Functional analysis of the SlGRAS10 gene in tomato fruit ripening
| Autor(a) principal: | |
|---|---|
| Data de Publicação: | 2021 |
| Tipo de documento: | Dissertação |
| Idioma: | eng |
| Título da fonte: | Repositórios Científicos de Acesso Aberto de Portugal (RCAAP) |
| Texto Completo: | http://hdl.handle.net/10451/53706 |
Resumo: | Tese de mestrado, Biologia dos Recursos Vegetais, Universidade de Lisboa, Faculdade de Ciências, 2022 |
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Functional analysis of the SlGRAS10 gene in tomato fruit ripeningedição génica por CRISPR-cas9fatores de transcriçãocarotenoides totaisexpressão génicaTeses de mestrado - 2022Departamento de Biologia VegetalTese de mestrado, Biologia dos Recursos Vegetais, Universidade de Lisboa, Faculdade de Ciências, 2022Fleshy fruits, like Solanum lycopersicum (tomato), comprise an indispensable commercial and nutritional food commodity, being an essential crop for agriculture and being part of the diet of a significant portion of the population. Tomato is a climacteric fruit; one feature of climacteric fruits is that these fruits continue their process of ripening after harvest making it much more challenging to store and distribute than other fruits due to easier fungal contaminations. This process of ripening is very complex and involves several mechanisms. The trigger to this reprogramming of fruit development and ripening comprises several transcription factors, among them the GRAS transcription factors have been suggested to play a role. In a previous work, the SlGRAS9 and SlGRAS10 have exhibited transcriptional up-regulation in post-breaker stage fruits (Grimplet et al., 2016a), suggesting these genes may be involved in tomato ripening. There are a few tomato ripening mutants that have been identified, and characterization of these mutants suggests that they may have defects in ripening regulatory systems. These mutations are the ripening-inhibitor (rin), nonripening (nor), and Colorless nonripening (Cnr) mutations. All three genes encode transcription factors, and the associated phenotypes suggest that the rin, nor and Cnr mutants are impacted in the central ripening phenomena. It is hypothesized that such genes might represent conserved regulators affecting ripening even in non-climacteric fruits, since homologs of the RIN-MADS gene have been found in climacteric species such as tomato but also in non-climacteric species, such as strawberry. The GRAS gene family is a plant-specific family of transcription factors with fundamental roles in the regulation of plant organ development and some GRAS genes have been identified as direct targets of RIN. By comparing the information available for tomato and grapevine GRAS genes, (Grimplet et al., 2016) identified candidate genes that might constitute conserved transcriptional regulators of both climacteric and non-climacteric fruit ripening, and that deserve further functional analysis, namely SlGRAS10 and its orthologue in Vitis vinifera PAT6. In our study, the SlGRAS10 mutant, developed by CRISPR-cas9 technology was stabilized from T0 to T1 with high mutation rates that were analyzed through TIDE analysis. The fruits from the T1 plants were collected in specific time-points and through Quantitative Real Time PCR (qPCR) the expression of a few genes involved in fruit ripening were analyzed. Most differences were found at fruits collected 10 days post Breaker (Br+10), where the relative expression of the RIN, PYL9, ACO1 and PSY was significantly lower than the Wild-type in specific knock-out lines. The analysis of total carotenoids content in the fruits pericarp also showed significant lower content of total carotenoids in the SlGRAS10 mutants, specifically at the Br+10 stage again, the fruits appear to have a concentration similar to the Wild-type at the Br+5 and Br+7 stages of ripening, which is a significant delay of 3 to almost 5 days. Interestingly, the 1A2, 2A2, 2A3, 1B1, 1B2 and 2B5 SlGRAS10 lines had an unanticipated phenotype of low seeds, that was related to smaller fruits in some lines. Due to this phenotype a germination assay was performed with pollen from selected lines. The most significant reduction in seed number (1A2 and 1B2) and most lines descendent from de 1A2 line had significantly lower pollen germination rates. In previous studies the