种间原生质体融合提高巴龙霉素单位产量的研究

将巴龙霉素产生菌与新霉素产生菌的高产变株进行了种间原生质体融合,融合频率为10^-4左右。在4l0株稳定的原养型重组体中,产生巴龙霉素者占58％。在200株产生巴龙霉素的种间重组体中,单位产量在1500μg／ml以上的约10％,获得了比巴龙霉素产生菌原始菌株(单位产量300μg／ml)单位产量高5—6倍的重组体菌株。核磁共振谱和质谱测定证明,高单位重组体所产抗生素确为巴龙霉素。结果表明,为了提高某一抗生素产生菌的单位产量,使之与另一生物合成途径相似的抗生素产生菌的高产变株进行种间杂交,是一值得探索的新途径。     

链霉菌原生质体种间融合提高细胞分裂素效价的研究

本文报道粉红孢类群的泾阳链霉菌与淡紫灰链霉菌不液化亚种的原生质体种间融合重组的结果。利用单亲灭活和双亲株的遗传标记筛选细胞分裂素的融合子,融合率为 10-4—10-2。在所得的融合菌株中,F1211菌株的CTK效价为292μg/L,F1613的CTK效价为857μg／L,比低产原始亲株提高2—7倍,比高产亲株提高0.3--3.0倍。在电子显微镜下观察到融合过程。     

原生质体紫外诱变选育高产抗菌脂肽菌株及其活性物质的研究

本研究以实验室自主分离的枯草芽孢杆菌mutHS-301为出发菌株,通过原生质体紫外诱变选育出高产抗菌脂肽突变菌株,并对其产生的抗菌脂肽提取物进行单组分分离纯化及对黄曲霉抑制作用进行初步研究。结果表明,在溶菌酶浓度为0.5 mg/mL,酶解时间为15 min,酶解温度为37℃条件下,获得原生质体的形成率和再生率效果最佳。采用紫外照射时间60 s进行该原生质体诱变,经筛选获得一株遗传性状稳定的高产抗菌脂肽菌株,命名为mutHS-539。研究表明,该突变株mutHS-539发酵上清液对副溶血性弧菌和金黄色葡萄球菌抑菌直径较原始菌mutHS-301分别提高了21.49%和21.05%,提取得到的抗菌脂肽产量较原始菌提高了40%。利用制备型硅胶板对发酵提取物进行分离纯化得到四种组分,分别为a、b、c和d;进一步检测对黄曲霉的抑菌活性,结果发现只有组分d对黄曲霉具有显著的抑制作用。经RP-HPLC分析及液质联用数据比对,该组分d的主要成分为杆菌霉素D。该抗菌脂肽提取物对黄曲霉抑制作用的研究显示,当抗菌脂肽浓度为0.2 mg/mL时能有效抑制黄曲霉菌丝的生长,抑制率达到了74.22%,且对黄曲霉孢子的致死浓度为0.8 mg/mL。     

透明颤菌血红蛋白基因在解淀粉芽孢杆菌中的表达及其影响

将强启动子P43与透明颤菌血红蛋白基因（vgb）通过重叠延伸PCR进行融合,克隆到芽孢杆菌整合表达载体pDG1730中,重组表达载体pDG-P43vgb转化促生防病解淀粉芽孢杆菌FZB42,Wsetern-Blot和CO差光谱分析表明重组菌株FZB42-VHb表达了有活性的VHb蛋白,VHb的表达对重组菌株菌体的生长及抗菌脂肽的产生都有促进作用.在相同培养条件下,重组菌最大菌体密度比原始菌株提高了14.49 %,抗菌脂肽fengycin的产量提高了1.74倍,抗菌脂肽surfactin的产量提高了3.14倍.     

种间双亲灭活原生质体融合选育高产Monacolin K红曲菌

为获得高产MonacolinK的红曲菌菌株,将经农杆菌介导转化获得的携带潮霉素抗性基因且以甘油为原料液态发酵高产MonacolinK的发白红曲菌H2和以大米为原料固态发酵产Monaco-1inK的烟色红曲菌9908作为亲本,对其原生质体分别进行热灭活及紫外灭活,然后对灭活双亲用PEG作融合剂进行原生质体融合。从融合子中选出有潮霉素抗性的突变株,通过发酵与亲本对比,筛选得到一株以大米为原料固态发酵高产MonacolinK的融合株F12．11,其MonacolinK产量达到8．73mg／g;较发白红曲菌H2与烟色红曲菌9908分别提高了100．23％和48．98％;一株以甘油为原料液态发酵高产MonacolinK的融合株F13-2,其MonacolinK的产量达到1752．46mg／L,较发白红曲菌H2与烟色红曲菌9908分别提高了32．98％和1979．33％。     

麦角固醇高产菌株的构建及其培养优化条件的研究

通过初筛、单倍体分离、诱变及酵母菌种间原生质体融合技术构建了2株麦角固醇产量明显提高的优良菌株YEF-21和YEF-29。并对YEF-21的培养优化条件进行了研究。结果表明在优化的实验条件下YEF-21的生物量及麦角固醇含量的综合值分别为原始亲株YE227和YE180的1.54和1.55倍。经遗传稳定性分析,没有发现标记基因的分离现象,从而证明获得的融合菌株是遗传稳定的,是具有一定实际应用价值的麦角固醇高产菌株。     

