Graduate Program

Biological Sciences

Degree Name

Master of Science (MS)

Semester of Degree Completion

2018

Thesis Director

Yordan S. Yordanov

Thesis Committee Member

Barbara S. Carlsward

Thesis Committee Member

Thomas Canam

Abstract

The NLG (Novel Leaf Gene) is a recently discovered gene from aspen (Populus tremula x alba). Previous research indicates that NLG impacts leaf shape. Our preliminary analyses indicate that NLG may function as a membrane bound protein with possible localization in plasma membrane and/or nucleus. Sequence and phylogenetic analyses in Arabidopsis thaliana and Populus trichocarpa identified four homologous genes in Arabidopsis and five in Populus. Gene expression analysis was done using online tools and indicated that NLG is expressed in multiple tissue types but predominately in stem and flowers. The protein coding region of the NLG orthologs genes from aspen and Arabidopsis was cloned, and four gene constructs used in both organisms. Constructs consist of an overexpression of NLG from poplar, overexpression of the Arabidopsis ortholog, two constructs consisting of gene fusion with GFP tags, one at the 5' and one at the 3' end of the poplar gene. An RNAi gene downregulation construct was also used for transformation of Arabidopsis. Escherichia coli was used for cloning and replication of the plasmids and Agrobacterium tumefaciens was used to transform Arabidopsis thaliana and Nicotiana tabacum (tobacco). Arabidopsis is transformed using floral dip and tobacco is transformed by leaf disc transformation. Multiple transgenic lines from both species were produced. Transgene presence was verified by PCR in most of the lines and the successful inheritance was recorded for the next generations. Tobacco was used to study the impacts of the gene on the plant phenotype. Gene overexpression of the orthologous genes from Populus and Arabidopsis shows clear impact on leaf and stem growth in tobacco. Leaf area is decreased while stem length is increased. Preliminary observations also indicate impact on stem and leaf anatomy influencing xylem and mesophyll development and growth.

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