SlGRAS9 and SlGRAS10 genes showed to have conserved expression patterns, which implied their retention of genetic redundancy. Therefore, it is suggested that SlGRAS9 and SlGRAS10 can compensate each other in a redundant functional way. Therefore, posteriorly to the SlGRAS10 mutants were established, other mutant plants were developed with a knock-out both in the SlGRAS10 and SlGRAS9 genes. These T0 lines were genotyped by Sanger Sequencing and later TIDE analysis to determine the type and mutation rate on those lines, the mutations rates for the SlGRAS10 were like the first single mutants developed and the mutation rates for the SlGRAS9 were slightly lower, but mutation rate is expected to grow on the T1 generation, since cas9 is still present. Altogether the data indicated that SlGRAS10 and SlGRAS9 play an important role in tomato fertilization and ripening. These mutants could be applied to produce seedless tomatoes with longer shelf-life as part of a breeding program to develop a new variety commercially valuable.Fortes, Ana Margarida da Costa Macedo, 1973-Repositório da Universidade de LisboaNeves, Catarina Leon Duarte2023-03-20T01:31:27Z202220212022-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10451/53706TID:202994392enginfo:eu-repo/semantics/openAccessreponame:Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)instname:FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologiainstacron:RCAAP2025-03-17T14:47:30Zoai:repositorio.ulisboa.pt:10451/53706Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireinfo@rcaap.ptopendoar:https://opendoar.ac.uk/repository/71602025-05-29T03:24:58.991678Repositórios Científicos de Acesso Aberto de Portugal (RCAAP) - FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologiafalse |
| dc.title.none.fl_str_mv |
Functional analysis of the SlGRAS10 gene in tomato fruit ripening |
| title |
Functional analysis of the SlGRAS10 gene in tomato fruit ripening |
| spellingShingle |
Functional analysis of the SlGRAS10 gene in tomato fruit ripening Neves, Catarina Leon Duarte edição génica por CRISPR-cas9 fatores de transcrição carotenoides totais expressão génica Teses de mestrado - 2022 Departamento de Biologia Vegetal |
| title_short |
Functional analysis of the SlGRAS10 gene in tomato fruit ripening |
| title_full |
Functional analysis of the SlGRAS10 gene in tomato fruit ripening |
| title_fullStr |
Functional analysis of the SlGRAS10 gene in tomato fruit ripening |
| title_full_unstemmed |
Functional analysis of the SlGRAS10 gene in tomato fruit ripening |
| title_sort |
Functional analysis of the SlGRAS10 gene in tomato fruit ripening |
| author |
Neves, Catarina Leon Duarte |
| author_facet |
Neves, Catarina Leon Duarte |
| author_role |
author |
| dc.contributor.none.fl_str_mv |
Fortes, Ana Margarida da Costa Macedo, 1973- Repositório da Universidade de Lisboa |
| dc.contributor.author.fl_str_mv |
Neves, Catarina Leon Duarte |
| dc.subject.por.fl_str_mv |
edição génica por CRISPR-cas9 fatores de transcrição carotenoides totais expressão génica Teses de mestrado - 2022 Departamento de Biologia Vegetal |
| topic |
edição génica por CRISPR-cas9 fatores de transcrição carotenoides totais expressão génica Teses de mestrado - 2022 Departamento de Biologia Vegetal |
| description |
Tese de mestrado, Biologia dos Recursos Vegetais, Universidade de Lisboa, Faculdade de Ciências, 2022 |
| publishDate |
2021 |
| dc.date.none.fl_str_mv |
2021 2022 2022-01-01T00:00:00Z 2023-03-20T01:31:27Z |
| dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/masterThesis |
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masterThesis |
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publishedVersion |
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http://hdl.handle.net/10451/53706 TID:202994392 |
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http://hdl.handle.net/10451/53706 |
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TID:202994392 |
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eng |
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eng |
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info:eu-repo/semantics/openAccess |
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openAccess |
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application/pdf |
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