Cloning And Functional Analysis Of Key Genes Involved In Chlorophyll Biosynthetic Pathway In Pseudosasa Japonica Cv.Akebobosuji

Plant photosynthesis is determined by chlorophyll (Chl) metabolism, which is an importantfactor of determining crop yield. The genes involved in Chl biosynthesis are numerous, and themutation of any of them may change the pigment level, causing abnormities in leaf color and eveninducing individual death. Pseudosasa japonica cv. Akebonosuji could present diverse leaf colorsuch as green and white stripes (the shade, shape, quantity, and location of which however aredifferent within different leaves), green and white. In our previous study, this ablusivephysiological phenomenon was speculated to be caused by the deviance of the biochemicalreactions catalyzed by NADPH: protochlorophyllide oxidoreductase (PORB) and chlorophyllide aoxygenase (CAO). Therefore, isolation and functional analysis of PORB-and CAO-encodinggenes were performed in this study. The major results were as follows:1. The full length cDNA (FL-cDNA) of PjPORB and PjCAO were isolated through rapidamplification of cDNA ends (RACE). The FL-cDNA of PjPORB were1567bp long with a1185bp open reading frame (ORF) encoding394amino acids while the FL-cDNA of PjCAOwere2070bp long with a1626bp ORF encoding541amino acids. The putative PjPORB proteinhad a cofactor binding motif and an active site motif, and the putative PjCAO containedfour domains: N-terminal region, A domain, B domain, and C domain containing a Reiske clusterand an iron-binding motif.2. In order to study the temporal and spatial expression of PjPORB and PjCAO in P. japonicacv. Akebonosuji, roots, stems and leaf buds including green-white striped, green, and white leafbuds (consisting of three overlapping immature leaves in different stages of leaf development)were collected to conduct real-time quantitative RT-PCR. Real-time quantitative RT-PCR analysisshowed that both PjPORB and PjCAO were ubiquitously expressed in root, stem and leaf, anddifferentially expressed in different stages of leaf development.3. Through adding restriction enzyme sites to PjPORB and PjCAO and then ligating to themultiple clone sites of pCAMBIA1301, the plant expression vectors pC1301-PjPORB andpC1301-PjCAO were constructed and transformed to Arabidopsis thaliana with anAgrobacterium-mediated floral-dip method. Transgenic lines were confirmed through GUSstaining, PCR and RT-PCR analysis. Through detecting chl content, it was showed that PjPORB-and PjCAO-overexpressing Arabidopsis had a higher level of chl than did those wild type ones.We thereby postulated that both PjPORB and PjCAO may play an important role in Chlbiosynthetic pathway of Arabidopsis, ultimately shedding a light on understanding of Chlsynthesis and photosynthesis of bamboo species.