Study On Mutating Genes Of Synthesizing PHA By Ion Beams And Molecular Mechanism Of High Yield Mutation Strains

Poly-B-hydroxyalkanoates (PHA), as a new type of macromolecular heat-plastic stuff, are accumulated in various bacteria as intracellular carbon and energy storage material and biodegradable, they are widely used in the area of industry, ariculture, medicine,food and environment. Low energy ions implantation were used to mutate the original strain pCB4 to improve the accumulation capability of PHA in this experiment and the parameters of implantation, breeding of PHA mutant and genetic stability were also studied. PCR technology and methods of bioinformatics were adopted to analysis the molecular mechanism of high-yield PHA strain pCBH4, the following main results were:1.The original strain pCB4 showed a "saddle type" dose- survival curve after dealt with low energy ions,. The best implantation energy and dose were determined to be 10ke V and 7.8×10₁₄N⁺/cm².2.A high-yield PHA strain named pCBH4 was bred from mutant strains after few rounds of screeing. and the results shown that the mutant strain had high genetic stability of synthesizing PHA after generations experiment. It comfirmed that high yield of PHA because of PHA relation genes' mutation by transforming plasmid extracted from pCBH4 containing synthesizing PHA genes to DH5a.3.The PHA in the sample was also analyzed by gas chromatography. The results indicated that the products of PHA were mostly PHB (41.33% in PHA), which was increased by 48.78% than of the comparison strain, while with few other components. The results demonstrated that they could mainly help to catalyse synthesizing PHB.4.phaA,phaB and phaC were amplified from pCBH4 using primers designed according to the original sequence and sequences analysis were investigated. It was found that there were 7 base sites and 5 amino acid sequences at the terminal of phaA changed and the same as phaC. Transversion was more than transition in the base mutation.The frequence of transversion was 78.57%,but that of transition was 21.43%.Bases and amino acids mutation of phaA and phaC caused variation of DNA and protein with secondry structure and patial properties. The change of base sequences in phaB were not observed.5.The experiment also studied the fermentation conditions of pCBH4 for accumulation of PHA.Higher biomass,PHA concentration and PHA content were achieved when pCBH4 cultured in stationary cultures for 36h after shaking for 24h with the initial pH(7.0),seed volume(8%)and nitrogen sources(peptone). The biomass, concentration of PHA, and PHA in biomass were finally determined to be 2.26g/L,1.81g/L and 80.08%, which were 82.26%, 196.72%, 62.80% higher than the control, respectively.