玉米黑粉菌DNA载体的构建和它的原生质球的转化和再生

THE CONSTRUCTION OF DNA VECTOR AND SPHEROPLAST TRANSFORMATION AND REGENERATION OF USTILAGO MAYDIS

我们用玉米黑粉菌(Ustilago maydis)热休克基因(Heat shock gene,hsp 70)的启动子和终止子与新霉素磷酸转移酶基因相连,构建成了有效的双功能质粒pDL1(在E。coli中用Amp作为选择标记,在玉米黑粉菌中用新霉素作为选择标记)。以分离的一株新霉素敏感的玉米黑粉菌作为受体菌,用构建的pDL1质粒作为供体DNA,对影响受体菌原生质球的形成条件(培养基、菌龄、各种酶、酶的浓度、作用时间和渗透压稳定剂)、再生条件和DNA转化条件进行了初步研究。对数前中期收集的菌体,以5mg/ml真菌溶壁酶处理30分钟左右,90%都形成原生质球,其再生率为60—80%,转化率为300—1000转化子/μg DNA。随机选出25个转化子,DNA杂交都为阳性。分析dot-blot和Southern blot DNA杂交结果,发现质粒在细胞中以整合形式存在,一个细胞可以有多拷贝的整合质粒,质粒可能以非完全同源性的重组形式,参入性地整合进染色体中。

We have constructed a eff ctive bifunction plasmid pDL1,which can be selected in Escherichia coli with ampicillin and in Ustilago maydis with neomycin,by ligating the promoter and terminator of the heat shock gene from U.maydis with neomycin phospho- transferase gene.The study on the conditions of formation,regeneration and DNA transfor- mation (media,growing time,enzymes,concentration of enzyme,digestion time,agents for stable osmotic pressure,etc.) was done using the isolated U.maydis strain sensitive to neomy- cin as the host strain and pDL1 plasmid as the donor DNA.Around 90% spheroplast forma- tion frequencies in the early logarithmic-phase cells in 5mg/ml fungous lysowall enzyme for about 30 minutes,60—80% regeneration frequencies and 300—1000 transformants per mic- rogram of DNA transformation frequencies were obtained.DNA hybridization was positive for 25 randomly selected transformants.The results of dot blot and southern blot hybridiza- tions revealed that he plasmid integrated into the chromosome in one or more than one copies and probably by the way of not perfect homolo ous recombination or integration into the ge- home.