genes/SLFs in Petunia inflata

Liu, Chen (2021) Expression analysis in self-compatible Petunia hybrida and promoter analysis of S-locus F-box genes/SLFs in Petunia inflata. PhD thesis, University of Nottingham.

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Abstract

Self-incompatibility (SI), a type of intraspecific reproductive barrier occurs widely in flowering plants and helps to avoid inbreeding and protects the genetic diversity of a population. When the pollen lands on a self-pistil, SI will prevent fertilization; when the pollen lands on the pistil of an unrelated plant, fertilization will not be affected. SI consists of two types: Sporophytic SI (SSI) and gametophytic SI (GSI). In GSI it is the haploid genotype of the pollen itself that controls the pollen phenotype. Transgenic studies in Petunia have shown that the pistil phenotype is determined by a stylar ribonuclease (S-RNase). In the fertilization process, Slinked F-box genes/SLFs that are expressed in pollen work together with S-RNases to determine the outcome of a pollination. Recently up to 20 SLF genes have been reported at the S-locus in Petunia, but the exact role of individual SLFs in controlling the phenotype is still not clear. Part of this project aims to investigate SLF expression between two closely linked varieties of Petunia hybrida, which have different phenotypes. A combination of RT-PCR and qRT-PCR using specific primers were used to determine the expression patterns of individual SLF genes in these two varieties. The results of RT-PCR and qRT-PCR indicate that all SLFs express in these two varieties, and most SLFs share significant differences in the expression levels comparison between these two closely linked varieties of Petunia hybrida. The partial DNA sequences of SLFs in these two varieties are proved that is identical. Therefore, the different expression level of SLFs might is the cause of the different SI between these two closely linked varieties of Petunia hybrida. The finding of this part of this project might be able to provide some evidence in the evolution theory of SI plants.

On the other hand, there are only two SLFs expression analysis papers been published to date from Kao et al. (2004) and Hua et al. (2007), which indicated SLFs express in pollen tube, pollen and anther of Petunia by RNA gel blot analysis and RT-PCR, but there is no paper to show the expression pattern of SLFs using GUS or GFP analysis. This project also aims to explore the promoter analysis of SLF genes that have recently been identified as part of a genome sequencing effort of Petunia inflata. The genomic sequence data were used to characterize the promoter regions to identify shared pollen promoter elements such as those identified for the gene LAT52 (a tomato pollen gene). Promoter fragments of three selected SLFs were cloned and used to prepare SLF promoter:GUS fusion constructs. Agrobacterium-based transformation was performed to transfer these constructs into Petunia hybrida. The GUS assay was used to test the SLF expression pattern in Petunia. The result of GUS analysis indicates SLF3 is expressed in pollen of stamens, which is consistent with the findings from Kao et al. (2004) and Hua et al. (2007). This part also provides a good attempt in other transformation experiments to use the SLF promoter of Petunia inflata. In addition, the results of both these two parts of this project support each other: the expression of SLFs in Petunia shows an exponential growth during the maturation of pollen.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Robbins, Tim
Wilson, Zoe
Keywords: Petunia, Self-incompatibility, SLFs, qRT-PCR, GUS
Subjects: Q Science > QK Botany > QK900 Plant ecology
S Agriculture > SB Plant culture
Faculties/Schools: UK Campuses > Faculty of Science > School of Biosciences
Item ID: 66045
Depositing User: LIU, Chen
Date Deposited: 08 Dec 2021 04:40
Last Modified: 08 Dec 2021 04:40
URI: http://eprints.nottingham.ac.uk/id/eprint/66045

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