Cloning and Preliminary Characterization of STY46 from Arabidopsis thaliana, a Candidate Protein Kinase Involved in the Plant Sugar Starvation Response

TL;DR

This study cloned and characterized the Arabidopsis STY46 protein kinase, exploring its potential role in plant sugar starvation response through genetic manipulation and molecular analysis.

Contribution

It reports the cloning of STY46 and initial steps towards functional analysis, including creation of overexpression lines and validation of gene expression.

Findings

Confirmed cloning of STY46 cDNA

Attempted knockout validation inconclusive

Developed overexpression construct

Abstract

To study how plants respond to conditions under which the carbohydrates needed for growth and development are exhausted, this project focuses on cloning and functional characterization of a cytosolic protein kinase, STY46 that may potentially be involved in the sugar starvation response (SSR). Generating A. thaliana STY46 knockout (KO) and overexpression (OE) lines is the main method in this project to determine the function of the protein. A SALK line (SALK116340) was confirmed to contain a TDNA insert in the STY46 gene and the aim was to prove that this reduced the expression of this gene. Highly intact RNA was isolated and reverse transcription PCR performed to determine the level of transcript of STY46 in STY46KO. However the results were inconclusive. This may be due to not enough amount of RNA use for the cDNA reverse transcription. To develop an overexpression line, STY46 was cloned from wild type Arabidopsis genotype. Primers were designed to flank the open read frame of the STY46. They also contained restriction enzyme recognition sites and a myctag engineered at their 5' ends to facilitate subsequent synthesis of a plant transformation construct. A cDNA fragment of the expected size was successfully amplified by PCR.