Studies On The Prodution Of PHB,A Biodegradable Plastic In Transgenic Plants--Isolation And Functional Analysis Of Seed Specific Promoter NapinB Promoter,and Obtainment Of Transgenic Brassica Napus

As a carbon reserve, Polyhydroxyalkanoates (PHAs) are produced in many species of bacteria when grown in an unbalanced nutrient condition. PHAs, with its water-resistance, good physical and piezoelectric properties, have the properties to rubber like materials, making them suitable polymers for a wide variety of uses. Especially, with the distinct advantage of being biodegradable and biocompatibility, PHAs are ideal alternatives to petrochemically derived plastics, which caused serious environmental pollution. Poly- P -hydroxybutyrate(PHB) is the best understood species of PHAs. The three enzymes involved in PHIB synthesis, phbA (encodes the first key enzyme of PHIB synthesis, 3- ketothiolase), phbB and phbC have been amplified and cloned from chromosomal DNA of Ralstonia eutrophus by PCR amplification. An effective way to reduce the harm to plant caused by accumulated polyester is to confine PHB in plastid of seed by seed-specific promoter and ctp gene (encoding chloroplastid transit peptide, CT?). 7s promoter and ctp gene were isolated and seed-specific expression vectors pSCB containing phbC and phbB and pSCAB containing phbC, phbB and phbA were constructed. cq gene led the enzymes involved in the PHB synthesis into plastids. Transgenic rapeseeds, which were confirmed by PCR and Southern analysis, were obtained. RT-PCR results showed that each gene has been expressed at the transcription level. In order to avoid the possibility of gene silence caused by high homology of promoter, it is necessary to isolate a new promoter to improve the constructed expression vectors. One of the napin genes, napinB, was isolated from Brassica napus (rape) by Mats et al. Radke et al have shown that 300 bp of the 5?flanking sequence in front of the translation initiation codon are sufficient to give a developmentally faithfhl expression of a chimeric gene construct in transgenic Brassica napus plants. Moreover, most of the important elements necessary for the expression of napin gene are contained in this region. A fragment of 286bp (nap3 00) in napinB promter region was isolated by PCR from genomic DNA of Brassica napus H 165. Nap300 and the corresponding sequence of napinB Ill promoter shared 97% homology at the nucleotide level, except for eight different bases. One in the TACACAT conserved motif (allowing one base mismatch) changed from A to T, which is more favorite in Brassica napus, two in B-box and the other five in non- consensus region which may be due to differences among varieties of Brassica napus and designed enzyme site. Two expression vectors harboring the nap300 promoter fused with GUS gene and phbA were constructed for functional analysis, respectively. Tobacco plants were transformed by Agrobacterium tumefaciens-mediated method. PCR and Southern results showed that nap300 had been integrated into genomic DNA of tobacco successfully. The assays of GUS and 3-ketothiokase activity in the seeds of transgenic tobaccos are still being carried out to identify the function of the promoter for the long growth period needed for tobacco plants to bear fruits. In our further work, the promoter would take the place of 7s promoter used repeatedly in the expression vectors pSCB and pCAB. We hopefully think that the transgenic plants with the improved expression vectors could produce high content of P1-lB and also provide scientific basis for the commercialization of PHB in soon early time. Transformatio...