Genes Cloning And Genetic Transformation Of Cellulose Synthase In Populus Ussuriensis Kom

Plant cellulose is the most important renewable resources that is most abundantly present in the biosphere. Therefore, it is almost paradoxical that we still know little about the molecular mechanism of cellulose biosynthesis in plants. Poplar is the model plant for tree molecular biology, so it is very important to study the mechanisms of cellulose biosynthesis in this plant.Two pairs of specific primers, synthesized according to conservative regions of CesA gene, were used to amplify genomic DNA from leaves of Populus ussuriensis Kom. by polymerase-chain reaction. According to the result of sequencing,5760 bp was named as PuCesA6. The full-length sequence has 13 extrons of 3812 bp and 12 introns of 1948 bp. Comparison of the nucleotide sequence of PuCesA6 with PtrcesA6 from Populus tremuloides showed 99% identity. Another PuCesAl fragment of 2341 bp been cloned has 98% identity to PtrCesA7.A full length (PuCesA6) cDNA clone encoding cellulose synthase (CesA) was isolated from Populus ussuriensis Kom. by using RT-PCR method. The full-length cDNA sequence of PuCesA6 is 3778 bp in length, including 66 bp of 5'UTR and 448 bp of 3'UTR. The ORF is 3264 bp encoding a deduced aminoacid sequence of 1087 residues with a moleeular weight of 122.51 kDa and an isoelectric point of 6.57. The protein contains the same domains and regions as angiosperm CesA genes:a zinc finger domain, a hypervariable region I (HVR-I), a hypervariable region II (HVR-Ⅱ), a conserved domains that are characteristic of the family 2 processiveβ-glycosyltransferases. And it is also predicted to contain eight transmembrane domains. The zinc finger domain and the C-terminal portion of the proteins are highly conserved between Populus ussuriensis Kom. and the nearest angiosperm CesA proteins. The result of phylogenetic analysis of comparing the protein encoded by PuCesA6 with 34 other CesAs indicated that it has a close relationship to that of PtrCesA6.The ORF sequence of PuCesA6 gene was recombined with the CaMV35S-sGFP-NOS vector. Recombinant plasmid was introduced into onion epidermal cells by the particle bombardment method with a PDS1000/He. Transformed cells were incubated for 2-4 h at 25℃in the dark and green fluorescence was monitored under a Laser scanning confocal microscope. After plasmolysis, PuCesA6 combined with sGFP was only located in plasma membrane of onion epidermal cell.The ORF sequence of PuCesA6 isolated with Xbal and Kpnl site was recombined with the pROKII vector, and the sense full-length expression vector of recombined PuCesA6 gene was named as pROKII-P6XbaIKpnI. Then, the pROKII-P6XbaIKpnI was transformed into tobacco by the mediated gene transfer of Agrobaeterium tumefaeiens(EHA105). The main process of tobacco transformation was that:determining the types of differentiation culture medium, pre-cultured 2 days, infected 5-10 min, and co-cultured 2-4 days. The prophase selection, delayed selection and anaphase selection were adopted. After the sample co-cultured, the selective pressure in selective culture medium is kanamycin of 100 mg/L, the ablastin of Agrobaeterium tumefaciens is carbenicillin with a concentration of 500 mg/L. The normal developing tobacco seedling on the selective culture medium was sampled to isolate DNA, and conducted PCR amplification by co-detection of NPTII gene and PuCesA6 gene. And then, the detection of Southern-blot was conducted, and the result is accordant with the relative result of PCR. The samples of positive seedings produced stronger hybridization signals, whereas the negative control of wild tobacco had not the hybridization signal. Those transgenic tobacco had " dwarf" phenotype, and the length of fiber cell was significantly shorter in length than that of wild tobacco by using statistic method.The cDNA fragment of PuCesA7 amplified with SacI and Kpnl site was inserted into the expresstion vector pROKII at anti-sense direction, and the recombined vector was named as pROKII-P7SacIKpnI. The pROKII-P7SacIKpnI was transformed into tobacco by the mediated gene transfer of Agrobaeterium tumefaeiens(EHA 105).