链霉菌种间原生质体融合的研究

金霉素链霉菌和龟裂链霉菌分别在含０．７５％，甘氨酸１．５０％的液体培养基中培养，对数生长中后期的菌丝体易于制备原生质体，当聚乙二醇（ＰＥＧ６０００）浓度为４０％，在５０℃下保温５ｍｉｎ，有效地诱导了两种原生质体的融合。用表型标记和间接选择的方法检出融合子，种间融合率为３．０５％。融合重组体与亲本在菌落形态，孢子形态，抗生素产量和抗噬菌体特性有显著的差异，不同重组体之间也表现了这些差异．     

产色链霉菌SY20-2与龟裂链霉菌SY20-4的种间原生质体融合

利用卡那霉素(250μg/mL)、链霉素(1 000μg/mL)和罗红霉素(150μg/mL)标记亲本菌株SY20-2、SY20-4,进行种间原生质体融合.试验结果表明,培养基中加入0.5%~1%甘氨酸可以提高菌丝体对溶菌酶的敏感度,有利于原生质体的释放.溶菌酶不仅影响原生质体的形成,而且影响原生质体的再生率,因此在35℃水浴条件下,SY20-2选择酶浓度2 mg/mL,作用时间30 min;SY20-4选择酶浓度4 mg/mL,作用时间40 min,最适于原生质体的再生.以45%PEG(分子量1 000)作助融剂,融合频率为0.124%,选出153个融合子,其中3株重组子的稳定性及抑菌活性较好,重组子C对烟草野火病菌的抑菌活性比对照要好.     

链霉菌种间融合及融合产物的分析

采用灭活龟裂链霉菌原生质体与金霉素链霉菌原生质体在聚乙二醇诱导下进行原生质体种间融合,利用金霉素链霉菌对链霉素的敏感性,建立选择培养基,提高了融合子的检出率。并采用同工酶谱分析及抗生素效价测定,对融合子进行了初步鉴定     

金色链霉菌原生质体的制备

对金色链霉菌原生质体制备的具体条件进行了优化研究 .发酵培养基中采用蔗糖浓度 12 %、甘氨酸浓度 0 .5 %、酶解条件采用菌龄为 48h的菌丝、溶菌酶浓度 6mg/mL、酶解时间为 1h制备原生质体 ,原生质体制备率可高达 6 0 %以上 .     

妥布霉素产生菌黑暗链霉菌F_(-211)发酵特性研究

从黑暗链霉菌Ｆ－２１１合成妥布霉素（Ｔｏｂｒａｍｙｃｉｎ）的发酵原理出发,研究黑暗链霉菌产生抗生素的特性,考察与生产妥布霉素紧密相关的种龄、周期、关键原料—油,和影响生物合成抗生素的各种主要因素,绘制出合理的发酵工艺控制图,优化发酵条件,使发酵单位提高了近一倍．     

林肯链霉菌原生质体形成、再生和融合的研究

林肯链霉菌生长在0.5%甘氨酸的S培养基中,能较好地被溶菌酶破壁形成原生质体。原生质体能在RB培养基上再生,但不能在R_2培养基上再生,这和已报道过的其他链霉菌不同。78-11菌株的再生频率约为25.7%,S-3菌株的再生频率约为20.5%。PEG(聚乙二醇)1000对诱导融合的效果较好,重组频率最高可达10~(-2)。重组子和亲本在培养特性、孢子形态方面无特殊差异,但经C_O~(60)诱变后重组子的正变率超过亲本。     

刺孢吸水链霉菌与淡紫灰链霉菌原生质体制备条件研究

通过对甘氨酸浓度、酶解温度、酶浓度、酶解时间等影响因素考察,及正交试验优化,确定了刺孢吸水链霉菌和淡紫灰链霉菌原生质体制备的最佳条件.实验结果表明,刺孢吸水链霉菌最佳原生质体制备条件是：甘氨酸浓度为0.5%,酶解温度28 ℃,溶菌酶浓度为3×10-3 g/mL,酶作用时间60 min;淡紫灰链霉菌：甘氨酸浓度为1.0%,酶解温度32 ℃,溶菌酶浓度4×10-3 g/mL,酶作用时间60min.有效的原生质体保存条件是：-20 ℃,在5 d内,原生质体再生率在15%以上.     

