COST-STSM FA0605-6828 Necla Pehlivan
Period: 2010-09-15 to 2011-01-15
STSM type: Regular (from Turkey to Italy)
STSM Applicant: Ms. Necla Pehlivan, Rize University, RIZE (TR), email@example.com
STSM Topic: The characterization of the site of insertion of transgene in the tomato plant genome
Host: Dr.Rina Iannacone, Metapontum Agrobios, Metaponto (MT) (IT), firstname.lastname@example.org
My aim for this action was to investigate for the characterization of the site of insertion of transgene ( a homeodomein leucine - zipper gene (ATHB7) which is a member of the homeobox transcription factor family in Arabidopsis and shows up-regulation, during water deficit conditions) in tomato plant genome, the expression of transgene and interaction with abiotic stress related proteins (by the use of Real time PCR, Northern Blott and the other molecular techniques which I wanted to become familiar with,) and the accumulation of metabolites related with drought stress (Proline, ABA, Jasmonic acid, Carotenoids).
Description of the work carried out during the visit
DNA from 4 transgenic tomato lines (T0, T1, T2, T6 - totally 220 plants) was isolated. The concentration of DNA was determined by a NanoDrop ND-1000 spectrophotometer and amplified by using ATHB7-07 F / ATHB7-07 R primer pair, in order to assess if gene insertion is ok. The observed results showed that 50 of them had that ATHB7 transgene correctly and by the light of this, total RNA was prepared from that 50 plants and after DNA was eliminated, samples were checked with 18S rRNA F / 18S rRNA R primers for genomic contamination by PCR. Than, cDNAs were constructed according to standart protocols and to observe gene expression patterns quantitative RT-PCR was performed using some samples which belong to different transformation events/ generations. 18S rRNA house keeping gene was used as an internal control and the efficiency values and relative expression levels was determined for the real-time PCR amplification of the gene of interest, ATHB7. According to results, high, medium, low or undetectable expression levels were selected for further experiments.
During my visit, I had a chance to join 54 th Annual Congress of SIGA (Italian Society of Agricultural Genetics). Furthermore; in the midst of my STSM, Rina and me went to Barcelona and met other COST members. I would like to thank everyone at there who created a positive atmosphere and also for their small and big advices and encouragements about my future career.
Last but not least; I would like to express my thanks to Giancarla and Rosita , (during the visit I have co-operated with technician Giancarla Taddonio and training student, Rosita Quinto) for their friendship and support, and all people for making Agrobios a nice place to work in.