hrvatski

Giberelin-20-oksidaza iz kukurijeka (Helleborus niger L.)

Lapovi kukurijeka (Helleborus niger L.), bijeli ili ružičasti tijekom cvjetanja, intenzivno ozelenjavaju i ostaju viabilni za vrijeme razvoja i sazrijevanja plodova. Proces ozelenjavanja lapova zakočen je u neoplođenim cvjetovima, ali može biti potaknut uslijed tretmana biljnim hormonima giberelinima što upućuje na činjenicu da endogeni giberelini mogu igrati važnu ulogu u metamorfozi lapova cvijeta kukurijeka nakon oplodnje. Poznato je da je enzim giberelin-20-oksidaza (GA 20-oksidaza) uključena u biosintezu bioaktivnih giberelina u biljkama. U ovom radu sintetizirana je djelomična cDNA gena za GA 20-oksidazu iz sjemenki kukurijeka koristeći degenerativne početnice, uklonirana u plasmid pRSET, te je rekombinantni protein proizveden u E. coli. Servisno su proizvedena poliklonalna antitjela specifična na rekombinantnu GA 20-oksidazu. Prisutnost GA 20-oksidaze u biljnom tkivu potvrđena je Western analizom. Bioinformatičke analize proteina GA 20-oksidaze napravljene su koristeci dostupne programe (http://www.expasy.ch/). The sepals of the Christmas rose (Helleborus niger L.) which are white or rose at anthesis, persist until the seeds are ripe and become intensely green which differs from reproductive development in commonly investigated model plants. This process is suppressed in unfertilized or depistillated flowers, but can be reinstated by treatment with gibberellins which indicates that endogenous gibberellins may play critical part in the metamorphosis of Christmas rose sepals. Enzyme gibberellin-20-oxidase (GA 20-oxidase) is known to be involved in biosynthesis of bioactive gibberellins in plants. Herein, partial GA 20-oxidase cDNA was generated from Christmas rose seeds by using degenerative primers, cloned into pRSET expression plasmid, and recombinant protein was produced in bacterial system. Whole cell proteins (containing GA 20-oxidase) were isolated and separated by SDS PAGE. Protein band corresponding to GA 20-oxidase was cut out of gel and used for custom service production of polyclonal antibodies by immunization of a rabbit. Obtained antibodies were purified and used for western blot analysis where a presence of GA 20-oxidase was screened in diverse developmental stages of Christmas rose flower. Bioinformatics analyses of GA 20-oxidase were mostly performed by using on line ExPasy programs (http://www.expasy.ch/).

giberelin-20-oksidaza; kukurijek; Helleborus niger L.

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