摘要
利用聚合酶链式反应 (PCR)技术 ,从类产碱假单胞菌YS1(PseudomonaspsuedoalcaligeneseYS1)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 :phaC1、phaC2基因。同时利用套叠PCR技术对 phaC1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phaC1)、pC3C2 (嵌合 phaC2 )和 pC3C1C2 (嵌合 phaC1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 (AgrobacteriumtumefaciensEHA10 5 )并且通过农杆菌介导的叶盘法转化烟草 (NicotianatobacumHonghuadajinyuan)。 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 phaC1和 phaC2。用氯仿 -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 2 5 6
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.
出处
《分子植物育种》
CAS
CSCD
2003年第1期58-65,共8页
Molecular Plant Breeding
基金
国家自然科学基金(30060005)
教育部优秀骨干教师项目资助。
