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玫瑰黄链霉菌Men-myco-93-63与变铅青链霉菌TK24的原生质体种间融合
Interspecific Protoplast Fusion between Streptomyces Roseoflavus Men-myco-93-63 and Streptomyces Lividans TK24
【作者】 刘莲娜；
【作者基本信息】 河北农业大学 ， 生物化学与分子生物学， 2011， 硕士
【摘要】 玫瑰黄链霉菌Men-myco-93-63对多种植物病原真菌和细菌有较强的抑制作用,温室和大田试验表明该菌株的代谢产物对棉花黄萎病和瓜类白粉病有较好的防效作用,其有效成分和作用机理正在研究中。双亲株灭活原生质体融合摆脱了遗传标记给育种工作带来的束缚,使融合子的检出变得直观,是值得推广的一种选育高产菌株的方法。本文对玫瑰黄链霉菌Men-myco-93-63和变铅青链霉菌TK24的原生质体分别进行热灭活和紫外灭活,在PEG 4000介导下融合,所得“融合子”以棉花黄萎病菌和马铃薯疮痂病菌为指示菌筛选抗生素高产优质菌种,传代培养检测高产“融合子”的稳定性,并观察“融合子”的培养特征和形态特征;利用基因组酶切和可溶性蛋白质电泳验证融合子与亲本的遗传关系;通过摇瓶发酵获得高产融合菌株抗生素粗品的产量,得出增产率;以融合子RH19为代表,采用亲株的发酵培养基对融合子RH19进行摇瓶发酵实验,利用薄层色谱(TLC)和高效液相色谱(HPLC)分析融合菌株抗生素粗品。通过玫瑰黄链霉菌Men-myco-93-63与变铅青链霉菌TK24原生质体的种间融合实验,得到了十四株对棉花黄萎病菌和马铃薯疮痂病菌具有抑制作用的融合子,融合子RH19抑菌活性明显强于玫瑰黄链霉菌Men-myco-93-63。经过十次传代发现,融合子的遗传稳定性良好。高产融合子在固体R2YE培养基及液体燕麦培养基上均产生黄绿色色素,并且培养特征和形态特征明显不同于亲株。对基因组DNA进行酶切发现融合子的酶切片段与亲本不同;对“融合子”RH19及亲本的可溶性总蛋白进行了SDS-PAGE分析,发现“融合子”RH19的可溶性总蛋白图谱与双亲有一定的相似性又产生了新的条带;摇瓶发酵实验结果显示融合子粗提抗生素的产量为0.18g/L,而玫瑰黄链霉菌Men-myco-93-63为0.07g/L,增产了159%;TLC和HPLC结果表明此菌株能够产生亲本不能产生的新的次级代谢产物。本研究证实了玫瑰黄链霉菌Men-myco-93-63和变铅青链霉菌TK24之间发生了基因重组。该研究结果为放线菌防治作物病害提供了新资源,具有广泛的应用前景和重要的应用价值。探索了玫瑰黄链霉菌Men-myco-93-63和变铅青链霉菌TK24的原生质体电击融合方法的可行性,研究结果发现,电击融合并不能得到具有优良性状的融合子,电击条件有待于进一步进行摸索。
【Abstract】 Streptomyces roseoflavus Men-myco-93-63 could inhibit many pathogen fungus and bacteria in lab. The fermentation liquid had a strong antibiosis activity against cotton verticillium wilt and cucurbits powdery mildew in greenhouse and field. The effective elements and function mechanism were under study. Parental strains of inactivated protoplasts fusion can get rid of the obstacles that genetic markers brought to breeding work, make the screening of fusants intuitive, and it is a good mearure of breeding high-yeilding strains and to be popularized.The research was carried out to inactivate protoplasts of Streptomyces roseoflavus Men-myco-93-63 and Streptomyces lividans TK24 using heat and UV, respectively, and two inactivated protoplasts were fused with PEG 4000. The high-yeilding fusants which have stronger inhibitory activity to Verticillium dahliae V41 and Streptomyces scabies H2 than Streptomyces roseoflavus Men-myco-93-63 were screened. The stability of fusants was detected by subcultured and the characteristics of culture and morphology was observed. The genetic relationship to parents was verified by genome digestion and electrophoresis of soluble proteins. The yield of antibiotics crude produced by high-yeilding strains was obtained by shaking fermentation and the increase rate was got. The shaking fermentation experiment of Fusant RH19 was carried out using the medium of parent, and the antibiotics crude of Fusant RH19 was analyzed with TLC and HPLC methods. We got 14 fusants which have inhibition effect to Verticillium dahliae V41 and Streptomyces scabies H2 through protoplast fusion between Streptomyces roseoflavus Men-myco-93-63 and Streptomyces lividans TK24. The inhibition effect of Fusant RH19 was significantly higher than Streptomyces roseoflavus Men-myco-93-63. The high-yielding fusant RH19 produced yellowish pigments in solid R2YE medium and liquid oats medium, and the characteristics of culture and morphology were different from parental strains. After subcultured 10 times, the fusants have stable heredity. The digestion of genome showed that the restriction fragments differ from that of parents. SDS-PAGE analysis showed that the soluble proteins of Fusant RH19 were different from that of parents, and noval bands were produced. The flasking fermentation experiment result showed that the yeild of antibiotics crude of Fusant RH19 was 0.18 g/L, and that of Streptomyces roseoflavus Men-myco-93-63 was 0.07 g/L, with 159% increase rate. TLC and HPLC results showed that Fusant RH19 produced new secondary metabolites that parent strain could not. This study confirmed that genome combination occured between Streptomyces roseoflavus Men-myco-93-63 and Streptomyces lividans TK24. The research results would provide new resources for prevention of crop diseases using actinomycosis, and it has a broad prospect of application and important application value.This study explored the feasibility of electroporation between Streptomyces roseoflavus Men-myco-93-63 and Streptomyces lividans TK24, the results showed that the method of protoplast fusion using electroporation could not obtain recombinants which has superior character, the condition of electroporation was to be groped.