诺西肽产生菌活跃链霉菌的原生质体制备与再生

目的研究诺西肽产生菌活跃链霉菌的原生质体制备与再生的最适条件。方法使用溶菌酶脱去细胞壁制备原生质体,并考察原生质体制备和再生的各种影响因素。结果确定了原生质体制备的条件:一级培养采用种子培养基,培养30 h,转种量的体积分数为5%;二级培养采用R2YE培养基,培养时间为32 h,最适甘氨酸质量浓度为6.0 g.L-1,最佳溶菌酶质量浓度为1.5 g.L-1,酶解时间为60 min,原生质体再生率达到5.3%。结论上述条件为活跃链霉菌原生质体制备与再生的最适条件,该条件的建立为活跃链霉菌原生质体的诱变育种奠定了基础。     

阿卡波糖产生菌原生质体的制备与再生

目的研究阿卡波糖产生菌原生质体制备与再生的条件。方法通过溶菌酶破壁的方法制备原生质体,考察影响原生质体制备与再生的因素。结果和结论确认原生质体的制备条件为:一级培养采用SM2液体培养基,培养时间为33 h,转二级培养的转种体积分数为15%;二级培养采用R2YE培养基,培养时间为20 h,甘氨酸质量分数为0.7%;溶菌酶作用质量浓度为3 g.L-1,作用时间100 min,最大原生质体制备量达到8×1010个.L-1。考察了原生质体再生培养基的组成,在优化的再生培养基上,再生率达到9.2%。     

刺糖多孢菌原生质体制备与再生条件优化

目的研究多杀菌素产生菌刺糖多孢菌(Saccharopolyspora spinosa)原生质体制备与再生的最佳条件。方法利用数理统计的方法研究了不同制备培养基、菌龄、甘氨酸浓度、溶菌酶处理条件以及再生培养基对原生质体制备和再生的影响,并考察了原生质体的适宜保藏温度。结果菌体在添加0.3%甘氨酸的EHC培养基中培养72h,用2mg/mL溶菌酶32℃酶解40min后,涂布在再生培养基R6上再生,原生质体制备率超过99%,再生数可达到107cfu/mL。刺糖多孢菌原生质体可置于4℃短期保存72h,长期保存需要放置于-80℃条件下。结论优化的结果为刺糖多孢菌原生质体融合育种和遗传转化体系建立奠定了基础。     

天蓝链霉菌2809原生质体的制备和再生

报道了天蓝链霉菌2809原生质体的制备,再生条件和再生株的产抗能力的变化;研究了甘氨酸浓度对菌丝体生长的影响;确定了溶菌酶作用的最佳条件;再生率为20%—30%;二次再生过程获得了高产菌株。     

普那霉素产生菌的原生质体诱变育种

普那霉素产生菌始旋链霉菌（Streptomyces pristinaespiralis ）11．2在含0．5％甘氨酸的种子培养基中培养到对数生长期，收集菌丝体，经2mg／ml溶菌酶在30℃下作用90min可获得大量的原生质体，其再生率为5．1％。始旋链霉菌11.2原生质体经UV诱变并在含普那霉素的再生平板上筛选普那霉素抗性菌株，从中获得一高产：突变株始旋链霉菌ZP-07，普那霉素产量达到1．59g／L，比出发菌株提高101．3％。     

黑暗链霉菌原生质体制备、再生及其DNA转化条件的研究

利用CP培养基培养黑暗链霉菌 (Streptomycestenebrarius) 990 4,采用二级培养即首先在37℃培养 48h ,然后按 10 %的转种量转种于新鲜的培养基中 ,同时补加 2 %甘氨酸 ,2 8℃培养 2 0h ,收获的菌丝体对溶菌酶敏感。在适宜的酶解条件下可形成 4 6 2× 10 9/mL原生质体 ,再生率为18%。利用经修饰的质粒DNA转化冻存的原生质体获得成功 ,转化率为 10 3～ 10 4 / μgDNA。     

