CONSTRUCTION OF CRISPR/CAS9 VECTOR FOR SILENCING CIF1 GENE OF TOMATO

Abstract

Tomato (Solanum lycopersicum) is a nutrient-dense food that contains various secondary compounds, as well as great health benefits. The sugar content of tomato fruit is partly controlled through the regulation and breakdown of sucrose in fruit set and development. Cell wall invertase (CWI) hydrolyzes sucrose into monosaccharides and transports it into the cytoplasm, which means the sugar content of tomato is regulated by CWI. Meanwhile, since  this gene repression is induced by a product of the CIF1 gene, inactivation of the CIF1 gene may enhance the sugar synthesis in tomatoes. Currently, CRISPR/Cas9 system is a state-of-the-art technology, has wide applications and high precision in gene editing. In this study, suitable gRNAs for the CIF1 gene were designed to build expression constructs. This expression system in the pRGEB31-CIF1G2 plasmid was introduced into DH10B Escherichia coli strain. Afterwards, the vector carrying this expression system was successfully transferred into the EHA105 Agrobacterium tumefaciens strain. Further, A. tumefaciens lines containing the vector pRGEB31-CIF1G2 can be used to generate desired traits in gene-edited Tiny-Tim tomato lines.