玫瑰黄链霉菌Men-myco-93-63活性组分的制备及相关基因克隆研究
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摘要
玫瑰黄链霉菌Men-myco-93-63是一种分离自马铃薯疮痂病自然衰退土壤中的重要生防菌,对黄瓜白粉病、棉花黄萎病等多种重要植物病害均有良好的防治效果。本文对玫瑰黄链霉菌Men-myco-93-63的生物活性组分及其相关基因进行了研究。
玫瑰黄链霉菌Men-myco-93-63在燕麦液体培养基进行摇瓶发酵培养,每1000ml发酵液可提纯获得172-2mg粗提抗生素,采用牛津杯法活性检测表明对棉花黄萎病菌、马铃薯疮痂病菌抑菌活性稳定。定量测定表明马铃薯疮痂病菌最敏感,0.3mg/ml粗提抗生素即可完全抑制其生长;粗提抗生素对变铅青链霉菌(TK24和TK54)的抑菌活性较低。薄层分析表明甲醇∶氯仿为1∶6时是粗提抗生素薄层层析的最佳展开条件,可分离出7个组分,其中组分Ⅲ、Ⅳ、Ⅶ对马铃薯疮痂病菌具有抑菌活性,组分Ⅶ还对棉花黄萎病菌有抑菌活性。抗生素组分Ⅲ对马铃薯疮痂病菌的抑菌活性最强。采用制备型薄层硅胶板对抗生素组分Ⅲ进行了大量制备,TLC和HPLC分析表明薄层分离制备的抗生素组分Ⅲ纯度较高,紫外光谱最大吸收波长为198.1nm和274.5nm,与先前用HPLC制备的组分13近似。
玫瑰黄链霉菌Men-myco-93-63对自身抗生素组分Ⅲ具有较高的耐受性,因而其基因组中有相应的抗性基因。采用鸟枪法基因克隆技术在变铅青链霉菌中克隆了玫瑰黄链霉菌Men-myco-93-63抗生素组分Ⅲ的抗性基因,定位于1.7kb基因片段上,该基因片段有4个ORFs,ORF1编码386aa,与灰色链霉菌和天蓝色链霉菌的β-半乳糖苷酶高度同源;ORF2编码83aa,与除虫链霉菌MA-4680、天蓝色链霉菌、山丘链霉菌核糖体大亚基L2蛋白RNA结合域以及南昌链霉菌、除虫链霉菌的葡聚糖内切酶高度同源,可能是链霉菌尚未发现的新基因。ORF3编码氨基酸与动球菌(Kineococcus radiotolerans SRS30216)的醛/酮还原酶一致性和相似性均为66%。在ORF3相应位置的互补链上(1697-1352bp)存在一个ORF4,编码115aa,与除虫链霉菌的分泌蛋白(multidrug resistance protein,SpcT-like efflux protein,450aa)有较低的同源性,这一编码区应进一步亚克隆研究其功能。
采用鸟枪法以质粒pIJ702为载体在链霉菌常用宿主菌中建立了玫瑰黄链霉菌Men-myco-93-63合成相关基因克隆文库,以马铃薯疮痂病菌为指示菌从6000个转化
Streptomyces roseoflavus Men-myco-93-63, which isolated from the potato scab decline field, was an excellent strain to control some plant diseases such as cucumber powdery mildew, cotton verticillium wilt and so on. The research was carried out to extract the active agent to inhibit the Streptomyces scabies from the fermentation and clone the genes related to antibiotic biosynthesis.S. roseoflavus Men-myco-93-63 was shake-flask culture in oatmeal medium and could produce 172.2 mg crude antibiotic in 1000 ml fermentation. The activity of the crude antibiotic was detected with oxford cup and the results showed that it could inhibit S. scabies S87 and Verticillium dahliaeV41 invariably. The quantitative assays also showed that 0.3mg/ml crude antibiotic was enough to inhibit S. scabies S87, but not to S. lividans. The crude antibiotic was analyzed by TLC and the results showed that it was the suitable developing solvent while the ratio of methyl alcohol and chloroform was 1:6 and 7 components could be separated on silica gel plate. The component Ⅲ, Ⅳ, Ⅶ had the antibacterial action to S. scabies S87 and the component Ⅲ was the most active agent. The component Ⅶ had the fungistatic action to V. dahliae V41 yet. Then the component III was prepared largely on preparative silica gel plate and the analysis of TLC and HPLC expressed it was very purity. The UV spectrum of the component Ⅲ showed that the maximum absorb wavelength (λmax) was 198.1nm and 274.5nm.The value of MIC (minimum inhibitory concentration) was determined and the results showed that S. lividans TK54 with pIJ702 could be inhibited when one milliliter MM medium had 80μl component Ⅲ. S. roseoflavus Men-myco-93-63 could be resistant higher concentration component III and the resistance gene was cloned by the shotgun method in S. lividans TK54 which located in the 1.7kb DNA fragment. This fragment had 4 ORFs, ORF1 encoded 386aa which was homologous to beta-galactosidase from S. grieus and S. colicolor. ORF2 which may be a new gene in streptomyces encoded a putative protein comprising 83aa. N-terminal of the putative protein was high homologous to RNA binding domain of ribosomal protein L2 from S. avermitilis MA-4680, S. collinus, S. coelicolor and C-terminal was very homologous to the endoglucanase from S. nanchangensis and S. avermitilis MA-4680. ORF3 encoded a putative protein