大豆根腐病生防菌KJB04-11的鉴定及其产生的脂肽类抗生素

从大豆根围筛选到1株对尖孢镰刀菌和立枯丝核菌都具有很好拮抗作用的菌株KJB04-11,经形态观察、生理生化特征和16SrDNA序列分析,属于枯草芽孢杆菌(Bacillussubtilis)。具有抗菌活性的KJB04-11发酵液无菌滤液对热和酸碱具有较强的稳定性。采用SephadexG-25柱层析、反相HPLC和冷冻干燥从KJB04-11发酵液中分离纯化了抗菌活性成分。由红外光谱、MALDI-TOF-MS、氨基酸组成及脂肽合成酶基因扩增结果推测该菌株产生的抗菌物质为C16、C17的mycosubtilin和C15的surfactin。田间试验表明,大豆种子经KJB04-11发酵液包衣处理对大豆根腐病防效为53.6%,大豆产量提高12.5%。     

Breeding of Monascus spp.with high monacolin K production by interspecific protoplast fusion of inactivated parental strains

为获得高产Monacolin K的红曲菌菌株,将经农杆菌介导转化获得的携带潮霉素抗性基因且以甘油为原料液态发酵高产Monacolin K的发白红曲菌H2和以大米为原料固态发酵产Monaco-lin K的烟色红曲菌9908作为亲本,对其原生质体分别进行热灭活及紫外灭活,然后对灭活双亲用PEG作融合剂进行原生质体融合.从融合子中选出有潮霉素抗性的突变株,通过发酵与亲本对比,筛选得到一株以大米为原料固态发酵高产Monacolin K的融合株F12-11,其Monacolin K产量达到8.73 mg/g;较发白红曲菌H2与烟色红曲菌9908分别提高了100.23％和48.98％;一株以甘油为原料液态发酵高产Monacolin K的融合株F13-2,其Monacolin K的产量达到1752.46 mg/L,较发白红曲菌H2与烟色红曲菌9908分别提高了32.98％和1979.33％.     

原生质体融合构建直接利用淀粉产衣康酸菌株

采用衣康酸高产菌株栖土曲霉T-730的原生质体,与葡萄糖淀粉酶产生菌黑曲霉Ni-5k的原生质体进行融合处理.在以30%PEG6000为促融剂,30℃保温20min的融合条件下进行融合,所得异核体经诱导获得3株稳定的融合株(F3,F9,F11).以生淀粉为唯一碳源,对F3,F9,F11的发酵性能测定表明,F3在发酵过程中积累葡萄糖淀粉酶和衣康酸;对F3进行发酵条件初步探讨的结果表明,以10%生淀粉为碳源,连续发酵6d,F3的衣康酸产酸率达40.9mg/mL,对供给淀粉的转化率为40.9%.     

原生质体融合子代的筛选和鉴定

介绍了绿色木霉N6和黑曲霉856原生质体融合子代的研究。经传代、发酵、筛选,从11株初筛的融合子中得到了3株纤维素酶活高且稳定的菌株AT23、AT16、AT34,其CMC酶活分别为亲本绿色木霉N6的2．2倍、1．4倍、1．2倍。并对其进行制霉菌素抗性试验和可溶性蛋白质凝胶电泳分析鉴定。试验结果证明了AT23、AT16、AT34是基因发生了重组的融合子且具有杂种优势。     

Preparation and fusion properties of protoplasts from mature pollen of Nicotiana tabacum

Summary A method to remove the exine from mature tobacco pollen and to release numerous intact pollen protoplasts has been developed. Post-anthesis binucleate pollen was treated with water, buffered with MES at pH 5.5, for two hours. Rupture of the exine was caused by the force of pollen hydration exposing the intine to subsequent enzymatic maceration. The high osmotic pressure (1000 mOsm·kg^-1 H₂O) of pollen protoplasts required a special maceration medium, 4% KCl (w/v). Action of an enzyme solution containing 1% (w/v) Macerozyme and 1% (w/v) Cellulase gave rise to viable protoplasts within 4 hours. When cultured in a tobacco mesophyll protoplast culture medium, the pollen protoplasts underwent regeneration of a cell wall, formation of various tube-shaped structures, and division of the generative nucleus into two nuclei. Using a PEG/Ca²⁺ method pollen protoplasts were fused with diploid mesophyll protoplasts. Evidence of transfer of chloroplasts into the pollen protoplasts was observed after one day of culture.Abbreviations BCP bromocresol purple - FDA fluoresceindiacetate - MES 2-(N-morpholino) ethanesulfonic acid - PEG polyethyleneglycol     

Laser-induced tobacco protoplast fusion

Laser tweezers can manipulate small particles, such as cells and organdies. When coupling them with laser microbeam selective fusion of two tobacco protoplasts containing some chloroplast was achieved. Physical and biological variables that affect laser trapping and laser-induced fusion were also discussed. The results show that the effect of chloroplast content and distribution on the yield of cell fusion is remarkable.     

