Studies On Improving The Genetic Manipulation System For A.meditteranei U32

This paper focuses on further development and improvement of the genetic manipulation system for A.meditteranei U32.First, to enrich the selective markers for U32, two resistance genes emr and cat, one report gene amy were introduced into U32 to test their expression. The results indicate that emr and amy can be selective markers for U32.Then with promoter-less amy, promoter-probe pZCamyP" was constructed and fusion expression of glnA promoter with promoter-less amy was achieved in U32. This result indicates that pZCamyP" is able to detect the promoter of U32 genes.At the same time, pULVK2.5GE carrying U32 glnA was constructed. Transformant of P32(glnA::Amr,Gln") with this plasmid restored the ability to grow on minimal medium. This result indicates that U32 glnA encodes GS.We also attempted to disrupt and delete U32 recA by homologous recombination but failed. This result indicates that U32 recA is critical to its survival. We found that U32 recA can implement JM109(DE3)(recA~).This result indicates that U32 recA can repair the damage by UV to some extent.Experiments show that pRLAm and pULVK2A have conspicuous differences in copy number and lost frequency. Sequence analysis indicates that the fragment which pULVK2A lost maybe encode two par gene par A and parB.