homologous to aldehyde/ ketone reductase from Kimococcus radiotolerans SRS30216. ORF4 was located in the complementary strand corresponding to ORF3 and encoded 115aa which was homologous lowly to multidrug resistance protein (SpcT-like efflux protein, 450aa) from S. avermitilis MA-4680. The coding sequence should be subcloned and identify the function.The clone library to isolate the gene related to the antibiotic biosynthesis was constructed by cloning Sau3A I partial-digested genomic DNA fragments ofS. roseoflavus Men-myco-93-63 into the plasmid pIJ702 in the host S. lividans TK54. This library was screened by inhibition to the indicate strain S. scabies S87. It was found that 3 clones had the ability to antagonize to S. scabies S87 and SA-3 was the strongest antagonist. The plasmid isolated from SA-3 was named pSA3 and about 8.5kb extrinsic fragment was detected. The function of pSA3 was identified and it was proved that S. lividans TK54 transformed by pSA3 could inhibit S. scabies S87, but no active to V. dahliaeWAl. The fermentation was prunosus when the transformant was shake-flask culture in oatmeal medium and the extraction with ethyl acetate could inhibit S. scabies S87. The HPLC analysis of the extraction demonstrated that they were very different between S. lividans TK54 transformed by pSA3 and by pIJ702. The analysis of sequence with Frameplot2.3 showed that the fragment included 10 ORFs and 2 incomplete ORFs. The results of blastp showed 2 ORFs were homologous to whiB (transcriptional regulator) and RNA/DNA helicase which had DEXDc (DEAD-like helicases superfamily) conserved domain and another ORF had HTHXRE (Helix-turn-helix, xenobiotic respohse element family of transcriptional regulators) and COG 1426 conserved domain, which was homologous lowly to DNA-binding protein. The putative proteins encoded by other 7 ORFs which had not homologous sequence were unknown about the functions. The fragment was likely related to regulation of the antibiotic biosynthesis. The extrinsic fragment gene sequence and the HPLC analysis of the transformant fermentation showed that this fragment was involved in the biosynthesis of the antibiotic in S. roseoflavus Men-myco-93-63.
玫瑰黄链霉菌Men-myco-93-63在燕麦液体培养基进行摇瓶发酵培养,每1000ml发酵液可提纯获得172-2mg粗提抗生素,采用牛津杯法活性检测表明对棉花黄萎病菌、马铃薯疮痂病菌抑菌活性稳定。定量测定表明马铃薯疮痂病菌最敏感,0.3mg/ml粗提抗生素即可完全抑制其生长;粗提抗生素对变铅青链霉菌(TK24和TK54)的抑菌活性较低。薄层分析表明甲醇∶氯仿为1∶6时是粗提抗生素薄层层析的最佳展开条件,可分离出7个组分,其中组分Ⅲ、Ⅳ、Ⅶ对马铃薯疮痂病菌具有抑菌活性,组分Ⅶ还对棉花黄萎病菌有抑菌活性。抗生素组分Ⅲ对马铃薯疮痂病菌的抑菌活性最强。采用制备型薄层硅胶板对抗生素组分Ⅲ进行了大量制备,TLC和HPLC分析表明薄层分离制备的抗生素组分Ⅲ纯度较高,紫外光谱最大吸收波长为198.1nm和274.5nm,与先前用HPLC制备的组分13近似。
玫瑰黄链霉菌Men-myco-93-63对自身抗生素组分Ⅲ具有较高的耐受性,因而其基因组中有相应的抗性基因。采用鸟枪法基因克隆技术在变铅青链霉菌中克隆了玫瑰黄链霉菌Men-myco-93-63抗生素组分Ⅲ的抗性基因,定位于1.7kb基因片段上,该基因片段有4个ORFs,ORF1编码386aa,与灰色链霉菌和天蓝色链霉菌的β-半乳糖苷酶高度同源;ORF2编码83aa,与除虫链霉菌MA-4680、天蓝色链霉菌、山丘链霉菌核糖体大亚基L2蛋白RNA结合域以及南昌链霉菌、除虫链霉菌的葡聚糖内切酶高度同源,可能是链霉菌尚未发现的新基因。ORF3编码氨基酸与动球菌(Kineococcus radiotolerans SRS30216)的醛/酮还原酶一致性和相似性均为66%。在ORF3相应位置的互补链上(1697-1352bp)存在一个ORF4,编码115aa,与除虫链霉菌的分泌蛋白(multidrug resistance protein,SpcT-like efflux protein,450aa)有较低的同源性,这一编码区应进一步亚克隆研究其功能。
采用鸟枪法以质粒pIJ702为载体在链霉菌常用宿主菌中建立了玫瑰黄链霉菌Men-myco-93-63合成相关基因克隆文库,以马铃薯疮痂病菌为指示菌从6000个转化
Streptomyces roseoflavus Men-myco-93-63, which isolated from the potato scab decline field, was an excellent strain to control some plant diseases such as cucumber powdery mildew, cotton verticillium wilt and so on. The research was carried out to extract the active agent to inhibit the Streptomyces scabies from the fermentation and clone the genes related to antibiotic biosynthesis.S. roseoflavus Men-myco-93-63 was shake-flask culture in oatmeal medium and could produce 172.2 mg crude antibiotic in 1000 ml fermentation. The activity of the crude antibiotic was detected with oxford cup and the results showed that it could inhibit S. scabies S87 and Verticillium dahliaeV41 invariably. The quantitative assays also showed that 0.3mg/ml crude antibiotic was enough to inhibit S. scabies S87, but not to S. lividans. The crude antibiotic was analyzed by TLC and the