来源于Pyrococcus furiosus的耐高温α-淀粉酶基因在衣藻叶绿体中的表达

Expression of the Gene Coding for a Thermostable α-amylase from Pyrococcus furious in Chlamydomonas reinhardtii Chloroplast

来源于Pyrococcusfuriosus的耐高温α-淀粉酶是一种重要的酒精工业用酶,在植物中表达耐高温α-淀粉酶可以大大降低用植物秸秆生产酒精的成本。选择衣藻叶绿体基因组同源片段clpP-trnL-petB-chlL-rpl23-rpl2和壮观霉素抗性基因,构建了来源于Pyrococcusfuriosus的耐高温α-淀粉酶基因的衣藻叶绿体表达载体p64A。通过基因枪将其导入衣藻叶绿体中,经壮观霉素抗性(100mg/L)筛选,获得了9个抗性衣藻转化子。转化子经过抗性继代筛选后,经PCR、Southernblot检测分析及暗培养,证实耐高温α-淀粉酶基因已整合到衣藻叶绿体基因组中并得到表达。酶活性检测表明,转基因衣藻表达产物具有耐高温α-淀粉酶活性,每克鲜重衣藻最高达77.5u。实验结果证明在植物叶绿体中表达工业酶制剂是可行的。

Thermostable α-amylase from Pyrococcusfurious is an important industrial enzyme in brewing and alcohol production. Eexpression of the thermostable α-amylase in plants can reduce greatly costs in the production of alcohol using crop plants. A chloroplast expression vector, p64A, containing the thermostable α-amylase gene from Pyrococcus furious, was constructed with clpP-trnL-petB-chlL-rpl23-rpl2 as Chlamydomonas reinhardtii plastid homologous recombinant fragments and spctinomycinresistant aadA gene as select marker. The plasmid p64A was transferred into the chloroplast genome of C. reinhardtii by the biolistic method. Nine independently transformed lines were obtained by 100mg/L spectinomycin selection. PCR amplification, Southern blot analysis of the transgene and cultivation in the dark all showed that the α-amylase gene had been integrated into chloroplast genome of C. reinhardtii. The activity of amylase expressed in the chloroplast of C. reinhardtii was detected by amylase activity assay and found to be as high as 77.5u/g fresh weight of cells. These experimental results demonstrated the possibility of using transgenic chloroplasts of plant as bioreactors for production of industrial enzymes.