Studies On Construction And Transformation Of PHA Plant Expression Vectors

To solve the problem asssocited with the effect of plasic pollution on the environment, considerable interests were put into the development and utilization of biodegrable plastics. Polyhydroxyalkanoates(PHAs), as a great polyester, is not only the storage carbon in many species of bacteria but also the raw material of biodegradable plastics. So PHAs rearch focused on producing PHAs by using the plant bioreactor at present.The genes were separated into phaA, phaB and phaC from the subclone of pseudomonas sp. producing PHAs,and then were orientationally inserted into a prokaryotic expression vector pBV220. By analyzing the bioactibity and function of their products, the biological function of phaA, phaB and phaC was confirmed. These have been done in our laboratory. To make better use of the three genes by plant bioreactor, two groups of different plant expression vectors that can be used to produce PHAs in different plants were constructed, and the transformation of PHA in Oxalis triangnlaris has been preliminarily reseached.The following is the main results:Three plant expression vectors pBI121-A,pBI121-B and ubquiting-C, which can be used to be transformed into Oxalis triangularis were respectively constructed by using the phaA, phaB and phaC. Moreover,the specific expression vectors of plant seed p7S-A,pBI121-S-B,and uBquiting-C used in maize trandforming were also constructed. And the results of analysis in restriction anduonclease digest and PCR amplification indicated that the expression vectors constructed is right.By reseaching the tissue culture of Oxalis trisngularis, regeneration system of Oxalis trisngularis was preliminaryly established and the optimum mediums were MS+2,4-D 1.5mg/L+6-BA 0.1 mg/L for inducing callus and MS+NAA 0.9 mg/L +6-BA 0.4 mg/L for inducing differentiation, and MS+2,4-D 0.5mg/L for inducing rooting. The optimum concentration of Kan selecting callus is 58 mg/L.The expression vector of PHA was transformated into Oxalis trisngularis by particle bombardment. 96 antibiotics plants were gotten by resistant selection of Kan, and the PCR test indicated that the rate of postive plants is 1.57%. In the 7 positive plants there is respectively one singular-transferplant of phaA and phaB, and there is one con-transferplant with phaA and phaB, and there is one con-transferplant with phaA and phaC, and there are two con-transferplants with phaB and phaC, and there is one con-transferplant with phaA, phaB and phaC. Then the analyses on the rapid propagation, the stability of heredity and erpression effect are still being reseached.