Construction of novel recombinant receptor proteins to improve disease resistance in wheat

Abstract

English: The aim of the current study was to construct novel recombinant RLK genes encoding chimeric receptor proteins to broaden disease resistance in wheat. Eight different RLKs were initially chosen as extracellular domain donors based on their ability to bind PAMPs/MAMPs or DAMPs. The kinase domain donor was LRK I0, an R protein that renders wheat resistant against Puccinia triticina. These recombinant genes were first designed in silico using a bio-informatic approach where the gene portions encoding the different extracellular domains were linked to the LRKIO kinase domain encoding gene fragment. Translation of the different recombinant genes confirmed that full length polypeptides will be formed. General PAMP binding capacity was therefore combined with the highly specific defence signalling ability of LRKIO within a single protein. Due to several problems encountered, only four recombinant genes were successfully produced, namely BRll-LRKlO, CERK-LRKIO, LecRKI-LRKIO and PERKI-LRKIO. To confirm the success of the approach, LecRKI-RLKIO was successfully transcribed and translated in vitro, thus producing the correct sized polypeptide. Future work will include the production of transgenie wheat to test the ability of these chimeric proteins to increase wheat resistance against pathogen infection.Afrikaans: Die doel van die huidige studie was om rekombinante RLK gene wat vir "n nuwe klas chimeriese reseptorproteïene kodeer te produseer om sodoende die siekteweerstand van koring te verbreed. Agt verskillende RLKs was aanvanklik gekies omdat hulle ekstrasellulêre domeine verskillende PAMPsIMAMPs en DAMPs bind. Die geenstreke wat vir die ekstrasellulêre domeine kodeer sal aan die geenstreek van LRKJO wat vir die kinasedomein kodeer, geheg word. LRKJO is "n weerstandsgeen wat aan koring weerstand teen Puccinia triticina verleen. Die rekombinante gene is in silica met behulp van bio-informatika ontwerp deur die ekstrasellulêre domein geenstreke aan die LRKJO kinasedomein spesifieke geenstreek te heg. Translasie van die verskillende rekombinante gene het bevestig dat elk van die gene vir "n volledige polipeptied kodeer. Die algemene vermoë om PAMPsIMAMPs en DAMPs te bind is dus binne 'n enkele proteïen met die spesifieke verdedigingsseinvermoë van LRK10 gekoppel. As gevolg van verskeie probleme, is daar slegs vier verskillende rekombinante gene geproduseer. Hulle was BRll-LRKJO, CERK-LRKJO, LecRKJ-LRKJO en PERKJ-LRKJO. Om die sukses van die benadering te toets, is die LecRKJ -RLKJO rekombinante geen suksesvol getranskribeer en getransleer om "n polipeptied met die korrekte grootte te vorm. Toekomstige werk sal die maak van transgeniese koring insluit om sodoende die vermoë van die chimeriese proteïene om aan koring weerstand teen patogeeninfeksie te verleen, te toets.