产黄青霉原生质体制备和再生影响因子分析

产黄青霉原生质体制备过程中,菌丝体最佳酶解条件与原生质体释放最适条件不同,菌丝体培养基含有0．05％L－天冬酰胺时,选取培养48h的菌丝体,在裂解酶(Lysing enzyme)浓度15mg/ml,0.7mol/L KCl作为渗透压稳定剂的条件下酶解,可获得大量原生质体;同时,再生培养基中含有25mmol/L Ca^2 离子、上层琼脂浓度1．1％,可获得较佳原生质体再生率。     

林肯链霉菌原生质体的形成、再生及其影响因素

林肯链霉菌林肯变种(Streptomyces lincolnensis var.lincolnensis)在含有0.5%甘氨酸的 S培养基上生长的菌丝体对溶菌酶敏感,酶解后产生10~8/ml 的原生质体。在适当的条件下可有20%的原生质体再生成细胞。原生质体在4℃贮存20h 后再生活力下降至原来的20%以下。原生质体在紫外光照射下比孢子更易发生突变。     

利用原生质体诱变筛选L-异亮氨酸高产菌株

在捧杆菌原生质体制备中,以单一甘氨酸代替青霉素与溶菌酶进行细胞脱壁处理,以含0.3M蔗糖的普通肉汤培养基作为再生培养基。L-异亮氨酸产生菌A6在肉汤中培养至对数期,加入0.3M蔗糖和3%甘氨酸,继续培养8h,原生质体的形成率达98%。原生质体经洗涤后的再生率为81.6%,不经离心洗涤直接涂皿的再生率为81.2%。A6菌株的原生质体经紫外线诱变处理后,再生菌株的L-异亮氨酸产量高达14.3mg/ml,比原菌株产量提高30%左右。     

利用原生质体诱变筛选L—异亮氨酸高产菌株

在棒杆菌原生质体制备中,以单一甘氨酸代替青霉素与溶菌酶进行细胞脱壁处理,以含0.3M蔗糖的普通肉汤培养基作为再生培养基,L-异亮氨酸产生菌A6在肉汤中培养至对数期,加入0.3M蔗糖和3%甘氨酸,继续培养8h,原生质体的形成率达98%,原生质体经洗涤后的再生率为81.6%,不经离心直接涂皿的再生率为81.2%.A6菌株的原生质体经紫外线诱变处理后,再生菌株在L-异亮氨酸产量高达14.3mg/ml,比原菌株产量提高30%左右。     

Genetic transformation ofStreptomyces caespitosus

Genetic transformation ofStreptomyces caespitosus by plasmid pIj 702 was carried out. Optimal conditions for the protoplast preparation ofStreptomyces caespitosus, its regeneration, and its transformation by pIj 702 were evaluated. Addition of 2% glycine to the culture broth was optimal for protoplast yield. Formation and regeneration of protoplasts were most efficient when the mycelium were harvested at between late log and stationary growth phase. The regeneration frequency of the protoplasts was 15% when the protoplasts were regenerated on R2YE agar media containing 0.5M sucrose. Under the best condition for protoplats regeneration, the optimal transformation frequency was achieved with 40% polyethylene glycol (M.W. 4,000) treatment for 2 minutes.     

Liquid culture enhances protoplast formation from the auxotroph (ser−) ofLentinula edodes

The optimal conditions for the production and regeneration of the protoplasts fromLentinula edodes were studied. Protoplast formation from the mycelia ofL. edodes which were cultured in liquid medium showed a significantly high yield compared with that of the mycelia which were cultured on cellophane covered agar media. A mixture of Novozyme 234 (15 mg/ml) and Cellulase Onozuka R10 (10 mg/ml) in 0.6 M mannitol (pH 4) was optimal lytic enzyme for the protoplast release. The optimal incubation time and mycelia age were 3.5-4 hours at 30 degrees C and 6-8 days, respectively. Regeneration frequency was 0.18% plated onto a medium containing 0.6 M sucrose, and 0.08% plated onto a medium containing mannitol. But hardly any regeneration was observed in the media containing NaCl, KCl, or MgSO(4). More than 90% of the protoplasts contianed nuclei and the nucleus number per protoplast was 1.1. The DNA content per nucleus was 5.1 pg. The diameter of the protoplast was 3-5 mum and it had a well defined cell structure.     

