秦岭链霉菌的原生质体再生与诱变育种

在秦岭链霉菌(Streptomyces qinlingensis sp. nov.)的菌种改良中, 应用原生质体再生并结合物理化学诱变能够得到产量较高、稳定性较好的菌株。筛选实验表明：秦岭链霉菌原生质体再生菌株R-72、诱变菌株NTG-1和H30-7对枯草芽孢杆菌的抗菌活性均提高了20%以上, 并且连续培养10代, 其遗传性状均比较稳定。进一步的生测实验表明菌株R-72、NTG-1和H30-7对5种病原细菌和5种植物病原真菌的抗菌活性相比原始菌株有显著提高。     

小麦白粉病生防菌G-1菌株原生质体诱变育种

以链霉菌G-1(Streptomyces sp.G-1)为出发菌株,通过研究菌株G-1原生质体形成与再生的条件,发现该菌株在含0.5%甘氨酸的菌丝体培养基中经过二次培养后,所得菌丝体用2 mg/mL溶菌酶在30℃下处理90 min,可获得大量原生质体,其再生率可达8.2%。菌株G-1的原生质体经紫外诱变和宁南霉素抗性筛选后,得到一高产突变株G-1-125,其有效组分A的产量达到794mg/L,较出发菌株提高了180%。     

纳他霉素产生菌原生质体的制备、再生及紫外诱变

研究了纳他霉素产生菌原生质体的最佳制备和再生条件,及此基础上的紫外诱变育种.初步确定了原生质体制备和再生的适宜条件为:菌龄48h,采用0.4%溶菌酶在30℃处理90min.原生质体再生后,73.3%的菌种产量得到了提高,其中5-12菌株增产74.7%,达到2121.2μg/mL.原生质体经紫外线诱变后得到的5株诱变菌株产量均有提高,其中菌株UV-2增产107.09%,达到2515.07μg/mL.     

链霉菌6803菌株对植物的化感作用

链霉菌属是绝大多数已知抗生素和一些重要活性物质的产生菌,它对高等植物是否具有化感作用尚缺乏研究. 从土壤中分离获得7个放线菌菌株,研究其对植物幼苗生长的抑制作用.结果表明： 链霉菌6803菌株在固体和液体发酵培养时均强烈抑制油菜根和稗草根的生长, 其液体发酵液的5倍稀释液对油菜和稗草苗生长的抑制分别达到60.7%和61.3%. 常规形态与生理生化试验表明,该菌株属于链霉菌属金色类群; 16S rRNA基因序列分析表明,该菌株为沙场链霉菌,基因序列相似性达到99.28%. 此菌株经过80～100 s紫外线照射筛选出的正突变菌株UV8024和UV100-2的10倍稀释发酵液对油菜幼苗生长的抑制作用比原始菌株分别提高了37.5%和38.1%, 经过1%硫酸二乙酯诱变50 min筛选出的正突变菌株D507的10倍稀释发酵液对油菜幼苗生长的抑制作用比原始菌株提高了29.8%. 链霉菌6803菌株对高等植物具有化感作用, 并可通过诱变育种提高其化感潜力.     

链霉菌11371原生质体的制备与再生

为选育链霉菌11371的高产Tetramycin菌株,摸索该菌株的原生质体的制备与再生。结果表明,链霉菌11371原生质体制备和再生的最佳条件为菌丝生长培养液中甘氨酸浓度为0．7％,培养温度为28℃,培养时间为42h,溶菌酶浓度为3mg／mL,酶解温度为37℃,酶解时间为90min。最佳再生培养基为R2YE培养基。     

链霉菌6803菌株对植物的化感作用

链霉菌属是绝大多数已知抗生素和一些重要活性物质的产生菌,它对高等植物是否具有化感作用尚缺乏研究.从土壤中分离获得7个放线菌菌株,研究其对植物幼苗生长的抑制作用.结果表明:链霉菌6803菌株在固体和液体发酵培养时均强烈抑制油菜根和稗草根的生长,其液体发酵液的5倍稀释液对油菜和稗草苗生长的抑制分别达到60.7％和61.3％.常规形态与生理生化试验表明,该菌株属于链霉菌属金色类群;16S rRNA基因序列分析表明,该菌株为沙场链霉菌,基因序列相似性达到99.28％.此菌株经过80 ～ 100 s紫外线照射筛选出的正突变菌株UV8024和UV100-2的10倍稀释发酵液对油菜幼苗生长的抑制作用比原始菌株分别提高了37.5％和38.1％,经过1％硫酸二乙酯诱变50 min筛选出的正突变菌株D507的10倍稀释发酵液对油菜幼苗生长的抑制作用比原始菌株提高了29.8％.链霉菌6803菌株对高等植物具有化感作用,并可通过诱变育种提高其化感潜力.     