results showed that it was the suitable developing solvent while the ratio of methyl alcohol and chloroform was 1:6 and 7 components could be separated on silica gel plate. The component Ⅲ, Ⅳ, Ⅶ had the antibacterial action to S. scabies S87 and the component Ⅲ was the most active agent. The component Ⅶ had the fungistatic action to V. dahliae V41 yet. Then the component III was prepared largely on preparative silica gel plate and the analysis of TLC and HPLC expressed it was very purity. The UV spectrum of the component Ⅲ showed that the maximum absorb wavelength (λmax) was 198.1nm and 274.5nm.The value of MIC (minimum inhibitory concentration) was determined and the results showed that S. lividans TK54 with pIJ702 could be inhibited when one milliliter MM medium had 80μl component Ⅲ. S. roseoflavus Men-myco-93-63 could be resistant higher concentration component III and the resistance gene was cloned by the shotgun method in S. lividans TK54 which located in the 1.7kb DNA fragment. This fragment had 4 ORFs, ORF1 encoded 386aa which was homologous to beta-galactosidase from S. grieus and S. colicolor. ORF2 which may be a new gene in streptomyces encoded a putative protein comprising 83aa. N-terminal of the putative protein was high homologous to RNA binding domain of ribosomal protein L2 from S. avermitilis MA-4680, S. collinus, S. coelicolor and C-terminal was very homologous to the endoglucanase from S. nanchangensis and S. avermitilis MA-4680. ORF3 encoded a putative protein
homologous to aldehyde/ ketone reductase from Kimococcus radiotolerans SRS30216. ORF4 was located in the complementary strand corresponding to ORF3 and encoded 115aa which was homologous lowly to multidrug resistance protein (SpcT-like efflux protein, 450aa) from S. avermitilis MA-4680. The coding sequence should be subcloned and identify the function.The clone library to isolate the gene related to the antibiotic biosynthesis was constructed by cloning Sau3A I partial-digested genomic DNA fragments ofS. roseoflavus Men-myco-93-63 into the plasmid pIJ702 in the host S. lividans TK54. This library was screened by inhibition to the indicate strain S. scabies S87. It was found that 3 clones had the ability to antagonize to S. scabies S87 and SA-3 was the strongest antagonist. The plasmid isolated from SA-3 was named pSA3 and about 8.5kb extrinsic fragment was detected. The function of pSA3 was identified and it was proved that S. lividans TK54 transformed by pSA3 could inhibit S. scabies S87, but no active to V. dahliaeWAl. The fermentation was prunosus when the transformant was shake-flask culture in oatmeal medium and the extraction with ethyl acetate could inhibit S. scabies S87. The HPLC analysis of the extraction demonstrated that they were very different between S. lividans TK54 transformed by pSA3 and by pIJ702. The analysis of sequence with Frameplot2.3 showed that the fragment included 10 ORFs and 2 incomplete ORFs. The results of blastp showed 2 ORFs were homologous to whiB (transcriptional regulator) and RNA/DNA helicase which had DEXDc (DEAD-like helicases superfamily) conserved domain and another ORF had HTHXRE (Helix-turn-helix, xenobiotic respohse element family of transcriptional regulators) and COG 1426 conserved domain, which was homologous lowly to DNA-binding protein. The putative proteins encoded by other 7 ORFs which had not homologous sequence were unknown about the functions. The fragment was likely related to regulation of the antibiotic biosynthesis. The extrinsic fragment gene sequence and the HPLC analysis of the transformant fermentation showed that this fragment was involved in the biosynthesis of the antibiotic in S. roseoflavus Men-myco-93-63.
引文
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