phaC1、phaC2基因双价植物表达载体的构建及其转基因烟草的获得

利用聚合酶链式反应(PCR)技术，从类产碱假单胞菌YSl(Pseudomonas psuedoalcaligenese YSl)染色体DNA中扩增并克隆了调控短链与中链PHA生物合成的两个关键酶基因：phaCl、phaC2基因。同时利用套叠PCR技术对phaCl基因进行了改造，经过基因拼接构建了植物表达载体pC3C1(嵌合phaCl)、pC3C2(嵌合phaC2)和pC3C1C2(嵌合phaCl和phaC2双基因)。将构建好的嵌合phaC1和phaC2双基因的植物高效表达载体pC3C1C2，用冻融法转入根癌土壤杆菌(Agrobacterium tumefaciens EHAl05)并且通过农杆菌介导的叶盘法转化烟草(Nicotiana tobacum Honghuadajinyuan)。2个月后获得了一批卡那霉素抗性烟草植株，抗性植株大田生长表型正常，生长速度相对缓慢。抗性植株经过PCR、PCR-Southern、Southern检测初步确定有32％烟草稳定整合了phaCl和phaC2。用氯仿一次氯酸钠直接从部分Southern检测阳性转化植株中抽提纯化得到PHA，在产物合成水平确证有25．6％的转基因植株。

Construction of Two Gene Plant Expression Vector Harboring phaC1、phaC2 Genes and Obtainment of Transgenic Tobacco

phaC1 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, phaC1 gene was modified using PCR. Three plant expression vectors: pC3C1 (containing phaC1), pC3C1 (containing phaC2), pC3C1C2 (containing phaC1 and phaC2) were constructed. The high performance two gene expression vector pC3C1C2 was transformed to Nicotiana tobacum Honghuadajinyuan plants mediated by Agrobacterium tumefaciens EHA105. A number of Kanamycin resistant transformants were obtained two months later. They were morphologically normal, but grew slower comparing with the control tobacco. 32% transformants were stable integration of phaC1 and phaC2 confirmed by PCR and PCR Southern. Moreover, extracting and purifying PHAs directly from positive transformants by chloroform hypochloric acid showed that 25 6%of transformed tobacco plants could produce PHAs.