广藿香原生质体制备、培养与融合技术优化研究

为建立高效稳定的广藿香原生质体培养与融合技术体系,该研究以广藿香愈伤组织悬浮细胞为材料,研究了原生质体制备的酶解条件和培养方法、细胞密度、激素种类和浓度等因素对原生质体培养的影响,并通过测定融合产物直径确立融合细胞筛选范围,进一步研究聚乙二醇浓度、细胞密度、融合时间及融合液加入量等因素对原生质体融合的影响。结果表明:制备原生质体的适宜条件为pH5.8,酶解温度25 ℃; 原生质体培养以铵盐减半的MS₁培养基进行海藻酸钠包埋、激素选用0.2 mg·L^-1 NAA、2.0 mg·L^-1 6-BA,培养密度2.0×10⁵个·mL^-1、蔗糖添加量1.0%、酸水解酪蛋白500 mg·L^-1的条件下原生质体分裂频率、植板率均较高,且开始分裂时间和细胞团形成时间都较短; 双细胞融合产物筛选范围为69.33～87.35 μm; 以40% PEG 6000化学促融30 min、加入0.5倍体积的融合液、细胞密度2.0×10⁵个·mL^-1的条件进行原生质体融合,聚合率可达57.19%; 获得的融合产物经海藻酸钠包埋培育2个月后可观察到再生愈伤组织。     

Interspecies protoplast fusion inLarix: Comparison of electric and chemical methods

Summary Larix was chosen for the study on interspecies protoplast fusion due to its ability to regenerate plants from protoplasts derived from embryogenic cultures.L. laricina line L2 was used in fusion experiments with eitherL. × eurolepis line L6 orL. × leptoeuropaea line L5. A method of unambiguous labeling of parental protoplasts prior to fusion was developed using vital fluorescent dyes. Of a number of dyes tested, only rhodamine B hexyl ester chloride (R6) and 3,3′-dihexylox-carbocyanine iodide (DiOC₆) stained the protoplasts in a consistent and uniform fashion. The fusion of mixed parental protoplasts that were internally labeled was carried out either in the presence of a 20% polyethylene glycol (PEG) solution or in an electric field. The progress of fusion was readily observed, taking only minutes under the experimental conditions. The fusion products could be identified by dual fluorescence several h after the onset of fusion. Heterofusion frequencies of approximately 18% and 6% in the presence of PEG and an electric field, respectively, were attained. Postfusion cultures betweenL. × laricina protoplasts and protoplasts ofL. × leptoeuropaea gave rise to cell colonies and betweenL. laricina andL. × eurolepis, to mature somatic embryos.     

Influence of the composition of the fusion medium on the yield of electrofused yeast hybrids

Abstract Electrofusion between cells of yeast strains with different genetic markers in isotonic sorbitol solutions leads to high yields of hybrids when 0.1 mM Ca²⁺ and 0.5 mM Mg²⁺ salts are aded. On average, 1000–2000 hybrids are obtained when electrofusion is performed (in a helical chamber) compared to a yield of about 40–120 in the absence of these bivalent cations. A further increase in yield can be achieved by the addition of 1 mg/ml albumin, which results in up to 4000 hybrids per experimental run. The entire fusion process leads to very reproducible results in the presence of these substances.     

Interspecific hybridization between Volvariella volvacea and V. bombycina by PEG-induced protoplast fusion

Interspecific hybridization between Volvariella volvacea and V. bombycina was studied using the protoplast fusion technique. The fusion frequency was found to be in the range of 0.032 to 0.333%. Protoplasts from various hybrids were released and regenerated to determine whether they were heterokaryons. In all regenerated colonies, both parental types could not be recovered at the same time. The nuclear DNA contents of hybrids were compared with their parents, and no diploid (parent 1 genome plus parent 2 genome) was found. Some hybrids revealed novel fragments in mitochondrial rDNA PCR profiles, which indicated that rearrangement of mtDNA could have occurred after fusion. Results from arbitrarily-primed polymerase chain reaction (AP-PCR) fingerprints also revealed that the majority of hybrids were similar to one parental type, but heterologous fragments were found in some hybrids.     

Genetic analysis of intraspecies recombinant formation by protoplast fusion with three species of Streptomyces

Abstract Protoplast fusion was shown to produce high frequencies of recombinant progeny in intraspecies crosses with auxotrophic mutants of Streptomyces canescens, Streptomyces griseus and Streptomyces limosus . The fused protoplasts were regenerated on non-selective media and the progeny spores subsequently analysed on selective media to allow detection of all possible genotypes. Prototrophic recombinants arose with frequencies of between 1% and 8%. All 4 possible genotypes were recovered in a series of 2-factor crosses and 6 of the 8 possible genotypes were detected in a 3-factor cross. In spite of attempts to equalise the ratios of parental protoplasts in the fusion mixture, there were noticeable deviations from unity in the ratios of parental genotypes in the progeny.