|
My summer research focused
on the evolution of genes that are involved in regulating how plants
grow, mainly genes that play a role in the development of the shoot
apical meristem (SAM). The CLAVATA (CLV) genes, which
are expressed in the shoot apical meristem, mediate normal
development and function of the SAM. Plants with clv1,
clv2, or clv3 mutations show overgrowth of the meristem
and develop more organs than normal. CLV3 encodes a 96 amino
acid protein that activates the CLV1/CLV2 signaling complex which
controls the balance between growth and differentiation in the
meristem. My research continued a project initiated by Jaime Wesker
which analyzed sequence diversity of the CLV3 gene from
Arabidopsis thaliana plants.
For this experiment, Jaime
and I used 27 accessions of A. thaliana from different parts
of the world, mainly from Europe. For each accession, we isolated
genomic DNA, used PCR to amplify the CLV3 gene, and directly
sequenced the PCR product.
I used a sequence analysis program to integrate the sequences
into an alignment which allowed me to compare our sequences with the
Colombia CLV3 accession, obtained from the Arabidopsis Genome
Initiative.
For comparison, I also
isolated the CLV3 gene from A. lyrata.
I performed PCR using primers specific to the A. thaliana
gene, inserted the PCR product into a plasmid, and sequenced three
clones. I used this method because A. lyrata is likely to be
heterozygous and therefore cannot be directly sequenced. The three
A. lyrata clones showed no differences among them.
I analyzed the data to
determine to what extent the CLV3 gene varies within A.thaliana.
Possible polymorphisms were determined by nucleotide
differences between accessions.
The level of nucleotide diversity in CLV3 (Pi
silent) was 0.006, which is similar to the average observed in other
Arabidopsis genes. Among
the A. thaliana sequences analyzed, I observed that the
accessions fell into three distinct groups of alleles. Out of 16
single nucleotide polymorphism sites found, there were 2 synonymous
and 2 non-synonymous mutations in the coding region and 13 mutations
at non-coding positions. This showed that more variation has
accumulated within the non-coding than the coding region.
|
