Functional characterization of an endosperm specific promoter p1062 from common buckwheat (Fagopyrum esculentum Moench) for driving tissue specific gene expression / Funkcijske lastnosti endospermskega promotorja p1062 navadne ajde ...

Abstract

Seed storage proteins of grain crops meet the major dietary protein requirement of over half of the world population. PCR based genome walking the 5’UTR of the gene coding for a lysine rich legumin type protein amplified a 1.1kb DNA fragment representing the promoter region of the gene. Clustal alignment of this sequence with other sequences in the Genbank database clearly showed 100 percent complementary base match of 282 bases at the 3’ end of the sequence, corresponding to nucleotide position 780-1062 with correspondingly similar number of bases on the 5’ end of the 1.7kb Bwleg gene.We detected one prolamin box and three RY-repeat motifs in the sequence. Seven deletion fragments of the putative promoter were generated by 5’ nested PCR and cloned in pCAMBIA1304 upstream of GUS gene after excising the CaMV 35S promoter from the vector. Arabidopsis plants plants harbouring the deletion construct pBwlDF1 to pBwlDF6 clearly showed seed specific expression of the reporter gene. Seeds harbouring the constructs pBwlDF3, pBwlDF4 and pBwlDF5 showed a nearly threefold decrease in GUS activity than those harbouring the construct with full length promoter.
 Key words: buckwheat, DNA, promoter, constructs
 
 Izvleček
 Založne beljakovine semen zrnastih poljščin ustrezajo glavnim potrebam po beljakovinah za več kot polovico svetovnega prebivalstva. S PCR in 5’UTR so za kodiranje kakovostnih beljakovin leguminskega tipa pomnožili odlomek 1,1 kb DNK, ki je promotorsko gensko območje. Vzporejanje te sekvence z drugimi sekvencami podatkovne baze genske banke jasno pokaže popolno komplementarnost 282 baz na 3’ koncu sekvence, kar ustreza pozicijam 780-1062 z ustreznim številom baz na 5’ koncu gena 1,7 kb Bwleg. V sekvenci smo odkrili eno prolaminsko škatljo in tri RY-ponovljene motive. Sedem delecijskih fragmentov putativnega promotorja smo generirali z 5’ PCR kloniranjem pCAMBIA1304 navzgor od GUS gena po izločitvi promotorja CaMV 35S iz vektorja. Semena s konstrukti pBwlDF3, pBwlDF4 in pBwlDF5 so izražali skoraj trikratno zmanjšanje GUS aktivnosti v primerjavi s konstrukti, ki so vsebovali polne dolžine promotorjev. 
 Ključne besede: ajda, DNK, promotor, konstrukti