International Network of Plant Abiotic Stress

Period: 2010-09-15  to 2011-01-15

STSM type: Regular (from Turkey to Italy)

STSM Applicant: Ms. Necla Pehlivan, Rize University, RIZE (TR), neclapehlivan@hotmail.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), riannacone@agrobios.it

Main goals

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.

Other comments

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.