妥布霉素高产菌的诱变育种

以黑暗链霉菌HA-1为出发菌株,应用紫外线诱变和紫外线复合吖啶噔诱变并结合自身产物梯度平板筛选的方法获得两株高产菌株UZ-90和UAZ-121.它们的总效价分别比出发菌株提高了60%和80%;产物中妥布霉素的含量分别比出发菌株提高了26%和35%.     

链霉菌702原生质体的制备和再生条件研究

为了确定链霉菌702菌株原生质体制备和再生较优组合条件,以链霉菌702菌株为试验材料,试验设计以单因素和多因素多水平的正交试验。在单因素试验结果中探索该菌的对数生长期为35 h~50 h,在菌丝体培养基中添加1.0%甘氨酸有利于该菌原生质体制备和再生;正交试验分别以影响该菌原生质体制备和再生的菌丝体培养时间、溶菌酶使用浓度、酶解温度和酶解时间的四因素三水平的L9(34)正交试验,试验表明:链霉菌702菌株原生质制备和再生较优组合为A2B2C3D1,即菌丝体培养时间为43 h,溶菌酶浓度为2.0 mg/mL,酶解温度为37℃,酶解时间60 m in,原生质体的制备率和再生率分别达到96.5%和27.8%。本试验为该菌进一步进行原生质体诱变打下良好的基础。     

自溶链霉菌遗传操作研究

放线菌新种自溶链霉菌能够产生一种称为自溶霉素的抗肿瘤活性产物.虽然自溶霉素具有成为抗肿瘤药物的良好前景,但是自溶链霉菌遗传操作方法的缺乏严重阻碍了对自溶霉素生物合成的研究.本工作对自溶链霉菌中各种遗传操作方法进行了探索.优化了PCR扩增的反应体系和程序,建立了外源DNA导入的方法,确立了构建基因敲除突变株的最佳方案.此外,也摸索了自溶霉素发酵和HPLC检测的适宜条件.利用所建立的方法对自溶链霉菌基因almRⅠ和almRⅡ的功能进行了初步研究,结果表明它们在自溶霉素生物合成过程中起正调控作用.自溶链霉菌遗传操作方法的建立为进一步开发利用放线菌资源奠定了基础.     

草菇原生质体的制备与融合研究

项目对低温草菇和高温草菇菌株进行了酶解制备原生质体及原生质体的融合进行了研究.研究结果表明:以融壁酶Novozym234对低温草菇和高温草菇酶解效果较好,酶解浓度为2.5%,稳渗剂以0.5Mol/L蔗糖为佳,pH值保持在8.5左右,以30%的助溶剂(PEG4000-6000)作为融合诱导剂进行草菇原生质体融合,并获得融...     

Enhancement and selective production of avermectin B by recombinants of Streptomyces avermitilis via intraspecific protoplast fusion

Among eight components of avermectin, B1 fractions have the most effective antiparasitic activities and the lowest level of toxic side-effects and are used widely in veterinary and agricultural fields. In-traspecific protoplast fusion between two strains of Streptomyces avermitilis, one an avermectin high producer (strain 76-05) and the other a genetically engineered strain containing the mutations aveDˉ and olmAˉ (strain 73-12) was performed for enhancement and selective production of avermectin B in the absence of oligomycin. Two recombinant strains (F23 and F29) were isolated and characterized with regards to the parental merits. F23 and F29 produced only the four avermectin B components with high yield and produced no oligomycin. The avermectin production of F23 and F29 was about 84.20% and 103.45% of the parental strain 76-05, respectively, and increased about 2.66-fold and 3.50-fold, re-spectively, compared to that of parental strain 73-12. F23 and F29 were genetically stable prototrophic recombinants and F29 was quite tolerant of fermentation conditions compared to avermectin high producer parental strain 76-05. The ability to produce avermectin B with high yield without the produc-tion of other avermectin components and oligomycin will make F23 and F29 useful strains for aver-mectin production. Strain F29's tolerance of fermentation conditions will also make it suitable for in-dustrial applications.     

