Three-piece-ligation PCR and application in disruption of chlorophyll synthesis genes in Synechocystis sp PCC 6803

Linear DNA, consisting of a drug-resistance marker and long flanking sequences, was synthesized by one-step polymerase chain reaction after a three-piece ligating reaction. Chlorophyll synthesis genes, chlH and chIL in Synechocystis sp. PCC 6803, were replaced by a kanamycin-resistance marker through double recombinations with flanking homology regions. Under LAHG conditions, the chIL but not chlH mutant stopped chlorophyll synthesis, while both synthesized chlorophyll in the light.

(1) Barnase、Barstar基因和TA29基因5'区调控顺序的PCR扩增、克隆和测序（2）水稻花粉核蛋白因子与TA29基因5'区调控顺序的相互作用

本文运用PCR技术，分别从烟草和B. amyloliquefaciens的基因组DNA中扩增出了TA29基因5'区调控顺序、Barnase和Barstar基因，克隆后进行序列分析，表明其核苷酸顺序与文献报道的一致，然后进行了初步的融合基因的构建。另外，凝胶滞后实验表明，水稻花粉（含绒毡层组织）细胞核中某种蛋白因子能够与TA29基因5'区调控顺序发生特异的结合。本文还对TA29基因5'区调控顺序及Barnase和Barstar基因的潜在应用价值和存在的问题进行了讨论。