Study On Pigment Composition, Structure Of Variegated Leaf And Related Genes From Four Ornamental Bamboos

The ornamental value of plant increased significantly with marking stripes on leaves.White or yellow bands on green leaves made foliage bamboo widely used in landscaping. Inorder to explore the mechanism on the formation of variegated bamboo leaves, the analysis onmorphology, structure, pigment constitution of leaves from four bamboos species(Hibanobambusa tranquillans f. shiroshima H.Okaura, Sasaell glabra f. albo-striata Muroi,Sasa auricoma E. G. Camu and Pleioblaseus kongosanensis f. aureo-striatus Muroi et Yuk.)was carried out, then a related gene encoding for the key enzyme of bamboo chlorophyllbiosynthesis was isolated and its function was identified in vitro, which was supposed toprovide basic knowledge for the mechanism on leaf color formation and breeding ofornamental bamboo. The main results are as follows:First, main pigments analysis. The content of chlorophyll and carotenoid showed it wasmuch higher in green sector than that in white sector of all the four bamboos’ leaves whichindicated that leaf variegation of four bamboos was due to the repression of photosyntheticpigment biosynthesis in leaves. Reversed phase high performance liquid chromatography(RP-HPLC) analysis of flavonoids in leaves showed that the retention time was agreed withpeaks of flavonoids in both green and white sectors, which indicated that the constitutes offlavonoids in leaves had little influence on the coloration of bamboo leaves.Second, morphology and structure analysis. The structure of chimeric leaves from fourornamental bamboos were observed under light-microscopy and electron-microscopy throughparaffin sections and ultra-thin sections respectively. The epidermis structure illustrated thatthere was no distinction between the green band and white band of H. tranquillans f.shiroshima and S. glabra f. albo-striata. However, different variation levels of chloroplast inmesophyll cells were found in both of the two bamboos, in which part of grana lamella weredissolved and amount of osmiophilic granules were formed. Otherwise, there was no visible distinction between green and yellow bands from S. auricoma and P. kongosanensisf.aureo-striatus. The results implied that structure variation of chloroplast resulted in leafvariegation in H. tranquillans f. shiroshima and S. glabra f. albo-striata but it was not the mainfactor for variegated leaves in S. auricoma and P. kongosanensis f.aureo-striatus.Third, POR gene isolation. The open reading frames (ORFs) encodingprotochlorophyllide oxidoreducatase were isolated from H. tranquillans f. shiroshima, S.glabra f. albo-striata, S. auricoma and P. kongosanensis f.aureo-striatus using RT-PCRmethod and named as HtPOR, SgPOR, SaPOR and PkPOR, respectively. Four ORFs allencode for394amino acids. The putative proptein consist of transit peptide (58residues at theN-terminal end) and mature protein (336amino acids). Homology analysis showed that themature proteins of bamboo were highly homologous to each other. Using the same primers,genomic sequences were obtained and sequenced respectively. Sequence analysis showed thatthe genomic sequences of POR from the four bamboos all contained three introns and fourexons.Fourth, HtPOR expression and enzymatic activity assay in vitro. The sequence encodingthe mature protein of HtPOR was subcloned into pMAL-P5X and transformed into Escherichiacoli ER2523cells. Fine soluble recombinant protein was induced by IPTG and the fusionprotein (1mg·mL-1) was purified with amylose resin column. The enzyme reaction wasoptimized and performed with pH7.2at room temperature under light. Spectra were recordedusing a UV-visable spectrophotometer. The result indicated that the fusion protein of HtPORexhibited enzymatic activity which could convert protochlorophyllide to chlorophyllide invitro.Fifth, Western blotting analysis. The recombinant protein was purified and used toimmunize rabbit to obtain anti-MBP-HtPOR antiserum as probe. The total protein was sampledfrom green band and white band of leaves of H. tranquillans f. shiroshima respectively. Theanti-MBP-HtPOR was used as probe. Western blotting analysis showed that HtPOR wasdetected in green band of leaves from H. tranquillans f. shiroshima, but not in white band, which agreed with that deficiency of grana lamella would suppress the chlorophyllbiosynthesis in white band of H. tranquillans f. shiroshima leaves.