Using Genetically Engineered Phb Gene Cloning And Tobacco Transformation

In response to prob1m associated with plastic waste and its effect on the environment, there has been considerable interest in the development and production of biodegradable plastics. Polyhydroxyalkanoates (PHAs) are polyesters that accumulate as a storage carbon in many species of bacteria and are biodegradable thermoplastics , polyhydroxybutyrate(PHB) is one kind of P1-lAs. With the development of molecular biology, people find out another way of using transgenic plants to produce P1-LB except for the traditional way of using the fermentation .To produce P1413 by genetic engineering in plant , genes from the bacterium Alcaligenes euirophus that encoding the three enzyme f3 -thiolase (PHBa), NADPH-depcndent acetyl-CoA reductase(P1-IBb) and poiy- 13 - hydroxybutyrte synthase (PHBc) required to convert acetoacetyl-coenzyme A to produce P1-lB are cloned .In order to target these enzymes into the chloplast,the genes are modified by addition of DNA fragments encoding a chloroplst transit peptide from the small ubunit of rubisco (TPSS) In this experiment, phbC gene primers and the chloroplast transit peptide gene primers are designed. Using PCR-technique, phb C gene is amplified from Alcaligenes eutrophus genome DNA, TPSS gene is amplified from Pissum satium eDNA The two genes are ligated at Xho I sites which are designed in primers.The modified TPSS-phbC is sequenced and cloned into pPOK2 which is a kind of plant expression vectors PhbA and phbl3 genes are cloned by Xiuling Ma. In this experiment, TPSS- phbA, TPSS-phbB are constructed into one pPQK2 vector. After direct trans feri ng pPOK2/TPS S-phbA, pPOK2/TPSS-phbB, pPOK2/TPS S-phbC, pPOK2/TPSS-phbA-TPSS-phbl3 ,pROK2 into Agrobacteriuin iunefaciens LBA4404 , then tobdcoo is transformed . PCR analysis shows the genes have been integrated into the genomc oftobacco. The analysis of southern bloting has get some results.