红霉素链霉菌原生质体的形成和再生及其对紫外光的敏感性

红霉素链霉菌的菌丝生长在含有0.8％甘氨酸的S培养基中,对溶菌酶的作用敏感,通过酶解,并利用一个包含10mM-MgCl_2和25mM-CaCl_2的高渗培养基能使其菌丝脱壁形成10~(10)/ml的原生质体。 原生质体能再生细胞壁,回复成为一个完整的细胞。最佳条件时再生频率达90％左右。 利用紫外光对红霉素链霉菌的孢子及原生质体分别照射3分钟,其致死率分别为97.95％和98.81％。     

顶头孢霉229与12的分生孢子及原生质体的形成和再生

头孢菌素产生菌顶头孢霉菌株229的沉没培养或斜面培养都可形成分生孢子,并可用普通滤纸将它们与菌丝及节孢子分开,但是它们成活率极低.这种成活的分生孢子的数量与培养基成分有关.菌丝培养基成分对制备顶头孢霉原生质体有显著影响.用一种MM培养基培养的菌丝,不经巯基化合物预处理,酶解(1%纤维素酶)3小时后,可得到大量原生质体.原生质体的再生频率为1.8～4.6%.与分生孢子形成的菌落相比,原生质体再生菌落的产抗生素能力显示出较大的变异性.本文还讨论了山梨醇与Nikkomycin对菌丝生长形态及原生质体形成的影响.     

Studies on protoplast formation and regeneration ofGanoderma lucidum

To obtain a new strain ofGanoderma lucidum by protoplast fusion technique, its protoplast formation and regeneration were studied. Several factors affecting the protoplast formation and regeneration were investigated to find their optimum conditions. The mycelium was grown for four days on the cellophane membrane placed onG. lucidum complete medium (GCM). When various commercial lytic enzymes were examined for protoplast isolation, the combination of Novozym 234 and β-glucuronidase was found to be effective. An osmotic stabilizer, 0.6 M sucrose in 20 mM phosphate buffer pH 5.8, gave the highest yield of protoplasts. Three-hour incubation in shaking incubator was most suitable for releasing protoplasts. To increase the protoplast yield, pretreatment with 2-mercaptoethanol was carried out. The regeneration frequency in GCM containing 0.6 M MgSO₄·7H₂O was shown to be 0.66%.     

巴戟天糖链对缺氧复氧损伤内皮细胞的血管形成作用研究

目的探讨巴戟天糖链(MOO)对缺氧复氧损伤内皮细胞的促血管形成作用。方法建立人脐静脉内皮细胞(HUVEC)缺氧复氧损伤(H/R)模型,实验分为6组:正常细胞组、H/R模型组、H/R+阳性对照组、H/R+MOO大、中、小剂量组。用倒置显微镜观察细胞形态;流式细胞仪测量在H/R条件下的细胞周期;采用transwell小室迁移实验和小管形成实验检测MOO对体外内皮细胞血管形成的作用。结果①与正常组相比,H/R模型组增加了G2+S期的细胞(P<0.05),与模型组相比,MOO大、中剂量减弱了这种趋势(P<0.05),使细胞状态更接近于正常细胞;②MOO各剂量组细胞迁移数量高于模型对照组(P<0.05);③MOO各剂量组小管形成的面积较模型对照组亦增多(P<0.05)。结论 MOO可以减轻H/R对血管内皮细胞的损伤,抑制细胞凋亡,有效促进H/R损伤的内皮细胞迁移和形成血管。