Literature published in the 1970’s document dioecious female Cannabis plants producing male flowers after treatment with colloidal silver (CS) during the flowering period (Sarath, 1979). While Van Der Straeten describes CS counteracting ethylene, a biosynthetic chemical expressed in female flowers, the gene expression changes from this treatment were not discussed (1991). What differential RNA expression occurs when transitioning female flowers to male flowers through CS treatment on Cannabis? My entire honor’s thesis will be performed in The Kane Lab using RNA donated from Skinny Pineapple LLC that will be prepared, sequenced, and analyzed on campus. This project will help better understand Cannabis’s gene expression patterns during vegetative
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Van Der Staten describes the basis of ethylene in higher plants as a chemical being highly involved throughout a plants life from seed germination to fruit ripening, as well as acting as a “stress response” (1991). After analyzing Van Der Staten’s publication, the CS could possible invoke a natural stress response allowing the female to self-pollinate to produce progeny, which would be considered an advantageous trait. Naturally, many Cannabis strains can produce male and female flowers to a varying degree, especially in response to temperature, light or mechanical stress (Moliterni, 2004). This supports the idea that there is a set of male genes that is conserved differently between the evolutions of each strain that can be expressed in response to stress. While it is proven to counteract ethylene’s hormonal effects, I argue this sets the conditions necessary for expressing this conserved set of male genes, which my goal is to determine which genes are being expressed. To determine any gene expression changes during this time, my professional investigator and I believe that RNA-sequencing and differential expression analysis will be the best approach to answer my question. To build the necessary skills, I began preparing for the data handling and analysis …show more content…
As a student within Dr. Kane’s Lab, I cannot handle high THC plant material, so it was necessary to have donated RNA of these Cannabis plants to be a starting point for this project. Through refining the experimental design, there will be two time points for extractions with 2 control and 2 treatment extractions coming from each plant. This should allow the RNA extracted from male and female flowers on the same plant to be analyzed to answer what changes in gene expression occurs between these two branches of the same plant. These RNA samples will be prepared through a cost-effective diluted SmartSeq2 library preparation kit with a modified protocol from Combs et al. publication (2015). Allowing for more samples to be prepared and sequenced with this protocol, I will produce reliable results and allow for further analysis of this data on subsequent projects through having greater replication of this experiment. The differential expression analysis will be performed through bioinformatics tools including HT-Seq, TopHat and other scripts that I will write in the bash programming language. This analysis will be performed on the supercomputer in Dr. Kane’s Lab since it has the processing power and memory to handle this amount of
Van Der Staten describes the basis of ethylene in higher plants as a chemical being highly involved throughout a plants life from seed germination to fruit ripening, as well as acting as a “stress response” (1991). After analyzing Van Der Staten’s publication, the CS could possible invoke a natural stress response allowing the female to self-pollinate to produce progeny, which would be considered an advantageous trait. Naturally, many Cannabis strains can produce male and female flowers to a varying degree, especially in response to temperature, light or mechanical stress (Moliterni, 2004). This supports the idea that there is a set of male genes that is conserved differently between the evolutions of each strain that can be expressed in response to stress. While it is proven to counteract ethylene’s hormonal effects, I argue this sets the conditions necessary for expressing this conserved set of male genes, which my goal is to determine which genes are being expressed. To determine any gene expression changes during this time, my professional investigator and I believe that RNA-sequencing and differential expression analysis will be the best approach to answer my question. To build the necessary skills, I began preparing for the data handling and analysis …show more content…
As a student within Dr. Kane’s Lab, I cannot handle high THC plant material, so it was necessary to have donated RNA of these Cannabis plants to be a starting point for this project. Through refining the experimental design, there will be two time points for extractions with 2 control and 2 treatment extractions coming from each plant. This should allow the RNA extracted from male and female flowers on the same plant to be analyzed to answer what changes in gene expression occurs between these two branches of the same plant. These RNA samples will be prepared through a cost-effective diluted SmartSeq2 library preparation kit with a modified protocol from Combs et al. publication (2015). Allowing for more samples to be prepared and sequenced with this protocol, I will produce reliable results and allow for further analysis of this data on subsequent projects through having greater replication of this experiment. The differential expression analysis will be performed through bioinformatics tools including HT-Seq, TopHat and other scripts that I will write in the bash programming language. This analysis will be performed on the supercomputer in Dr. Kane’s Lab since it has the processing power and memory to handle this amount of