摘要
从动物肠道优势需氧菌中筛选适于原生质体制备和再生的菌株,确定其原生质体制备与再生的最佳条件,为今后作为与厌氧菌原生质体融合的亲本奠定基础。研究发现,供试20株不同动物肠道优势需氧菌中,只有来自ICR小鼠肠道的M3,M4,M5和M6以及来自猪肠道的Z5对溶菌酶较为敏感。进一步研究发现,5株溶菌酶敏感菌株以来自ICR小鼠肠道的M6(即弗氏埃希氏菌株Escherichia fergusonii M6)原生质体制备率和再生率均最高。进而以菌株M6为试材,研究不同菌龄、酶浓度、酶解时间、渗透压稳定剂以及添加不同化学组分等对菌株M6原生质体制备与再生的影响。通过本研究确定的菌株M6原生质体制备与再生的最佳条件为:菌龄5h,溶菌酶浓度20mg/mL,脱壁时间45min,渗透压稳定剂为0.7mol/L山梨醇,向再生培养基中同时添加0.8%牛血清蛋白和0.1mol/L N-乙酰氨基葡萄糖,此时原生质体平均制备率与再生率分别为87.80%和85.42%。
In this study, bacterium suitable for protoplast preparation and regeneration was se- lected from dominant aerobic intestinal bacteria isolated from different animals. The optimal conditions for protoplast preparation and regeneration of the selected predominant aerobic in- testinal bacterium were determined, the study of which will provide research basis for the usage of the protoplast of the selected bacterium as one parent in protoplast fusion with that of an an- aerobic bacterium. The result showed that only 5 of the 20 predominant aerobic intestinal bac- teria were relatively sensitive to lysozyme, which included bacterial strains M3, M4, M5 and M6 isolated from intestinal tract of ICR mouse, and Z5 isolated from pig. Further studies indi- cated that among all of the 5 lysozyme sensitive bacteria, strain M6 （i. e. Escherichia ferguso- nil M6） had the highest protoplast preparation rate and protoplast regeneration rate. There- fore, bacterium Escherichia fergusonii M6 was chosen for further study. Different factors, in- cluding bacterial age, enzyme concentration, enzymolysis time, osmotic pressure stabilizer andaddition of different chemical compounds were studied. The optimal conditions for the proto- plast preparation and regeneration of strain M6 were as follows： bacterial age 5 h, lysozyme concentration 20 mg/mL, enzymolysis time 45 min; and in the regeneration medium, osmotic pressure stabilizer 0.7 mol/L D-sorbitol, 0.8% bovine serum albumin and 0.1 mol/L N-acetyl- glucosamine were added. The average protoplast preparation and regeneration rate was 87.80 % and 85.42% under the optimal conditions.
出处
《河北农业大学学报》
CAS
CSCD
北大核心
2013年第4期70-75,共6页
Journal of Hebei Agricultural University
基金
国家自然科学基金(30570035
31170058)
河北省百名优秀创新人才基金(CPRC027)
关键词
优势需氧菌
弗氏埃希氏菌
原生质体
制备
再生
predominant aerobic bacteria
Escherichia fergusonii
protoplast
preparation
re-generation
