Cloning And Characterization Of Cellulose Synthase From Poplar (Populus Tomentosa Carr.)

Poplar is the model plant for tree genetics and breeding, and tree molecular biology. Cellulose microfibrils provide strength and flexibility to plant tissues and are also of great importance to wood, paper, textile, and chemical industries. So it is very important to study the mechanisms of cellulose biosynthesis in plant. In this research, the full length of PtoCesA1, fragments of PtoCesA2 and PtoCesA3 were obtained from Chinese white poplar by RT-PCR. And their functions were analysised by transgenic approach. The main results are as follows: 1) A full-length of cellulose synthase gene, PtoCesA1, was cloned from cDNA prepared from secondary xylem zone of Chinese white poplar (Populus tomentosa). Sequencing analysis showed the length of PtoCesA1 is 3,215bp, with ORF from nucleotides 52 to 2,985, and shared 98% identity with PtrCesA1 from Populus tremuloides 2) Four different fragments of PtoCesA1, amplified with specific primers, were inserted into the binary expresstion vector pBI121 at anti-sense direction, and the resulting vectors were named as pBIPF, pBICC1, pBIPR, pBIBR. The four vectors were transformed into tobacco and poplar via Agrobacterium tumefaciens systems, and transgenic poplar plantlet and mature tobacco plants T1 were obtained. Among them, the transformed tobaccos with pBIPF showed identical phenotype with wild tobacco. While transformed tobaccos with pBICC1, pBIPR or pBIBR showed obvious changes with wild tobacco. They had small leaves, "dwarf"phenotype, with less thickness of secondary xylem and fiber cell wall, and less cellulose content. It indicated that the growth of transgenic tobaccos was restrained because of the antisense expresstion of pBICC1, pBIPR or pBIBR, and suggested that PtoCesA1 might involved in cellulose biosynthesis of secondary cell wall. No significant differences were observed among the transgenic tobaccos with pBICC1, pBIPRor pBIBR. It showed that the fragment length may not affect the inhibit effect of antisense transformation of PtoCesA1. 3) The pBIPA1vector, a sense full-length expresstion vector with PtoCesA1, was constructed by introduction BamH I site in PtoCesA1 with synonymous mutation. This vector was transformed into tobacco and poplar by Agrobacterium tumefaciens systems, and transgenic tobacco plants and poplar plantlet were obtained. Those transgenic tobacco had small leaves, "dwarf"phenotype, with a loss of thickness of xylem and fiber cell wall. In addition, no significant differences were observed between the phenotypes of transgenic tobaccos with pBIPA1 and tobaccos with anti-PtoCesA1. 4) The two DNA fragments , PA2(PtoCesA2)and PA3(PtoCesA3), were cloned from cDNA prepared from secondary xylem of poplar. Sequencing results indicated the length of PA2 was 815bp with 98% identity with ptrCesA2 from Populus tremuloides, and PA3 was 381bp with 99% identity to ptrCesA3. The expression vectors were named pBIPA2 and pBIPA3 that the two fragments were inserted into pBI121 at antisense direction. Futhermore, the tandem of PA2 and PA3 was cloned into pBI121 at antisense direction, resulting vectors was named pBIPA2+PA3. The transformed poplar plantlet and mature tobaccos of the three vectors were obtained with Agrobacterium tumefaciens systems seprately. All of those tansgenic tobaccos showed the same phenotypes including small leaves, "dwarf"phenotype, and decrease of thickness of xylem and fiber cell wall. It showed that the growth of tansgenic tobaccos, especially the development of secondary xylem, was restrained because of the antisense expression of the three fragments respectively. 5) The analysis results showed that they had no distinct difference among the phenotypes of transformed tobaccos with the anti-PtoCesA1, anti-PtoCesA2 or anti-PtoCesA3 respectivly. It indicated that the three genes involved in the same rosette. Our research results supplied the important evidence to explain the interactional machinism of the CesA1, CesA2 and CesA3 in poplar. Our research also gives a new pathway to study the cellulose biosynthesis machinism in forest.