Construction Of GhCEL Gene Expression Vector And Its Transferring Into Tobacco

Cellulose has become the worldwide hotspot in biological research field in the past decades due to its great abundance in the nature and its remarkable commercial value in the world. With the development of genomics, recent studies have highlighted the cellulose synthase complex and the role the genes involved in cellulose biosynthesis pathway in plant. The findings will be the guidance of the production and application of cellulose. Previously, according to an expressed sequence tag (EST) of cotton fiber a full-length cDNA was isolated (which is designated GhCEL, GenBank accession number: AY574906) from a fiber cDNA library of Gossypium hirsutum L. by the PCR screening by our laboratory, which is highly homologous to KOR gene of Arabidopsis thaliana. In this research GhCEL expression vector was constructed and transformed into tobacco. We also analysed the change of contents of cellulose of the transgenic plants.1 .Construction of expression vector: The special primer pair was designed to amplify a full-length of GhCEL gene from cloning vector by the PCR reaction. GhCEL gene was inserted into a cloning vector SK-pBlueScript+, and the resulting recombinant vector was named as SK-pBlueScript+: GhCEL. At the same time CaMV35 promoter digested from pFGE5941 vector was inserted into expressing vector pCAMBIA 1302, and the resulting vector was named as pCAMBIA 1302: CaMV35. GhCEL digested from intermediate vector SK-pBlueScript+: GhCEL was inserted into pCAMBIA 1302: CaMV35, and then GhCEL-GFP fusion gene expressing vector was obtained.2.The tobacco transformation: The tobacco leaf discs which were infected by agrobacterium tumefaciens with GhCEL gene was put into MS cocultured media. After 3 days, they were transferred into the selective media with hygromycin (25mg-L^-1) and cefotaxime (500mg-L^-1). The leaf disc without GhCEL gene were transferred into the selective media as the control. The callus around the leaf disc were transferred to the subculture media. When the bud were more than 3 centimeters,they were cut and transferred into MS rooting media and the young rooted buds were planted into soil.