Study On Cloning And Functional Expression Of PHT1Gene In Camellia Oleifera

Oil-tea{Camellia oleifera) is an important woody edible oil tree in south of China. At present there is3.67106ha oil-tea planting area in China and4.5105tons of oil was produced annually, average oil output per ha is only45-90kg. Oil-tea is mainly distributed in South China where acidic soils are predominant, and phosphorus deficiency is the main factor limiting the yield of oil-tea. Phosphorus is the inverse concentration of active transport processes across the plasma membrane of the epidermis and cortex cells, whereas the process is mainly regulated by the gene family of high affinity phosphate transporters in root cell membrane. So the study of PHT1gene has an important significance on explored the molecular mechanism of oil-tea in phosphorus starvation and screened germplasm resources with high phosphorus efficient. This thesis carried out a number of studies in gene cloning of PHT1from oil-tea, the relative expression in different tissues and eukaryotic expression in tobacco. The main results of the research are as follows:1. Molecule Cloning and bioinformatics analysis of PHT1gene from oil-tea. The phosphate transporter named CoPhtl was isolated from oil tea (Camellia oleifera ’Huoshuo’) with the method of RT-PCR and RACE. The open reading frame of CoPhtl is1626bp in length and it codes a predicted protein of542amino acids. The estimated molecular weight and isoelectric point of the putative protein were59.64KD and8.67. The analysis of fat coefficient and total average hydrophilicity show that CoPhtl is a hydrophobic protein. The amino acid identity compared with other phosphate transporters is highly conserved and shows the highest similarity (88%) with the CoPhtl of Catharanthus roseus. Putative secondary structure and topology of the encoding protein have the main feature of transmembrane protein, and is consistent with PHT1encoding protein in other species. As the template, genomic DNA was extracting from oil-tea leaves. Cloning CoPhtl genome sequence used PCR reaction. The sequencing result showed that CoPhtl has no introns.2. The relative expression of PHT1Gene from C.oleifera in different organs under different phosphorus levels. The use of qRT-PCR standard curve to optimize system, the relative expression of CoPhtl in different organs under different phosphorus levels was detected. In0.1mmol/L P level, the relative expression of CoPhtl in the tender roots was the largest and the following is the root, stem, new leaf, old leaf. When P levels was in the1mmol/L, the relative expression of CoPhtl was root> tender root> stem> leaf> new leaf. The study also found that, in the phosphorus level was0.1mmol/L, the relative expression of Camellia tender roots in PHT1was significantly higher than the levels of phosphorus was1mmol/L, indicated that low phosphorus may induce PHT1, which is consistent with studies in other species.3. The expression of PHT1from C.oleifera in tobacco. The plant expression vector pCAMBIAl304-35s-CoPhtl was constructed successfully by DNA recombinant technique, which was transformed into Agrobacterium tumefaciens GV3101. Transgenic tobacco plants recovered from leaf discs applying Agrobacterium mediated gene were selected on solid medium containing hygromycin. PCR amplification and GUS staining were the common method for identification of transgenic plants. Total DNA of transgenic plants as a template, with the non transgenic plantlets as negative control group, plasmid DNA as positive control group, primers were designed by GUS sequence in expression vector for PCR amplification. The results showed:extended strip in transgenic plants was consistent with target strip in plasmid, and the untransformed plants did not amplify any bands. The roots of the transgenic and untransformed plant as the samples were stained with GUS. The results show that:the roots of transgenic plants by GUS stained into blue, and non infected root color was white.