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Characterization of novel stem cell regulators in Arabidopsis thaliana

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  1. Tez No: 402623
  2. Yazar: BUĞRA ÖZDEMİR
  3. Danışmanlar: PROF. DR. JAN LOHMANN
  4. Tez Türü: Yüksek Lisans
  5. Konular: Biyoloji, Biyomühendislik, Biology, Bioengineering
  6. Anahtar Kelimeler: Belirtilmemiş.
  7. Yıl: 2014
  8. Dil: İngilizce
  9. Üniversite: Ruprecht-Karls-Universität-Heidelberg
  10. Enstitü: Yurtdışı Enstitü
  11. Ana Bilim Dalı: Belirtilmemiş.
  12. Bilim Dalı: Belirtilmemiş.
  13. Sayfa Sayısı: 94

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Özet (Çeviri)

miRNA-Induced-Gene-Silencing (MIGS) is a recently-developed RNAi-based method that permits efficient knockdown of genes which are targeted for silencing via ta-siRNA pathway by 5' terminal fusion of miR173 target site (miR173ts) (Felippes, et. al., 2012). miR173 is expressed in most Arabidopsis tissues and functions in production of ta-siRNAs by selectively targeting mRNAs carrying the miR 173 target site. The produced ta-siRNAs mediate the gene repression through destabilization of the mRNA. In the original protocol ofMIGS, fragments of genes to be knocked down, were fused at their 5' ends with a miR173 target site, and the resulting construct was ectopically expressedin plants under the 35S promoter. The purpose of this study was the adaptation of MIGS as a tissue-specific forward genetics screen that would enable the discovery of genes active in a specific tissue/organ. By random insertion of pCL V3: :miR173ts fusion construct across the Arabidopsis genome, silencing of random endogenous transeripts tagged by miRl 73ts was intended. Afterwards, extraordinary phenotypes w ere search ed for through screening of large numbers of plants. This is, therefore, a promoter tagging screen with a knockdown effect. Advantages of promoter-tagging method, such as do minance of the knockdown effect, are also present in our method, accompanied by the further strengths such as knockdown effect and ab sence of mi sexpres si on. During o ur screens, a wide range of traits were detected in T -1 plants. Some of these traits are strongly linked to aberrant meristem function/organization. This might indicate silencing of genes active in SAM. 4 out of 51 lines grown inT -2 gencration showed phenotypes in the second gencration as well. Two lin es with strong phenotypes were selected for mapping the T -DNA insertion. Due to time limitation, only one out of 4 T-2-confirmed lines was chosen for mapping. For this line, the mapping identified a sequence of vector backbone which appeared to be integrated in plant genome. The other candidate selected for mapping was a T -1 plant (M72) with a strong phenotype, showing a segregation pattem indicative of two T-DNA insertions. For this plant, mapping of one of the two T -DNA insertions identified the fo11owing gene as a putative target ofMIGS: CYTOCHROME P450, FAMILY 97, SUBFAMILY B, POLYPEPTIDE 3 (CYP97B3).

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