Caracterização do gene bZIP em tomateiro: deleção de uma uORF conservada e análise in silico de correlações do gene SlbZIP1

Abstract

The cultivated tomato, Solanum lycopersicum, belongs to the family Solanaceae that has many species of economic relevance. Due to the characterization the tomato, besides being a commercial product, has also been used as a model plant for the development of research to investigate plant physiological and metabolic processes. Because of the high consumption and to meet the demand for productivity, resistance and other characteristics, analyses of conditions that can be used for tomato breeding have been requested. Some sugars, like sucrose, that confer sweet taste to tomatoes, are also signaling molecules that can regulate gene expression, by the process of sucrose-induced repression of translation, in bZIP (basic leucine zipper) transcription factors. The bZIPs are associated with fundamental functions in environmental and developmental signaling, acting in central carbon metabolic regulation. In the knowledge that modifications in specific regions of S-class bZIPs can alter post-transcriptional regulation, the use of genetic engineering tools to produce targeted mutagenesis has been used as a strategy. The aim of this work was to characterize the SlbZIP1 gene, using the CRISPR/Cas9 technique for the deletion of a uORF (upstream open reading frame) region involved in the regulation of this gene and the identification of an expression correlation network of SlbZIP1-related genes obtained through RNA-seq libraries to understand their interactions. Plant transformation was performed via Agrobacterium tumefaciens, and the obtained events were selected through resistance to the selective agent kanamycin and validated by PCR. For the molecular characterization of the gene editing, the fragments were amplified and sequenced. A 42 bp deletion of uORF was observed, compatible with the target region and the suggested proposal. This result demonstrates that the gene editing strategy presented in this study is effective and usable. As for the in-silico analyses, a search in already published studies of RNA-seq data in tomato plants was performed. A total of 193 libraries sorted into different tissues and subjected to various conditions were selected. The data were processed, aligned, and the expression correlation between all obtained transcripts and the SlbZIP1 gene was established by Spearman's test, with a correlation coefficient ρ≥ 0.8. A total of 85 sequences of various functions were considered, where other transcription factors and protein kinases stood out. These results agree with other studies, which indicate that protein kinases act synergistically with specific transcription factors and perform important activities for tolerance and response to various environmental conditions. Further studies can be conducted to increase the knowledge about the SlbZIP1 gene.