糖多孢红霉菌同源片段长度与染色体重组率关系的研究

为了探索同源片段长度与糖多孢红霉菌染色体同源重组率的关系，化学合成或用重叠PCR合成带有突变位点、在突变位点两侧长度为（26bp+27bp）、（500bp+576bp）和（1908bp+1749bp）的同源序列，克隆于糖多孢红霉菌同源重组载体pWHM3后，分别构建了pWHM1113、 pWHM1116和 pWHM1119质粒。以PEG介导转化糖多孢红霉菌A226原生质体，3个质粒分别获得每皿30个、69个和170个转化子，但pWHM1113质粒不能与染色体有效整合，pWHM1116质粒与染色体整合率为转化子的2%，而pWHM1119质粒与染色体整合率达到转化子的19%。 pWHM1116和 pWHM1119质粒均可进行有效的染色体二次重组，将突变位位点引入染色体。因此，同源片段长度为（500bp+576bp）或更长时，可与糖多孢红霉菌染色体进行有效的单重组和双重组。

Study on Relationship Between Length of Homologous Sequences and Chromosomic Recombination Rate in Saccharopolyspora erythraea

In order to study the relationship between lengths of homologous fragments and chromsomic recombination rate in Saccharopolyspora erythraea, three homologous sequences, with mutant loci and different flanking sequences, (26bp+27bp),(500bp+576bp)and(1908bp+1749bp), were synthesized by chemical reaction or PCR amplification, and cloned into pWHM3 to construct homologous recombination plasmids, pWHM1113, pWHM1116 and pWHM1119 When the plasmids were transformed into protoplast of Saccharopolyspora erythraea A226 under PEG mediated, on an average 30, 69 and 170 transformants grew on each plate for the three plasmids respectively, but chromosomic integration frequency were 0, 2% and 19% among corresponding transformants. Both pWHM1116 and pWHM1119 could take double crossover recombination, and exchange the mutant loci in the chromosome. It was concluded that when the flanking sequences were equal or more than (500bp+576bp), they could take effective single and double recombination with Saccharopolyspora erythraea chromosome.