Bianca Corriea, visiting exchange-student researcher working in the Wyatt Lab, is using antisense RNA (asRNA) to verify the candidate gene responsible for the unique phenotype of the gps3 mutant in response to GPS treatment.
To test the gps3 candidate gene, Corriea must silence the expression of this single gene and verify that this targeted transformation results in the gps 3 phenotype. The gene silencing method that Corriea will deploy is to sequence the candidate gene and then splice a copy of the gene into agrobacterium. Agrobacterium is used in plant biology as a method of introducing genes into the genome of test plants through a process known as agrobacterium-mediated transformation mechanism. This mechanism takes advantage of the agrobacterias natural process of invading host organisms and semi-randomly inserting its DNA into the host genome.
Once Correia has spliced a copy of the candidate genome into the agrobacterium plastid, she will allow the bacteria to infect the plant – effectively introducing her gene into the plant. Once in the plant, the gene will express in reaction to gravity persistent signaling (GPS) treatment, and Corriea will leverage the plants own RNA interference (RNAi) biological processes to silence the gene of interest.
RNA interference (RNAi) inhibits gene expression by destroying specific messenger RNA (mRNA) which mutes the corresponding protein. This is accomplished by using Dicer – a biological process that keeps cells tidy by eliminating duplicate or mutant transcripts.
Genes are composed of two discrete strands – a coding gene and a non-coding gene that is complementary to the coding gene. Normally, the coding gene is the only gene expressed resulting in mRNA which then enters the ribosome and results in a specific protein expression. Corriea will add a promoter to the non-coding strand of DNA that will cause it to express as the complementary coding strand to the one the coding DNA expresses. Since the resulting strands of RNA will be complimentary they will bind together – effectively blocking the expression of the gene. Furthermore, the double-strand of RNA will trigger the Dicer mechanism to destroy it. Andrew Fire and Craig C. Mello first demonstrated RNAi role in silencing gene expression and shared the 2006 Nobel Prize in Physiology or Medicine for this work.
In this sense, Corriea is standing on the shoulder of giants in her gps3 candidate gene research. By silencing the candidate gene in the gps3 mutant responsible for its GPS treatment phenotype, Bianca will be able to determine if the gene is indeed responsible for the behavior.
Identifying the gps3 mutant gene will contribute to the growing knowledge of the genes responsible for plant reaction to gravitropism, helping pave the way for sustaining plants in microgravity conditions.
Currently, Corriea is finalizing her sequencing of the candidate gene. Once this is complete, she will begin splicing the sequence into agrobacterium to introduce into mature arabidopsis gps3 mutants.