紫外线诱变原生质体选育核黄素高产菌株

以产核黄素生产用菌——阿舒假囊酵母RS-89为出发菌株,用对致期生长的菌丝体,经0.5%蜗牛酶+0.5%纤维素酶作用2h可获得大量原生质体,其再生率为6.1%。对经UV诱变的原生质体再生株进行初筛、发酵复筛,从中获得了菌落大、色素深、产量高于出发菌株30%的高产稳定株——UP-91。     

产黄青霉ds RNA病毒通过原生质体融合的转移

将国内青霉素产生菌(Penicillium chrysogenum)的黄孢子系统及绿孢子(包括淡绿,灰绿)系统的十多个菌株,经过病毒提取、电镜观察、奥氏免疫双扩散、凝胶电泳及放射免疫测定,证明黄孢子系统的菌株含有不同滴定度的、直径40nm的球形病毒,而绿孢子系统中检查不出病毒。从营养要求、孢子颜色不同的带病毒和无病毒菌体中分离原生质体,进行不同组合的原生质体的融合杂交,获得营养互补融合的异核体。异核体1中,病毒通过胞质融合转移到原来无病毒的灰绿孢子菌株及细胞核融合后的杂合二倍体中。灰绿孢子的病毒量接近二倍体的1/3。二倍体菌落生长稳定,低温保存二年后经0.01—0.02M对氟苯丙氨酸(PFA)诱发和分离,产生亲本类型的分离子,分离子及二倍体仍然含有病毒。异核体2作亲本性分离,黄孢子仍有病毒,淡绿孢子及细胞核融合后产生的二倍体均无病毒,表明非感染性为显性。此种淡绿孢子的突变体中存在非感病菌系,它不支持病毒的复制。提取各杂交组二倍体内的病毒所特有的dsRNA时,可看出dsRNA的存在和病毒的存在一致。多数杂合二倍体的青霉素产量比亲本高。     

高等担子菌双重营养缺陷突变菌株的分离

带有单一营养缺陷的凤尾菇和裂褶菌的单核体菌株经亲和性交配,各自交配产生后代,从中分离出遗传特性稳定,生理特征表型正常的双重缺陷营养害变型菌株,为原生质体融合育种研究提供了可靠的亲本菌株。     

原生质体紫外诱变选育灵芝新菌种的研究

对灵芝原生质体进行了紫外诱变处理 ,经过粗筛和精筛后 ,从中选出多糖含量和产量明显高于原始菌株的两株诱变株 430 2 0 #和 430 2 6#。经过 1 0代PDA斜面继代培养及其摇瓶试验和连续 3次 3t罐的中试试验 ,表明所得诱变株为比原始菌株更优秀的稳定高产、高多糖含量的灵芝生产菌株。本研究为选育适合发酵的灵芝生产菌种提供了一种快速、有效的方法     

黑曲霉原生质体诱变选育果胶酶高产菌株

通过UV和NTG诱变筛选获得了2株高产果胶酶突变株。以果胶酶产生菌黑曲霉EIM6为诱变材料,采用1．5％的溶壁酶和1．5％的纤维素酶处理其对教生长期菌丝体2h获得高质量的原生质体。采用UV25S或50μg／mL NTG诱变30min,构建原生质体突变库,经刚果红果胶平板筛选获得果胶酶突变株,通过液体深层培养复筛获得高产突变株EIM6-U11、EIM6-N5,酶活力分别从46598．08、46598．08U／mL提高至68596．57、68879．56U／mL,分别提高了47．21％、47．82％。连续8次传代经发酵测酶活力表明高产突变株EIM6-U11、EIM6-N5具有较高的遗传稳定性。     

