枯草芽胞杆菌基因组混组方法

Genome shuffling method of Bacillus subtilis

摘要:

基因组混组作为一种育种方法，通过循环原生质体融合等手段，使得不同菌株来源的基因组能够得到充分重组，增加将正向突变整合到同一重组子中的机会。使用4株带有4种不同标记的枯草芽胞杆菌亲本为初始菌株，通过循环转化、循环转导或循环原生质体融合的手段进行基因组混组，统计后代中非亲本类型占整个群体的比例，以衡量基因组混组的效果。分别经过5轮循环原生质体融合、循环转化或者循环转导，结果显示，重组程度较高者在后代群体中的比例较低，带有4种标记的后代未出现，带有3种标记的后代最高分别为4.53×10?4、1.64×10?4、4.47×10?3，明显低于文献报道的天蓝色链霉菌中同样实验的结果：带4种和3种标记的后代分别占2.5％、17％。对比上述实验的结果和文献报道的天蓝色链霉菌、乳杆菌基因组混组的结果，并结合计算机模拟循环融合过程，分析后认为:要达到较充分的基因组混组，需要有能够实现微生物细胞间高频重组的操作技术作为基础，重组频率应该不低于10?3~10?2数量级。

关键词:基因组混组，枯草芽胞杆菌，分子育种，原生质体融合，转化，转导，计算机模拟

Abstract:

Genome shuffling methods were explored for Bacillus subtilis strain molecular breeding. Recycling protoplast fusion, recycling transformation and recycling universal transduction were used for genome shuffling in B. subtilis. Four strains with different nutrition-deficiency markers were used as initial strains. After five rounds protoplast fusion, transformation or transduction, the descendant with 4 markers had not been detected, and the rate of descendant with 3 markers were 4.53×10?4, 1.64×10?4, 4.47×10?3, respectively. A computer program was made to simulate the recycling fusion process. Based on simulation result and comparing the genome shuffling result of B. subtilis in this experiment and that of Streptomyces coelicolor reported in references, effective genome shuffling needs a high recombination rate of at least between 10?3 and 10?2.

Key words:genome shuffling, Bacillus subtilis, molecular breeding, protoplast fusion, transformation, transduction, in silico simulation