香菇菌丝原生质体制备及融合条件的研究

讨论不同菌龄、酶解时间、酶解温度、渗透压稳定剂对香菇原生质体产率的影响。结果表明 ,香菇菌丝制备原生质体时最适菌龄为 6天 ,酶解时间为 3h ,酶解温度为 34℃ ,渗透压稳定剂用 0 .6mol/L甘露醇为最佳。在此基础上 ,以 35 %的PEG为融合诱导剂进行香菇B0 1、L2 6种间原生质体融合 ,并用显微镜观察到融合子的形成     

木本植物原生质体培养与融合研究进展

为了全面了解模板植物原生质体培养和融合的现状，找出原生质体培养的规律，概述了原生质体的分离、培养及再生植株的条件和影响因素，介绍了国内外原生质体培养和融合及无性系变异筛选及遗传转化的研究动态。     

Enhancement of monacolin K production via intergeneric protoplast fusion between Aspergillus terreus and Monascus anka

Intergenric protoplast fusion between Aspergillus terreus CA99 and Monascus anka M-3, the high and low producers of monacolin K respectively,was performed for enhancement of monacolin K production. The 24-hour-old mycelia of A. terreus CA99 and M. anka M-3 were treated with 0.5% lywallzyme, 0.3% snailase and 0.3% cellulase at 34℃ for 5 h and at 30℃ for 3.5 h, and their protoplasts formation reached 1.76×107/mL and 1.68×107/mL respectively. Parental protoplasts were irradiated with a 30 W UV light away from 30 cm for 3 min and then mixed. The mixture was incubated with 30% PEG 6000 for 15 min. The reviving fusants were isolated on the regeneration plates. Of the 363 fusants isolated, over 100 showed enhanced monacolin K production compared with the parental strain M. anka M-3. Ten of them produced monacolin K about 1.6-fold of that M.anka M-3 does and the monacolin K titer of two fusants (F49 and F104) increased by about 1-fold. The monacolin K yields of F49 and F104 were 460 μg/mL and 457 μg/mL respectively. In optimized fermentation medium, the monacolin K titer of F49 reached 1216 μg/mL.     

Enhancement of monacolin K production via intergeneric protoplast fusion between Aspergillus terreus and Monascus anka

Intergenric protoplast fusion between Aspergillus terreus CA99 and Monascus anka M-3, the high and low producers of monacolin K respectively，was performed for enhancement of monacolin K production. The 24-hour-old mycelia of A. terreus CA99 and M. anka M-3 were treated with 0.5% lywallzyme, 0.3% snailase and 0.3% cellulase at 34℃ for 5 h and at 30℃ for 3.5 h, and their protoplasts formation reached 1.76×10⁷/mL and 1.68×10⁷/mL respectively. Parental protoplasts were irradiated with a 30 W UV light away from 30 cm for 3 min and then mixed. The mixture was incubated with 30% PEG 6000 for 15 min. The reviving fusants were isolated on the regeneration plates. Of the 363 fusants isolated, over 100 showed enhanced monacolin K production compared with the parental strain M. anka M-3. Ten of them produced monacolin K about 1.6-fold of that M.anka M-3 does and the monacolin K titer of two fusants (F49 and F104) increased by about 1-fold. The monacolin K yields of F49 and F104 were 460 μg/mL and 457 μg/mL respectively. In optimized fermentation medium, the monacolin K titer of F49 reached 1216 μg/mL.