【摘要】 原生质体融合(protoplast fusion)技术是将遗传性状不同的亲本原生质体融合杂交的基因重组技术。从健康桑树根系中分离到一株产油脂内生真菌MOD-1,采用酶解方法制备内生真菌MOD-1和产纤维素酶菌株绿色木霉TP4、T44及长柄木霉TL的原生质体,获得4个菌株的原生质体产量分别为7.02×10~6个/m L、4.43×10~6个/m L、6.35×10~6个/m L和4.35×10~6个/m L,再生率分别为4.90%、3.26%、4.51%和4.12%。优化亲本原生质体灭活条件为:MOD-1菌株经紫外线照射10 min或加热15 min,TP4菌株经紫外线照射25 min或加热20 min,T44菌株经紫外线照射25 min或加热25 min,TL菌株经紫外线照射30 min或加热25 min。在上述条件下各亲本原生质体的灭活率均达到100%。将灭活的MOD-1菌株原生质体分别与不同方式灭活的TP4、T44、TL菌株原生质体融合,经初筛和复筛获得了一株兼具MOD-1与TP4双亲优良性状的融合菌株MP-1,其油脂产量、油脂含量(质量分数)分别为0.082 4 g/L、14.46%,显著高于亲本菌株MOD-1。融合菌株MP-1传代培养10代后,油脂产量与油脂含量的遗传稳定性分别达到92.84%和95.00%。更多还原

【Abstract】 Protoplast fusion is a kind of gene recombination technology which fuses parental protoplasts that have different genetic traits to achieve the purpose of hybridization. A lipid-producing endophytic fungus MOD-1 was isolated from healthy mulberry root. Using enzymatic hydrolysis method,protoplasts of mulberry endophytic oleaginous Macrophomina phaseolina MOD-1,cellulase-producing strains including Trichoderma viride TP4,T44 and Trichoderma longibrachiatum TL were obtained. The protoplast yield of MOD-1,TP4,T44 and TL strains reached 7. 02×10⁶m L^-1,4. 43×10⁶m L^-1,6. 35×10⁶m L^-1and 4. 35×10⁶m L^-1respectively,and the regeneration rate reached 4. 90%,3. 26%,4. 51%and 4. 12% respectively. The optimized inactivation conditions of parental protoplasts were as follows: MOD-1,UV-irradi-ation for 10 min or heat-inactivation for 15 min; TP4,UVirradiation for 25 min or heat-inactivation for 20 min; T44,UV-irradiation for 25 min or heat-inactivation for 25 min;TL,UV-irradiation for 30 min or heat-inactivation for 20 min. Under these conditions,the inactivation rate of parental protoplasts all reached 100%. MOD-1 inactivated protoplast was fused with T44,TP4 or TL inactivated protoplast by different ways,and a fusant MP-1 from MOD-1and TP4 was obtained through primary and secondary screening. Its lipid yield and lipid content（ mass fraction） achieved to 0. 082 4 g/L and 14. 46%,being significantlyhigher than those of parental strains. The genetic stability of lipid yield and lipid content reached 92. 84% and 95. 00% respectively after 10 generations of subculture.更多还原