吸水链霉菌NND-52-C基因工程宿主载体系统的构建

吸水链霉菌NND-52-C菌株是大环内酯类抗生素-阿扎霉素B的高产菌株。采用原生质体转化技术,将来自变铅青链霉菌TK24菌株的pU702质粒转化吸水链霉菌NND-52-C菌株的原生质体,建立了吸水链霉菌NND-52-C菌株的基因工程宿主载体系统。确定了NND-52-C菌株原生质体制备和再生的条件,其原生质体形成率达到10^8／mL,再生率约为0．2％,转化率为10^2-10^3个转化子／μg质粒DNA。     

原生质体技术选育桃红侧耳优良菌株

研究了桃红侧耳原生质体的制备及再生,并进行了原生质体再生株的筛选工作。实验表明,培养5天的桃红侧耳菌丝体30℃酶解3h原生质体数目可达8.4×107/mL。原生质体再生率在1号再生培养基上最高,为6.9%。再生菌株经筛选后,得到长速显著快于出发菌株的优良菌株H120和H247。经F3代栽培实验证明:H120和H247的生物学效率明显优于出发菌株,且差异极显著。酯酶同工酶分析表明:H120和H247酶谱均发生了变化。     

几种诱变因子对龟裂链霉菌原生质体的影响

作者应用通电、ＵＶ、ＮＴＧ和亚硫酸氢钠等诱变因子对土霉素产生菌——龟裂链霉菌１３８—ｌ原生质体进行处理。结果表明,各种诱变因子在对原生质体致死率５０％左右时具有较好的诱变效应。经亚硫酸氢钠＋ＬｉＣｌ处理获得的Ｃｌ１８菌株通过１６５００１发酵罐试验,最高发酵水平达３３３３０ｕ／ｍｌ,平均发酵水平为３０５４２．５ｕ／ｍｌ,比出发菌株１３８—１提高２０．６％。     

林肯链霉菌双亲灭活原生质体融合的研究

分别以紫外线、热灭活林肯链霉菌 94 7和 95 0 2原生质体 ,然后进行灭活双亲的原生质体融合 ,从 1 6株融合子筛选到林肯霉素高产株。用双亲的互补营养缺陷型对林肯链霉菌原生质体的制备、融合、再生的部分条件进行了研究。发现含 0 .4 %Gly和 34 %蔗糖的SM培养基最适于实验菌株原生质体的制备、再生。聚乙二醇 (PEG)分子量对原生质体融合影响不大 ,其在P缓冲液中的浓度却很重要。含 5 0 %PEG的P缓冲液最有利于原生质体融合     

利用原生质体诱变育种选育富硒能力强的酵母菌株

利用原生质体诱变育种技术选育富硒能力强的酵母菌株,从13株啤酒酵母中筛选出一株富硒量高的诱变出发菌株,采用溶壁酶进行破壁,确定了原生质体制备的最适条件为酶浓度1g／100mL,酶解处理时间为120min,原生质体形成率为95．2％,再生率为21．8％,诱变后筛选出富硒量为821mg／kg,酵母干菌体收获量为0．88g／100mL的酵母菌Al。     

~(60)Coγ射线对不同细胞形态出芽短梗霉的辐射诱变效应

使用~(60)Coγ射线辐照诱变的方法处理出芽短梗霉AureobasidiumPullulansAP92菌株的原生质体、菌丝体片段、分生孢子悬液,经初筛、复筛与对突变株的遗传稳定性研究,发现采用原生质体进行诱变,所获突变株的正突变率、单株产多糖的提高幅度、正突变株的产多糖遗传稳定性均明显高于菌丝体与分生孢子。比较出发菌株AP92与经原生质体诱变获得的正突变株A81的性能,有如下明显改善：产多糖能力从16．35g／L提高到29．69g／L,糖转化率从32．7％升到61．4％,残糖从13．269／L降到3．06g／L,而发酵周期则可缩短约24小时。结果证明,对原生质体进行~(60)Coγ射线诱变,是优化出芽短梗霉菌种的有效途径。     

