Genomic Cloning And Sequence Analysis Of The Fragments Of Pha Synthesis Gene Isolation

Poly-l3-hydroxyalkanoates(PHAs) are accumulated in various bacteria as intracellular carbon and energy storage material ,these bacterial PHAs are expected to become attractive alternatives for petrochemically based plastics,since they are biodegradable thermoplastics. Now PHAs research focuses on using plant bioreactor to produce PHAs. This dissertation is mainly to analyze the DNA sequence that included PHA synthesis genes by bioinformatics,to find genes correlative to PHA synthesis from this sequence and to amplify and separate them by PCR. The result indicates:1 The full length of the sequence is 9118bp(Accession Number:AB085816 submitted in DDBJ).Five open reading frame were extracted from this sequence by nucleic acid analysis software. BlastP program was used to compare all five ORFs with published sequences in GenBank. It was found that ORF1 ORF2 ORF3 were high identity to that of PHA synthesis genes in GenBank,but ORF4 and ORF3 were little identity to them.2 By multiple sequence alignment of ORF1 ORF2 ORF3,the result indicates they are highly conservative to PHA synthesis genes. ORF1 ORF2 and ORFS are respectively considered to be PHA synthase (PhaC) .,3-ketothiolase(PhaA) and acetoacetyl-CoA reductase (PhaB). ORF1 maybe be a new type PHA synthase because it had much more difference in amino acid sequence than other Pseudomonas PHA synthase.3 Through searching promoter sequence region by promoter prediction software http://www. fruitfly. org/cgi-bin/seq_tools/promoter. pi,the most possible promoter sequence region were found in the sequence. The result showes that PhaC PhaA and PhaB are controlled by the same promoter ,they are in one operon,ORF4 and ORFS iscontrolled by another promoter Pro4.4 To summarize,Predicting PHA synthesis molecular mechanism:Firstly 6- ketothiolase(PhaB) condenses acetyl-CoA molecules to acetoacetyl-CoA,then which is reduced by acetoacetyl-CoA reductase(PhaA) dependent on NADPH to D-(-)-J3-hydroxybutyryl-CoA. Subsequently polymerization to PHA is catalyzed by PHA synthase(PhaC).5 phaC phaA and phaB are specifically amplified by PCR and cloned successfully to pGEM-T vector.6 After plasmid p7S and pBI121 were digested with Hindlll and Sad,7S promoter purification fragment was ligated with pBI121 purification fragment without CaMV35S promoter and recombinants were transformated into JM109. The specific expression vector of plant seed was constructed successfully.