Screening Of P.pseudoalcaligenese YS1 BAC Library And Genetic Stability Analysis Of Transgenic Tobacco

Polyhydroxyalkanoates (PHAs), a family of intracellular polyesters synthesized by many bacteria, have a lot of promising applications in agriculture, medicine, macromolecule material, tissue engineering, environmental protection and packaging, due to their excellent biodegradability and biocompatibility. In the PHAs biosynthesis pathway, PHA synthases are found to be the key enzymes for the determination of most of PHA properties, such as monomer compositions and molecular weights. Until now, more than 59 PHA synthases have been cloned from about 45 different bacterial strains.Strain Pseudomonas. Pseudoalkaligenese YS1 was capable of producing PHAs containing monomer of HB and mcl HAs in certain medium. With specificly designed primers according to polyhydroxybutyrate( PHB, one type of PHAs) synthase and PCR amplification, a probe for screening of the BAC liberary was obtained from chromosomal DNA of P. pseudoalkaligenese YS1. The fragment was sequenced and closely related to a part of PHB synthase gene (phbCj) of Alcaligenes eutrophus with 99% similarity. Probes obtained by PCR and a 24bp Oligo DNA fragment from Pseudomonas sp. 61-3, phbC sequence were hybridized with P. pseudoalkaligenese YS1 BAC library based on Dot blotting and Southern blotting. A positive transformant named as pH22-E5 whose foreign fragment is about 5kb was subcloned.Another part of this thesis is to test the genetic stability of the PHA gene tansgenic plant. Two transgenic tobacco plants and their progenies which were introduced two key polyhydroxyalkanoates polymerase genes phaC1 , phaC2 were detected. The results showed that the PHA synthase gene can inherit stably in transgenic tobacco plants. Positive transformants were detected by Kan resistant screening in field and Southern blotting. Moreover, extracting and purification of PHAs directly from 74 positive transformants by chloroform-hypochloric acid showed 61 transformants produced the PHAs. The ratio of positive transformed tobacco and their progenies is 67.7%.