原生质体激光诱变选育少根根霉脂肪酶高产菌株

对少根根霉BUCT-11原生质体制备、再生条件及激光诱变育种进行了研究.结果显示,少根根霉BUCT-11原生质体形成及再生最佳条件为:菌龄24 h,混合酶由27 mg/mL的蜗牛酶和53 mg/mL的纤维素酶组成,酶解时间1.5 h,酶解温度30 ℃,渗透压稳定剂为0.6 mol/L NH4Cl、0.02 mol/L ...     

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。     

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.     

Protoplast isolation and regeneration from <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Gracilariales,Rhodophyta)

The tropical agarophyte Gracilaria changii has been much researched and documented by the Algae Research Laboratory, University of Malaya, especially with regards to its potential as a seaweed bioreactor for valuable compounds. Protoplast regeneration of this seaweed was developed following the optimization of protoplast isolation protocol. Effect of the concentration and combination of isolating enzymes, incubation period, temperature, enzyme solution pH, tissue source on the protoplast yields were used to optimize the isolation protocol. The enzyme mixture with 4% w/v cellulase Onozuka R-10, 2% w/v macerozyme R-10 and 1 unit mL^-1 agarase was found to produce the highest yield of protoplast at 28°C and 3 h incubation period. Thallus tips gave higher yields of protoplasts than middle segments. Freshly isolated G. changii protoplasts were cultured in MES medium. Regeneration of protoplast cell walls after 24 h was confirmed by calcofluor white M2R staining under UV fluorescence microscopy. The protoplasts with regenerated cell walls then underwent a series of cell division to produce callus-like cell masses in MES medium. Following this, juvenile plants of G. changii were obtained.     

桃褐腐病菌(Monilia fructigena)原生质体制备及再生条件

以桃褐腐病菌(Monilia fructigena)为供试菌株,研究了酶系组成、液体培养基、菌龄、酶解温度、酶解时间对原生质体制备的影响,以及等渗液、固体再生培养基、酶解时间对原生质体再生的影响。结果表明:Fries(1/2)液体培养基培养24h,在10mg/mL崩溃酶+5mg/mL纤维素酶+20mg/mL蜗牛酶+10mg/mL溶菌酶的混合酶液中28°C酶解4h为桃褐腐病菌原生质体制备的最佳条件。采用液体再生涂布平板法,以含Ca2+的STC为等渗液的液体培养基和含蔗糖及Ca2+的Fries(1/2)固体培养基为桃褐腐病菌原生质体再生的最佳条件。经过观察与测定,再生菌株保持了原有的培养性状和致病性,接种桃果实后发病率为100%。     

Plant Regeneration from Protoplasts of Savoy Cabbage(Brassica oleracea L. var. subauda)

Protoplasts of savoy cabbage (Brassica olleracea L. var. subauda), "SA61" (SV), were isolated from leaves and hypocotyls of seedlings grown in vitro, in enzyme mixture containing 2% cellulase (Onozuka R-10) and 0.8% macerozyme RI0. Good results of protoplast collection were obtained by using 18% and 17% sucrose solution floating leaf protoplasts and hypocotyl protoplasts respectively, and centrifugalizing with the rate of 500 r/min. All the collected protoplasts were cultured in 5 different liquid media from which the best results were observed on DPD1 medium for leaf protoplasts and on MS1 medium for hypocotyl protoplasts, with the highest cell division rate and planting efficiency. About 2 weeks of cultures, many cell clusters and a few embryo-like structures were visualized. The cell clusters developed into visible microcalli in 20-30 days and grew up to 1 mm or so in dimeter about 40 days of culture. For growth, the calli were transferred to 7 different agar media and from which two suitable media, MB2 and MB3, were selected. Cultured for 40-50 days, the calli grew up, and were transferred to 4 solid media for organ differentiation. Ideal results of shoot regeneration were obtained on MS, medium. About 2 weeks after rooted on the MS medium without any auxin, intact plants were regenerated.