Cloning And Expression Analysis Of Histone H3.3Gene, PpH3.3, From Peach

As an important biological phenomena, the formation and release of bud dormancy iscritical for perennial woody plants. Researches on the mechanism of bud dormancy have beencarried out in several species, referring to numerous fields including physiology, cytology,molecular biology and so on. The role of chromatin remodeling in dormancy process isattractive. As a key component of chromatin, Histone H3is one of the four histones, alongwith H2A, H2B and H4, which forms the eukaryotic nucleosome octomer core, and plays animportant role in maintaining structure and function of chromosome. The reseach had beencarried out in horticultural experimental station and laboratory of protected pomology ofShandong Agricutural University from2010to2013with ‘Shuguang’ peach (Prunus persicavar. nectariana cv. Shuguang) as the experimental material, and a series of studies have beenconducted on the isolation, sequence and expression analysis, function identification of a H3variant gene. The main results were as follows:(1) According to the suppression subtractive hybridization library (SSH) created by ourlab, a novel histone H3variant gene was isolated from dormant peach floral buds with rapidamplification of cDNA ends (RACE), namely PpH3.3, and the genebank accession wasKC907628. The full-length cDNA of PpH3.3is821bp, and encodes a136amino acid proteinthat contains all the plant H3conserved function domains, N-terminal tail and H3fold domain.Amino acid sequence alignment revealed that PpH3.3shared high identity with H3inArabidopsis and Vitis vinifera. Four introns were found in the region of genomic sequence,and more interestingly, one intron was located between the5′UTR and the first exon.(2) One1547bp promoter sequence was obtained using hiTAIL-PCR. Sequence analysisshowed that there were some light responsive elements in the promoter region, such as3-AF1binding site, Box4, C-box, G-box, CATT motif, GT1motif, GTGGC motif, TCT motif andSP1. GUS staining showed that PpH3.3expressed high in root、leaf、flower and fruit podsof Arabidopsis. (3) Real-time PCR provided a relative quantitative estimation of PpH3.3expressionduring natural dormancy process. Expression level of PpH3.3was high in dormancy periodand low in dormancy induction period and dormancy release period, respectively. Comparedto hydrogen cyanamide (HC), heat shock (HS) led to remarkably higher transcription,however both HS and HC led to similar expression patterns.(4) A sense expression vector pROKII-PpH3.3was constructed, and transferred into thepoplar84K. PCR identification showed that PpH3.3had been expressed successfully in thepoplar84K.