Cloning And Expression Of The Brazilian Rubber Tree Dead Skin Gene Hbf

CIoning and Expression of Hbf associated with TappingPanel Drvness of H6vea brasmensiSAbstractIn previous study, a cDNA fragment Hb-1 of H6vea brasgfen5iS was obtained byDDRT-PCR.The objective of this study was to cIone and express this cDNA.Degenerate primers were designed based on the sequence of Hb-1 to amplify thecorresponding fuII cDNA of Hb-1 by 5'-RACE. The fuII-length cDNA, called Hbf (for HE veabrasHfensiS faCtor), was 1204 bp in Iength with a 930 bp open reading frame. A5'-UTR(untransIated region) of 45 bp and a 3'-UTR of 209 bp was fOund. It deduced aminoacid sequence shows similarlties 59% with F16N14.2 and pre5ents a common DNA bindingmotif with F16M14.2, Ibbf, Ibbfl, Mybltf and MybSt1. The resuIts of protein domain predictionindicate that there may be a N-glycosyIation site, three CAMP- and cGMP-dependent proteinkinase phosphorylation sites, a Protein kinase C phosphorylation site, nine Casein kinase IIphosphoryIation sites, flve N-myristoyIation sites, an Amidation site and an ATP/GTP-bindingsite. The resuIt of PSORT (PrediCtion of Protein LocaIization Sites) suggests that hbf mayencode a nucIear protein.The DNA sequence of Hbf was obtained using PCR. The product was 1277 bp in lengthwith a M4 bp intron, which contain apparently a donor splice site, an acceptor splice site anda branch point.The result5 of Southern anaIysis indicated that Hbf has Iow copies in the H6veabrasHtensiS gename and there was no diff6rence in copies between the heaIthy trees and theTPD trees.The resuIts of Northern analysis indicated that Hbfwas expressed significantIy higher inheaIthy trees than in TPD trees and it was expressed highest in bark (near cambium) andlowest in Ieaf. Moreover, Hbfwas expressed f8r Iower in the Iatex of the tapping line than inthose of f8r from the tapping Iine. Those f8Cts indicated that the leveI of expression Hbf isrelated to TPD of Hevea brasHfen5iS.The 5' Ieader sequence of Hbfwas obtained by using GenomeWaIker. It might contain apromoter region.The gene fragment of Hbf was obtained by PCR method and inserted into pBI121 vectorat the site of Xba I and Sac I to construCt an expression vector.