Full text
| |
| Author(s): |
João Henrique Servilha
Total Authors: 1
|
| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Biociências (IBIOC/SB) |
| Defense date: | 2024-04-22 |
| Examining board members: |
Cristiane Paula Gomes Calixto;
Felipe Klein Ricachenevsky;
Silvana Aparecida Creste Dias de Souza
|
| Advisor: | Cristiane Paula Gomes Calixto |
| Abstract | |
Rice is one of the most consumed foods in the world, but heat stress affects its yield and grain quality. To identify mechanistic solutions to improve this crop under climate change threats, the molecular responses of thermotolerance must be identified. Alternative pre-mRNA processing is one of the mechanisms involved in plant response against stresses, but little is known about how the variation of transcribed isoforms is regulated by temperature changes. The rice genes LOC_Os02g40900 and LOC_Os05g30140 are candidates for heat-sensitive splicing regulators. To investigate this hypothesis, it was generated plasmids based on the CRISPR/Cas9 methodology to knock out these genes in rice plants. Using the dual-targeting strategy, it was selected two target sequences in the coding region of each gene. These target sequences were inserted in plant expression vectors via PCR, sub-cloning, and Golden Gate Assembly. The correct plasmids were confirmed by Sanger sequencing and they were then inserted into Agrobacterium and confirmed by colony PCR. For plant transformation, rice calli were obtained by inducing undifferentiated embryo division in rice seeds. After calli induction and subculture, occurred the transformation via Agrobacterium, enabling that the calli resistant to the selective agent proceeded to the regeneration medium, generating transgenic plants, confirmed via PCR and Sanger sequencing for at least one of the expected editions. (AU) | |
| FAPESP's process: | 21/12014-2 - Establishment of the CRISPR-Cas9 methodology and obtaining knockout plants in candidate genes for regulators of alternative RNA processing in heat stress in rice |
| Grantee: | João Henrique Servilha |
| Support Opportunities: | Scholarships in Brazil - Master |