el-Sheekh M M
Botany Department, Faculty of Science, Tanta University, Tanta 31527, Egypt.
Folia Microbiol (Praha). 2000;45(6):496-504. doi: 10.1007/BF02818717.
The chloroplasts of Chlamydomonas reinhardtii were transformed using a vector (paadAGUS4.1) that contained a spectinomycin-resistance gene (aadA) as a selectable gene, and bacterial uidA (GUS) as a reporter gene, and pea 4.1 kb D-loop containing sequence. The vector was introduced into the alga through particle gun bombardment. The transformed colonies were screened for the presence of foreign genes by Southern hybridization using GUS, aadA and 4.1 pea Ori probes. Expression of aadA and GUS genes was detected in all colonies that were grown on spectinomycin. A detailed restriction analysis followed by southern hybridization of total genomic DNA using pea 4.1 kb D-loop as probe indicated that the D-loop sequence can serve in site-specific integration of foreign DNA due to high homology. Restriction analysis of different colonies showed that the foreign DNA was probably present in a mixture population of autonomous segment and integrated in the native chloroplast genome.
使用一种载体（paadAGUS4.1）对莱茵衣藻的叶绿体进行转化，该载体含有作为选择基因的壮观霉素抗性基因（aadA）、作为报告基因的细菌uidA（GUS）以及包含豌豆4.1 kb D-环的序列。通过粒子枪轰击将该载体导入藻类。使用GUS、aadA和4.1豌豆Ori探针通过Southern杂交筛选转化菌落中是否存在外源基因。在所有在壮观霉素上生长的菌落中均检测到aadA和GUS基因的表达。使用豌豆4.1 kb D-环作为探针进行详细的限制性分析，随后对总基因组DNA进行Southern杂交，结果表明由于高度同源性，D-环序列可用于外源DNA的位点特异性整合。对不同菌落的限制性分析表明，外源DNA可能存在于自主片段的混合群体中，并整合到天然叶绿体基因组中。
