M.Sc Thesis

M.Sc StudentMozesh Oded
SubjectToward Production of Salt Tolerant Plants: Identifying Genes
Associated with Salt Tolerance in Thellungiella
DepartmentDepartment of Biotechnology
Supervisors PROFESSOR EMERITUS Peter Neumann
Full Thesis textFull thesis text - English Version


Thellungiella halophila (Th) is a plant model for genetic research of halophytes. It is a small plant with a short life cycle, produces thousands of seeds, can easily be genetically transformed and tolerates extreme salinity (>500 mM NaCl). Thellungiella halophila is from the same plant family as its salt susceptible relative, Arabidopsis thaliana (At), Thus; they share high sequence identity (90%-95%) at the cDNA level. Because small differences in gene expression may underlay the differences in salt tolerance between T. halophila and A. thaliana, they are good candidates for comparisons of gene expression.

I therefore looked for genes highly expressed under salt stress in T. halophila as compared to A. thaliana. I used the suppression subtraction hybridization (SSH) method in which transcripts of Arabidopsis subjected to salt stress were subtracted from transcripts of salt stressed Thellungiella, to provide a cDNA library of differently (highly) expressed genes of the subtracted (Thellungiella) cDNA (tester). Sequencing of ~150 cDNAs of the SSH results provided ~80 different Thellungiella genes which were differentially expressed. Northern blot analysis was performed to support the SSH results for some candidate genes reported by the SSH.   Three of the genes tested by Northerns gave signals and supported the SSH results: 1. A gene for UBX domain containing protein (pux4). 2.  A gene for protein kinase C 3.A gene for auxin/aluminum-responsive protein. In addition, phenotypes were examined for Arabidopsis T-DNA knockouts of genes revealed by the SSH, in order to determine whether the knockouts changed the plant responses to salt stress. The growth kinetics of the roots and shoots of the knockout plants under normal and salt stress conditions were compared to wild type response under the same conditions. Out of eight T-DNA knockout lines, two showed different shoot growth kinetics compare to WT: Thus, leaves of knockout plants for the protein kinase C gene promoter and for 5’ UTR grew faster than WT under salt stress. Five knockouts showed different root growth kinetics: roots of knockouts for the UBX domain containing protein (pux4) gene, and the nonspecific lipid transfer protein grew slower than WT under salt stress. In contrast, roots of knockouts for the dehydration-induced protein (ERD15) gene grew faster than WT roots under salt stress and the roots of knockout plants for the dehydration-induced protein (ERD15) gene grew faster than the WT under optimal growth conditions.