Development of gene constructs targeting fruit ripening transcription factors of previously characterized by incomplete repression.

With an increasing world population, challenges arise in producing and distributing enough food to feed the rising numbers of hungry mouths. One such challenge is maintaining proper nutrition where there is a lack of food. The time it takes to deliver produce to such areas may not be sufficient to ensure that it is fresh, or simply not be profitable with the given restraints of shelf life. One potential contribution to aid the resolution of this problem is understanding the function of genes involved in flower and fruit development, such as the MADS box genes. These genes are highly conserved transcription factors involved in plant organ development. Their role in flower and fruit development was best explained in Arabidopsis that have dry siliques as opposed to fleshy fruit. Identifying MADS box genes involved in fleshy fruit development and ripening, may lead to insights affecting the ripening process of fruits such as tomatoes.
The goal was to knock out potential MADS box tomato orthologs and define their role in fleshy fruit development and ripening. CRISPR-Cas9 is a novel technology developed from a bacterial defense mechanism and modified for gene silencing in eukaryotes. We anticipated to create CRISPR lines for the four candidate genes by designing constructs that contain two or three guide RNAs. The RNA guides will direct the Cas9 protein to the specific genomic region to generate a double stranded DNA break leading to the silencing of the gene function. Moving forward, the Giovannoni lab can use the constructs to observe the individual and combined knockout phenotypes to evaluate the gene functions and compare with previous RNAi phenotypes.

My Experience

My PGRP internship experience has been very eventful. I really appreciated all the exposure to different kinds of research going on at the weekly seminars. Also, digging into my research topic, learning how to write a proposal and present a poster were all new experiences that will help me later on. While writing, I was forced to consider the connection between my research and potential impacts on both agriculture and human health, which was something I never directly looked at before. The bioinformatics courses also provided an intriguing introduction to Unix and R, both of which I will come across again in furthering my education. Learning how to use programs to identify potential guides and align sequences was also something I had been hoping to learn. Overall, I am so grateful to have been here this summer!