Pseudomonas Ys1 Short Chain And Medium-chain Pha Synthase Phac1, Phac2 Bivalent Genes Into Tobacco

Polyhydroxyalkanoates (PHAs) are intracellular polymers in bacteria. It can be classified into three categories, such as short chain length (scl) PHAs, medium chain length (mcl) PHAs, and PHAs containing monomer of HB and HAs (hydroxyalkanoates). PHAs, as one of the most interesting biodegradable material have a lot of promising applications in medicine, agriculture, environment and food industry.Strain Pseudomonas psuedoalcaligenese YS1 was capable of producing PHAs containing monomer of HB and mcl HAs in certain medium. phaCl and phaC2, two key polyhydroxyalkanoates polymerase genes of PHA biosynthesis were amplified and cloned from chromosomal DNA of Pseudomonas psuedoalcaligenese YS1 using PCR. At the same time, phaCl gene was modified by PCR. Three plant expression vectors containing Kanamycin photransferase gene: pC3Cl (containing phaCl), pC3Cl (containing phaC2), pC3C!C2 (containing phaCl and phaCT) were constructed. The high performance two-gene expression vector pC3C!C2 was transformed to Nicotiana tobacum Honghuadajinyuan plants mediated by Agrobacterium tumefaciens EH A105 according to the leaf disc procedure. Transformed shoots were selected on solidified medium containing l00mg/L Kanamycin. A number of Kanamycin resistant transformants were obtained two months later. They were morphologically normal, but grew slower comparing with the control tobacco. One hundred resistant transformants were checked by PCR and PCR-Southern, and 32% transformants were confirmed to be stably integrated phaCl and phaC2 genes. Positive transformants were also detected by Kan resistant re-screening on solid medium or directly detection in field and Southern blot. Moreover, extracting and purification of PHAs directly from 32 positivetransformants by chloroform-hypochloric acid showed that 25 transformants obtained the product. Band of C=O group around 1724 cm"'was suggested for the characteristic band of PHA were observed on the FT-IR spectra of both of the fresh tobacco leaves and their extractions. Twenty-five percent of transformed tobacco plants were detected to produce PHAs. Qualitative and quantitative detection of plants PHAs using gas chromatography (GC) indicated that HD were the major composition of PHAs and the maximum PHA accumulation was 22 mg/g dry weight.