ISOLATION AND CHARACTERIZATION OF PROMOTERS OF GENES INVOLVED IN POLYAMINE METABOLISM IN

STRAWBERRYÁkos Mendel, László Kovács, Norbert Hídvégi, Erzsébet Kiss

Institute of Genetics and Biotechnology, Szent István University, Gödöllı

Figure 3. E. coli colony PCR

Figure 5. A. tumefaciens colony PCR

�Promoter regions of two genes SAM-decarboxylase and Sperimidne-synthase(showing alteration in gene expression during fruit ripening, hence supposed to be ripening-specific) were isolated and characterized by bioinformatic methods.

�Polyamines – similarly to ethylene – can regulate numerous plant-physiologycalprocesses, additionally the SAM (S-adenosyl-methionine) is a common precursor of ethylene- and spermidine-biosynthesis.

�The Spermidine-synthase is the gene encoding the key enzyme of the polyamine-biosynthesis.

�Increased spermidine and spermine level in plant cells extends abiotic stress-resistance, and influence fruit ripening, researches showed.

�Isolation of promoters of Spermidine-synthase (SSper) and SAM-decarboxylase (SAM DC1, DC2) genes, and sequence-analysis of the promoters.�Integration of the promoters and the sGFP riporter gene (in pGWB 604 binaryvector).�Transformation of Agrobacterium tumefaciens strain C58C1 with theseconstructions, and infiltration of Agrobacterium tumefaciens strain C58C1 containing the constructions into Nicotiana benthamiana model plant in order totest transient expression.�Examination of the operation and inducibility of the promoters using sGFPriporter gene.

RESULTS AND DISCUSSION

- Control (emptypGWB 604+C58C1)

+ Control(2*35S+mGFP+C58C1)

SSper Pro 2300+pGWB604+C58C1

- Control (emptypGWB 604+C58C1)

+ Control(2*35S+mGFP+C58C1)

SSper Pro 2300+pGWB 604+C58C1Sam DC2 Pro 2700+pGWB 604+C58C1

Sam DC1 Pro 4100+pGWB 604+C58C1

Research was supported by OTKA K101195 and the TÁMOP-4.2.2.B-10/1 „Development of a complex educational assistance system for talented students and prospective researchers at the Szent István University” projects. We would like to thank ABC (Gödöllı) for providing us the construction applied as „+ Control”.

Figure 6. Transient expression analysis in Nicotiana benthamiana

MATERIALS AND METHODS�Primary, genomic clones of these promoter regions were amplified from DNA of Fragaria vesca (Figure 1.).

�The length of the promoter regions were specified as below: 2321 bp in case of Spermidine-synthase, 4100 bp (SAM DC1) and 2714 bp (SAM DC2) in case of SAM-decarboxylase (as two different gene-variation were identified).

�Secondary, these promoter-fragments were ligated into Gateway pENTR cloningvector, than these vectors were carried into the compatible pGWB 604 binaryvector which contains sGFP riporter gene and were developed exactly for testing of promoters.

�The ready-made constructions were transferred into E. coli strain JM109, andfor proving the success of the transformation, the constructions were transferredinto Agrobacterium tumefaciens strain C58C1 too (Figure 2., 3., 4., 5.).

�After the checkout of the colonies, transient expressional examination wereperformed on Nicotiana benthamiana model plant by infiltration of Agrobacteriumcultures (Figure 6.).

Figure 1. Genomic clones of promoters region

Figure 2. E. coli colony PCR

INTRODUCTION OBJECTIVES

Figure 4. A. tumefaciens colony PCR

�Genomic clones of promoter regions were amplified, ligated into pENTR cloningvector, than into pGWB 604 binary vector.

�At first, the constructions were transferred into E. coli strain JM109, than intoAgrobacterium tumefaciens strain C58C1.

�Agrobacterium colonies containing the promoter constructions were infiltrated intoNicotiana benthamiana mode plants

�Specificity of the promoters have not proven yet, further examinations needed inthese fields, but as we can see on Figure 6, they show different expressionpattern comparing to the 2*35S contitutive promoter.