The Mechanism of the Silencing of a Transgene, NCED3-LUC, in Arabidopsis Thaliana

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علوم بحتة وطبيعية

الملخص

The Arabidopsis thaliana NCED3-LUC transgenic line was constructed by severalgroups to study the regulatory network of the NCED3 gene, the protein of whichcatalyzes the rate-limiting step of ABA biosynthesis under drought. The transgenicluciferase gene is expressed when the plants encounter drought stress. Intriguingly, thistransgenic luciferase gene is silenced after propagation for several generations. Todetermine 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 ethanemethyl sulfonate (EMS). One mutant line, denoted as #73, with recovered luciferaseactivity was selected for further study. Analysis of the methylation status by bisulfitesequencing revealed that the transgenic NCED3 promoter in the #73 mutant had lessmethylation than the wild type. Demethylation was also evident for the endogenousNCED3 promoter and retrotransposon AtSN1 in the #73 mutant. The phenotype of #73mutant includes small size, rapid dehydration rate, altered morphology, and a thinepicuticular wax layer. By use of map-based cloning, the region containing the mutatedgene was delimited to a contig of two BAC clones, F11F19 and F9C22, on chromosome2. Our results indicate that NCED3-LUC gene silencing results from hypermethylation ofits promoter region, but additional study is required to determine the exact position ofthe mutated gene and to fully understand the mechanism of NCED3-LUC silencing.4ACKNOWLEDGEMENTSI would like to take this opportunity to thank my committee chair, Professor Jian-KangZhu, who is also the supervisor of my master’s thesis, for his guidance throughout thecourse of this research. I also would like to thank my committee members, ProfessorLiming Xiong and Professor Samir Hamdan, for their patience and support in reviewingmy thesis.My appreciation also goes to Dr. Zhenyu Wang for taking time to teach me basicexperimental skills and for providing suggestions to my project. Meanwhile, mygratitude goes to my friends and lab mates and the department faculty. Thank you allfor 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 supportand encouragement.

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رقم الوثيقة

fc9f9294-f68c-4530-8efc-3a2385b868f2

رقم العقد

0

نوع الوسائط

Crawler

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الرسائل العلمية

صيغة المصدر

رسائل ماجيستير

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pdf text

أسماء الملفات

1700988_1.pdf

بيانات النشر

ألقاب المؤلفين

[{"name_ar":"جونسونج زاهاو","title_ar":"اعداد","title_en":"Preparation"},{"name_ar":"جيان كانج","title_ar":"اشراف","title_en":"Supervision"}]

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English

المصدر والدورية

اسم المصدر

The Mechanism of the Silencing of a Transgene, NCED3-LUC, in Arabidopsis Thaliana

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0

كلمات الباحثين

dc.contributor.advisor Zhu, Jian-Kang dc.contributor.author Zhao, Junsong dc.date.accessioned 2011-07-17T09:35:30Z dc.date.available 2011-07-17T09:35:30Z dc.date.issued 2011-06-20 dc.identifier.doi 10.25781/KAUST-0J766 dc.identifier.uri http://hdl.handle.net/10754/136192 dc.description.abstract The Arabidopsis thaliana NCED3-LUC transgenic line was constructed by severalgroups to study the regulatory network of the NCED3 gene, the protein of whichcatalyzes the rate-limiting step of ABA biosynthesis under drought. The transgenicluciferase gene is expressed when the plants encounter drought stress. Intriguingly, thistransgenic luciferase gene is silenced after propagation for several generations. Todetermine 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 ethanemethyl sulfonate (EMS). One mutant line, denoted as #73, with recovered luciferaseactivity was selected for further study. Analysis of the methylation status by bisulfitesequencing revealed that the transgenic NCED3 promoter in the #73 mutant had lessmethylation than the wild type. Demethylation was also evident for the endogenousNCED3 promoter and retrotransposon AtSN1 in the #73 mutant. The phenotype of #73mutant includes small size, rapid dehydration rate, altered morphology, and a thinepicuticular wax layer. By use of map-based cloning, the region containing the mutatedgene was delimited to a contig of two BAC clones, F11F19 and F9C22, on chromosome2. Our results indicate that NCED3-LUC gene silencing results from hypermethylation ofits promoter region, but additional study is required to determine the exact position ofthe mutated gene and to fully understand the mechanism of NCED3-LUC silencing.4ACKNOWLEDGEMENTSI would like to take this opportunity to thank my committee chair, Professor Jian-KangZhu, who is also the supervisor of my master’s thesis, for his guidance throughout thecourse of this research. I also would like to thank my committee members, ProfessorLiming Xiong and Professor Samir Hamdan, for their patience and support in reviewingmy thesis.My appreciation also goes to Dr. Zhenyu Wang for taking time to teach me basicexperimental skills and for providing suggestions to my project. Meanwhile, mygratitude goes to my friends and lab mates and the department faculty. Thank you allfor 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 supportand encouragement. dc.language.iso en

إشراف وإعداد

الإشراف

جيان كانج

الإعداد

جونسونج زاهاو

الاقتباسات الببليوغرافية