Genetic recombination by protoplast fusion inNocardia asteroides

Summary Fusion and regeneration of protoplasts ofNocardia asteroides strains ATCC 3318, IMRU W3599 and HIK B971 have been used to study genetic recombination in this species. Protoplasts were produced by treatment with lysozyme, following incubation with glycine. Mutants of ATCC 3318 were grown in peptone yeast extract medium at 32°C prior to protoplast production to maximize protoplast frequency, whereas mutants of IMRU W3599 and HIK B971 were grown in trypticase-soy broth. Glycine concentrations favoring protoplast formation varied from 1.5% to 5% depending on strain. For all strains, protoplast formation was complete 1 h after addition of 5 mg/ml lysozyme. Protoplasts were fused by addition of 50% polyethylene glycol-1000. In general, 25% of the protoplasts could be regenerated. The incidence of recombinant recovery was increased up to 750-fold. The distribution of recombinant phenotypes in matings was similar for protoplast fusion and conventional crosses.     

有益芽孢杆菌受体菌研究

采用酸性茚三酮法测定了30株有益芽胞杆菌的赖氨酸产量,然后在不同的溶菌酶浓度下,对赖氨酸产量超过0．07g/L的21株菌进行原生质体转化质粒pUB110,测定原生质体形成率、原生质体再生率及转化频率,结果6103,6104,6120,6129四株菌的转化频率较高。最后,采用经典遗传学方法选育AEC抗性突变株,使赖氨酸积累提高。其中,B.licheniformis 6104诱变菌株610401能积累赖氨酸2．91g/L ,比出发菌株提高了17倍左右,转化率也提高了一个数量级。通过质粒的再转化试验及传代稳定性试验,进一步证实B.licheniformis 6104及其 突变菌株610401是较好的受体菌,尤其是用于赖氨酸合成酶基因的表达。     

Intraspecific protoplast fusion of Candida tropicalis for enhancing phenol degradation

Auxotrophic mutants and phenol-degrading defective mutants were separately isolated in a phenol-utilizing strain of Candida tropicalis M4, and were hybridized through protoplast fusion. Some protoplast fusants with phenol-degrading ability were obtained and were very stable. Two of the fusants exhibited slightly higher rates of growth than did the wild strain when the cells were grown on phenol medium, and they possessed about 1.9 and 2.2 times respectively higher phenol hydroxylase activity than the wild strain.     

Effect of Chloramphenicol on Host-Bacteriophage Relationships in the Lactic Streptococci

Chloramphenicol (CM)-resistant mutants of Streptococcus lactis strain ML3 were obtained either as a consequence of continuous transfer of the bacteria in broth containing increasing amounts of CM or by selecting for high-level resistant derivatives after mutagenic treatment of the bacteria. Some CM-resistant cells obtained by the first method were also resistant to the homologous bacteriophage. Cells trained to grow in the presence of CM developed resistance to some heterologous attacking phages but not to phage ml(3). Mutants selected for phage resistance were not resistant to CM. There appear to be two different loci for CM resistance on the bacterial chromosome: the one for high-level resistance is associated with the phage-resistance locus and the other is independent of it. A concentration of CM (280 mug/ml) that was bacteriostatic for ML3 inhibited the intracellular growth of ml(3) phage in strain ML3-CM(r)I, which had been trained to grow in the presence of that CM concentration, despite the fact that cells of this strain were not phage-resistant per se. The drug had no direct virucidal action and did not prevent adsorption to or penetration of phage into the bacterium. Lysogenization did not occur. It is concluded that the block in phage development probably involves inhibition of synthesis of phage components, either involving deoxyribonucleic acid at an early stage or the phage coat protein at a later one.     

灵芝原生质体分离与再生研究*

首次详细、系统地探讨了灵芝原生质体分离与再生的条件,结果表明：用0．6mol／L甘露醇配制成含2％溶壁酶和0．5％崩溃酶的复合酶,在30℃,pH为6.0时酶解3小时,可得到最高的原生质体得率。但考虑到原生质体的再生,以酶解2．5小时为最适。酶解2．5小时所得原生质体经精制,用纤维二糖培养基（以0.6mol／L甘露醇为渗透压稳定剂）进行双层平板（上层平板含0．2％琼脂）培养法再生,原生质体的再生率最高。本研究为以后进行灵芝原生质体的融合以及灵芝的转基因研究打下了基础。