链霉菌原生质体的再生

（续１９９８年第３３卷第１期第４１页）３原生质体的再生链霉菌原生质体的再生是指经去壁的原生质体在高渗的再生培养基上,一方面再生出原有的细胞壁,恢复菌丝原来的完整形态,另一方面恢复细胞的生理功能,保证细胞萌发、生长和分裂。原生质体再生过程有以下几个步骤...     

链霉菌原生质体的制备

原生质体是细胞去除了坚韧细胞壁后对渗透压极为敏感的球质体,最外层是裸露的细胞质膜,失去了细胞壁的原生质体,染色体ＤＮＡ在诱变剂作用下,更易引起死亡突变,敏感性提高。菌种的敏感性越高,诱发突变的机会越大。因此,用原生质体代替孢子、菌丝细胞作为诱变育种的...     

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。     

褐黄孢链霉菌原生质体制备与再生

以纳他霉素产生菌株褐黄孢链霉菌SG-2002为出发菌株,考察了菌丝体培养基、甘氨酸浓度、培养时间、溶菌酶浓度、酶解温度、酶解时间及再生培养基对原生质体形成与再生的影响.原生质体形成和再生的最佳条件为S培养基中添加1%的甘氨酸,菌丝体培养 30 h,溶菌酶浓度 40 mg/(g菌体干重),酶解温度 30 ℃,酶解时间 60 min.褐黄孢链霉菌的原生质体形成量达到4.0×106 个/mL,再生培养基选择R5′培养基,再生率为9.0%.     

天兰淡红链霉菌原生质体再生株激光辐照的研究

本文首次报导天兰淡红链霉菌原生质体再生株铜蒸气激光辐照的研究结果,从中得到生产罐中柔红霉素发酵单位比其出发株提高１２．９％的高产株Ｃ－８２,其最高发酵单位达国内最好水平,已在国内应用。     

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.     

应用原生质体技术选育天兰淡红链霉菌

本文报道柔红霉素产生菌——天兰淡红链霉菌原生质体的制备和再生方法,并从再生菌落中筛选得到摇瓶发酵单位提高５％的高产菌株３０＃。将再生高产株经铜蒸气激光辐照,得到在生产罐中发酵水平比对照出发菌株提高１２．９％的高产菌株８２＃。     

链霉素生产菌—灰色链霉菌和热灰紫链霉菌的原生质体融合的研究

本文采用原生质体融合技术,把链霉素生产菌——灰色链霉菌No．45 Streptomyces griseus No.45(Lin,Rif‘)同耐高温的不产生抗生素的热灰紫链霉菌T272 Strep-griseus thermogriseoviolaceus T272(Lin^s,Rif^f)进行了原生质体融合。以抗性为选择标记,以PEG6000为助融剂,选出了融合体。制备超薄切片后在电镜下观察了原生质体融合的详细过程。在链霉素生物合成受抑制的高温(37℃)下,测定了融合体的抑菌括性,从形态上与两亲株不同的46株融合体中发现6．3％的融合体既有耐高温的特性也有抑菌的活性。     

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     

不吸水链霉菌梧州新亚种基因文库的构建

以不吸水链霉菌梧州新亚种为材料,提取的总基因组经Sau3AI不完全酶切,回收3~9 kb DNA酶切片段,用T4连接酶将回收片段按一定比例与经BamHI酶切、南极热敏磷酸酶去磷酸化处理后的pUC18质粒载体连接,热激法转化受体菌E.coliDH5α,在含有IPTG/X-gal和Amp 的LB平板上筛选重组子,所得克隆数为9×103个,以不吸水链霉菌染色体基因组大小为7 Mb计算,概括了不吸水链霉菌梧州新亚种约99%的基因组,达到了建库要求的理论值。随机挑取白色菌落,提取重组质粒进行酶切电泳分析,均有外源片段插入。基因文库的构建,为进一步深入研究梧宁霉素生物合成基因结构及功能奠定基础。     

Characterization of Streptomyces D3 derived from protoplast fusion between Streptomyces cyaneus 190-1 and Streptomyces griseoruber 42-9

