The Arabidopsis thaliana NCED3-LUC transgenic line was constructed by several
groups to study the regulatory network of the NCED3 gene, the protein of which
catalyzes the rate-limiting step of ABA biosynthesis under drought. The transgenic
luciferase gene is expressed when the plants encounter drought stress. Intriguingly, this
transgenic luciferase gene is silenced after propagation for several generations. To
determine the mechanism of this gene silencing, we used a forward genetics approach.
The seeds of NCED3-LUC (referred as the ‘wild type’) were mutagenized by ethane
methyl sulfonate (EMS). One mutant line, denoted as #73, with recovered luciferase
activity was selected for further study. Analysis of the methylation status by bisulfite
sequencing revealed that the transgenic NCED3 promoter in the #73 mutant had less
methylation than the wild type. Demethylation was also evident for the endogenous
NCED3 promoter and retrotransposon AtSN1 in the #73 mutant. The phenotype of #73
mutant includes small size, rapid dehydration rate, altered morphology, and a thin
epicuticular wax layer. By use of map-based cloning, the region containing the mutated
gene was delimited to a contig of two BAC clones, F11F19 and F9C22, on chromosome
2. Our results indicate that NCED3-LUC gene silencing results from hypermethylation of
its promoter region, but additional study is required to determine the exact position of
the mutated gene and to fully understand the mechanism of NCED3-LUC silencing.
4
ACKNOWLEDGEMENTS
I would like to take this opportunity to thank my committee chair, Professor Jian-Kang
Zhu, who is also the supervisor of my master’s thesis, for his guidance throughout the
course of this research. I also would like to thank my committee members, Professor
Liming Xiong and Professor Samir Hamdan, for their patience and support in reviewing
my thesis.
My appreciation also goes to Dr. Zhenyu Wang for taking time to teach me basic
experimental skills and for providing suggestions to my project. Meanwhile, my
gratitude goes to my friends and lab mates and the department faculty. Thank you all
for making my time at King Abdullah University of Science and Technology so wonderful.
Finally, my heartfelt gratitude is extended to my parents for their everlasting support
and encouragement.
| Date of Award | Jun 20 2011 |
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| Original language | English (US) |
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| Awarding Institution | - Biological, Environmental Sciences and Engineering
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| Supervisor | Jian-Kang Zhu (Supervisor) |
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