@phdthesis{
author = "Timotijević, Gordana",
year = "2009",
abstract = "Dve parcijalne cDNK (FeAP9 i FeAP12) koje kodiraju aspartične proteinaze heljde su dobijene primenom metode PCR sa degenerativnim prajmerima dizajniranim na osnovu konzervisanih sekvenci biljnih aspartičnih proteinaza (AP). Korišćenjem 5’ i 3’ RACE (rapid amplification of cDNA ends) pristupa, odgovarajući 5’ i 3’ krajevi sekvence FeAP9 su amplifikovani i rekonstruisana je kompletna sekvenca. Utvrđeno je da FeAP9 sadrži otvoreni okvir čitanja od 1521 bp koji kodira prekursorni protein od 507 ak. Poređenje aminokiselinskih sekvenci sa ostalim biljnim AP pokazalo je da FeAP9 i FeAP12 imaju veliku homologiju sa tipičnim aspartičnim proteinazama i da sadrže PSI (plant specific insert) sekvencu karakterističnu samo za biljne AP. Uočena je i karakteristična struktura: signalna sekvenca, prosegment i nizvodna polipeptidna sekvenca sastavljena od dva lanca zrelog enzima koja su razdvojena PSI regionom. Dodatno je iz gDNK heljde izolovan i genomski klon FeAP9 dužine 5374 bp koji sadrži 12 introna i ova strukturna organizacija pokazuje sličnost sa većinom biljnih AP. Dodatno je otkriveno prisustvo lider introna koji je lociran samo 1 bp uzvodno od ATG u 5’UTR regionu AP gena. Kompjuterska analiza ovog introna ukazuje na prisustvo potencijalnih regulatornih sekvenci koje mogu biti uključene u odgovor na različite stimuluse. U cilju ispitivanja ekspresije gena za FeAP vršena su analize iRNK različitih organa heljde u normalnim fiziološkim stanjima ili u uslovima različitih faktora abiotičkog stresa. Primenjene su metode Northern blot, RT PCR i Real-time PCR uz upotrebu proba ili prajmera specifičnih za FeAP9/FeAP12. Pokazano je da se FeAP9 eksprimira u semenu, listu, cvetu i korenu heljde, a maksimum ekspresije u semenu dostiže se u fazi 19-23 dana nakon cvetanja. Osim toga, uočeno je da nivo ekspresije FeAP9 dramatično raste u senescentnom listu. Ekspresija FeAP12 vezana je samo za seme, a profil ove ekspresije se razlikuje u odnosu na profil ekspresije FeAP9. Utvrđeno je takođe da se nivo FeAP9 transkripta povećava u listovima biljaka koje su izlagane suši, mraku, mehaničkim povredama i salicilnoj kiselini, što ukazuje na moguću ulogu ovog gena u odgovoru na stres. Takođe je proizveden rekombinanti FeAP9 protein korišćenjem pMAL ekspresionog vektora, međutim protein je pretežno bio lokalizovan u inkluzionim telima, a različite metode renaturacije nisu dovele do proteinazne aktivacije. Napravljena su primarna poliklonska antitela koja prepoznaju 14 ak dug oligopeptid koji odgovara N terminusu FeAP9 proteina i korišćena su za imunodetekciju u analizi Western blot. Pri redukujućim uslovima antitelima smo detektovali protein od 47 kDa u svima analiziranim tkivima, osim u cvetu gde je bio prisutan protein od oko 52 kDa. U prisustvu β-merkaptoetanola detektovan je polipeptid of 32 kDa u stablu, listu, korenu i semenu, dok je polipeptid od 26 kDa uočen u cvetovima, što je verovatno posledica post-translacione obrade. Imunodetekcijom je pokazano da se FeAP9 akumulira u semenu tokom sazrevanja i da je prisutan u početnim fazama germinacije. Osim toga, FeAP9 je bio zastupljen u svim fazama razvića listova, kao i u tučkovima razvijenih cvetova heljde., Two partial cDNAs (FeAP9 and FeAP12) encoding for buckwheat aspartic proteianse were obtained by PCR with degenerate primers designed for conserved sequences of plant aspartic proteinases (APs). Using rapid amplification of cDNA ends approach, 5’ and 3’ends appropriate to FeAP9 has been amplified and full length sequence was reconstructed. It was found that FeAP9 cDNA contained open reading frame of 1521 bp encoding precursor polypeptide of 507 aa. Alignment of deduced amino acid sequences with other plant APs demonstrated that FeAP9 and FeAP12 contained a plant specific insert of 104 amino acids (aa), a unique sequence of plant APs. Derived amino acid sequence comprised: signal sequence, prosegment and downstream polypeptide sequence that includes two chains of the mature enzyme separated by PSI. In addition, we have isolated genomic clon FeAP9 of 5374 bp from buckwheat gDNA. Structural organization of aspartic proteinase AP9 gene was similar to majority of plant APs containing 12 introns. Also, we have reveled presence of the leader intron located 1 bp ustream of the ATG in 5’UTR region of AP gene. Computer analiysis of the leader intron predicted the existence of regulatory sequences that could be involved in responses to different stimuli. In order to investigate expression of the buckwheat FeAP, Northern blot analysis was performed with gene specific probe and total RNA isolated from different buckwheat organs, as well as from the seeds throughout development. It was shown that AP is expressed in buckwheat seeds, leaves, flowers and roots. Expression reached maximum at stage of 19-23 days after flowering. Also, it was detected that the level of AP expression dramatically increases during leaf senescence. In order to distinguish precise difference in expression of FeAP9 and FeAP12 genes we have designed primers from 3’ UTR of FeAP9 gene and gene specific primers from coding FeAP12 sequence. RT PCR experimentes indicate that FeAP9 is ubiquitinously expressed in all analyzed tissues, while FeAP12 expression is seed specific and its profile of expression differs in relation to FeAP9 profile. Furthermore we monitored FeAP9 expression changes in response to a variety of abiotic stresses using Real-time PCR. We found that the expression is upregulated by exposure to drought, darkness, wounding and SA, indicating a likely role for the gene in stress responses. Finally, we have produced recombinant FeAP9 protein using pMAL expression vector, but protein was located mostly in inclusion bodies and different refolding methods did not result in activation of the proteinase. Therefore, primary polyclonal antibodies against oligopeptide corresponding to the N-terminal 14 residues of FeAP9 was produced and used in Western blot analysis. Under nonreducing condition protein of 47 kDa corresponding to FeAP9 was detected in all analyzed tissues except in the flowers, where the protein of 52 kDa was present. In the presence of β-mercaptoethanol we detected polypeptide of 32 kDa in steams, leaves, roots and seeds, while 26 kDa polypeptide was noticed in flowers. Imunodetection showed that FeAP9 was accumulated during seed development and it was present in early stages of germination. Moreover, FeAP9 was detected in all stages of leaf development, as well as in pistils of fully opened buckwheat flowers.",
publisher = "Univerzitet u Beogradu, Biološki fakultet",
title = "Molekularno kloniranje i analiza gena za aspartičnu proteinazu heljde (Fagopyrum esculentum Moench), Molecular cloning and analysis of the aspartic proteinase gene from buckwheat (Fagopyrum esculentum Moench)",
url = "https://hdl.handle.net/21.15107/rcub_nardus_10621"
}