Streptomyces D3, derived from protoplast fusion between Streptomyces cyaneus 190-1 and Streptomyces griseoruber 42-9, has the ability to produce high levels of xylose isomerase when grown on hemicellulosic materials such as xylan as the carbon source. Comparison between the partial nucleotide sequences of the 16S ribosomal RNA genes from S. cyaneus 190-1, S. griseoruber 42-9, and fusant D3 showed that the 16S rRNA gene of fusant D3 was identical to that of S. cyaneus 190-1. Partial sequence analysis of the xylose isomerase genes also indicated that the gene of fusant D3 was identical to that of S. cyaneus 190-1. The partial DNA fragments for the xylanase genes (xlnA and xlnB) of fusant D3 were amplified by PCR, and subjected to Southern hybridization analysis. The results revealed that the xlnB gene of fusant D3 was similar to that of S. cyaneus 190-1, but that the xlnA gene of fusant D3 was similar to that of S. griseoruber 42-9. These results suggest that the majority of the genome of fusant D3 may be derived from S. cyaneus 190-1.     

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.     

食用菌原生质体巨大化及其再生的初步观察

通过观察几种食用菌原生质体的巨大化过程,发现含有细胞壁裂解酶的高渗营养液是比较理想的巨大化反应液;巨大化反应不同时间的原生质体再生率有“高→低→高”的起伏变化;巨大化后原生质体再生率普遍提高。最后测定了巨大化时间、渗透压稳定剂、培养基及其碳源等对巨大化原生质体再生率的影响。     

影响枯草杆菌原生质体转化的因素

原生质体转化是将外源基因导入细菌的主要方法之一,其转化频率受制于多种因素。本研究以激BacillussubtilisDB104为宿主菌,以枯草杆菌的高表达型质粒pNQ122为外源DNA,研究了原生质体再生率、原生质体浓度和用于制备原生质体的细胞生长期对转化频率的影响,获得了在该系统中实现高频率转化的条件。该转化条件使外源基因在多个B.subtilis菌株中的转化成为可能,并使从B.subtilis中筛选中性蛋白酶基因获得成功。     

Genome shuffling enhances biocontrol abilities of Streptomyces strains against two potato pathogens

Aims: To employ the genome shuffling technique for improving the phenotype of a biocontrol control agent of the genus Streptomyces. Methods and Results: Two rounds of genome shuffling (GS) were carried out with Streptomyces melanosporofaciens EF‐76, a geldanamycin producer. Six fusants that showed optimized in vitro antagonistic activity against Streptomyces scabies or Phytophthora infestans, two important pathogens of potato crops, were selected. All selected fusants retained the capacity to produce geldanamycin, but none overproduced this antibiotic. The higher antagonism ability appeared to result from a diversification of secreted metabolites. Seven or eight metabolites were detected in the HPLC profiles of parental strains, whereas 12–15 were detected in fusant strains. Biocontrol assays revealed that four of six fusants protected tubers more efficiently than parental strains. Conclusions: GS emerged as an elegant and rapid tool to optimize the antagonistic ability of Streptomyces strains. Optimization of the in vitro antagonistic activity against plant pathogens appears to be an effective approach to select for improved biocontrol agents. The enhanced phenotype did not depend on an overproduction of a specific antibiotic but rather on the secretion of a wider variety of secondary metabolites. Significance and Impact of the Study: Improved capacities of a biocontrol agent compensate for the lack of efficient chemical control of potato scab.     

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%。     

Optimization of transformation procedures in avermectin high-producing Streptomyces avermitilis

The optimal pH conditions for efficient transformation of protoplasts and intact cells were established in avermectin high-producing mutants, ATCC31780 and L-9. Among all factors tested, protoplast buffer pH was elucidated as the most important factor influencing transformation efficiency. The optimal pH of the protoplast buffer for the regeneration of ATCC31780 was 6.5, and using this condition, 4.5 × 10⁶ transformants per g of pIJ702 were produced. At pH 6.3, the maximal number of L-9 transformants was 1.6 × 10⁵ per g of the same plasmid. However, the protoplasting process decreased avermectin productivity to half or one-sixth of ATCC31780 or L-9, respectively. To avoid the productivity loss, electroporation of intact cells without lysozyme treatment was developed for these mutant strains even though this method was approximately 100-fold less efficient. In this method, the initial pH of culture medium was elucidated as a critical factor and optimized for both transformation efficiency and avermectin productivity.