【作者】 魏颖颖；

【导师】 吴元华；

【作者基本信息】 沈阳农业大学 ， 植物病理学， 2009， 博士

【摘要】 嘧肽霉素是本研究室研发的具有自主知识产权的新型抗病毒农用抗生素,由不吸水链霉菌辽宁变种(Streptomyces ahygroscopicus var.Liaoningensis)产生,对多种植物病毒病害、真菌病害都有很高的防效,为了从分子生物学角度研究嘧肽霉素的生物合成,本文建立了不吸水链霉菌的遗传转移系统,克隆获得了嘧肽霉素的部分生物合成相关基因,并对其功能进行了研究,获得的结果如下:1.利用紫外诱变方法,获得了1株嘧肽霉素生物合成阻断突变株UV2136和1株嘧肽霉素高产菌株,前者为互补克隆策略研究嘧肽霉素生物合成基因提供了转化受体,后者为嘧肽霉素的生产提供了高产菌株。2.针对嘧肽霉素的生物合成阻断突变株UV2136,本文研究了其最佳的菌体培养、原生质体制备和再生条件。采用改良的含15%蔗糖、0.5%甘氨酸的YEME液体培养基振荡培养36h,获得细腻丰富的菌丝体;对不同的酶浓度、酶解温度和酶解时间进行3因子4水平的正交试验,筛选最佳酶解条件为:溶菌酶浓度2mg/ml,28℃,作用90min,可获得最高的制备率99%;再生培养基以R2YE为最佳,采取直接涂布方式,25.5℃培养可达到48%的再生率;3.利用原生质体转化法建立了嘧肽霉素生物合成阻断突变株的质粒转移系统,本试验为后续的互补克隆提供了方法,也是首次建立不吸水链霉菌的基因转移系统。UV2136对硫链丝菌素高度敏感,选择含有该抗生素标记的质粒pIJ702和pWHM3,探索其转化UV2136的可能性。结果表明,UV2136含有较强的限制修饰系统,采取热衰减法获得原生质体、原生质体热处理、冷处理、EDTA处理、质粒DNA变性等,均不能实现质粒pIJ702向UV2136的转化。来自非甲基化宿主E.coli ET12567的pWHM3也无法实现转化,而源于E.coli DH5α的甲基化pWHM3,可以低频转化菌株UV2136,转化效率为4转化子/μg pWHM3,经UV2136自身修饰的pWHM3可实现高效转化,达到10~4转化子/μg pWHM3。4.利用接合转移实现了温敏感型质粒向不吸水链霉菌辽宁变种的转化,为研究嘧肽霉素生物合成相关基因的功能分析提供了载体。利用BclI酶切质粒pIJ702,回收其中含有硫链丝菌素抗性基因的1,055bp的BclI-BclI片段,插入质粒pKC1139的BglⅡ位点,构建pKC1139-tsr质粒;设计引物,以pIJ702为模板扩增了其中含硫链丝菌素的目的基因,经ApaI、NheI双酶切,插入到pSET152的ApaI-NheI酶切位点,构建pSET152-tsr质粒;pKC1139-tsr质粒可通过接合转移实验转入不吸水链霉菌辽宁变种,为后续的基因功能研究提供了载体,但pSET152-tsr质粒不能倒入,表明嘧肽霉素产生菌中可能不含有attp位点。5.采用互补克隆策略获得了3个可能的嘧肽霉素生物合成相关基因,cytoA、cytoB和cytoC,cytoA、B分别编码双组分系统的传感激酶和反应调节因子,cytoC编码保守假定蛋白,并分别对其进行了功能研究,表明cytoA、B可能参与嘧肽霉素生物合成的调控,cytoC直接参与嘧肽霉素的生物合成。利用EcoRI、HindⅢ对野生型菌株04-2-2基因组DNA进行不完全酶切,回收3-15kb DNA片断,分别与经EcoRI、HindⅢ酶切的pWHM3连接,转化嘧肽霉素生物合成阻断突变株,筛选硫链丝菌素抗性菌落,对其进行嘧肽霉素活性测定,从EcoRI酶切产物中获得1个恢复嘧肽霉素产生能力的克隆,定名为UV2136-R,对其中的插入片段进行分析,结果表明,该片断含有3个开放阅读框,分别定名为cytoA、cytoB和cytoC。其中,cytoA大小为1464bp,编码一个由487个氨基酸残基组成的蛋白质,数据库比较结果表明cytoA编码的氨基酸序列属于组氨酸蛋白激酶超家族,与始旋链霉菌中的传感激酶有较高的同源性,一致性可达66%,说明cytoA的编码产物可能为组氨酸激酶;cytoB大小为570bp,编码一个由189个氨基酸残基组成的蛋白质,数据库比较结果表明cytoB编码的氨基酸序列与灰色链霉菌中的反应调节因子有很高的同源性,一致性可达86%,表明cytoB的编码产物为反应调节因子,与cytoB编码的传感激酶共同组成二组分系统,基因登陆号为GQ149124,两者的阻断使嘧肽霉素的产生能力降低,可能为正调控基因;cytoC大小为783bp,基因登陆号为GQ250103,编码一个由260个氨基酸残基组成的蛋白质,数据库比较结果表明,cytoC的编码产物属于DUF899超家族,与固氮菌和始旋链霉菌中的保守假定蛋白的同源性高,一致性可达85%,该假定蛋白与硫氧环蛋白相似,其阻断使菌株丧失嘧肽霉素产生能力,说明其是参与嘧肽霉素生物合成的一个重要基因。6.利用同源克隆获得了一个嘧肽霉素生物合成基因cytoD,可能编码葡萄糖基转移酶,功能验证表明其直接参与嘧肽霉素的生物合成。嘧肽霉素与杀稻瘟菌素S有相同的组成部分,即胞啶咛,推知可能有相同或相近功能的生物合成基因存在,以灰色产色链霉菌中催化胞啶咛合成的blsD基因的部分序列为探针,对嘧肽霉素产生菌04-2-2的完全酶切片段进行Southern杂交,从XbaI完全酶切的基因组DNA中获得一条2.1kb左右的杂交片段,利用基因小库构建、菌落原位杂交等克隆得到了含有杂交片段的菌落,其中的质粒定名为pUC18-CGA,测序结果表明其中含有1个完整的开放阅读框,cytoD由981bp组成,编码326aa组成的蛋白质,Blast结果表明,其编码产物与灰色链霉菌中的葡萄糖基转移酶有较高的同源性,一致性可达85%,说明cytoD可能为葡萄糖基转移酶基因。cytoD的阻断使菌株丧失嘧肽霉素产生能力,说明其是参与嘧肽霉素生物合成的一个重要基因。以上工作对克隆获得完整的嘧肽霉素生物合成基因簇、研究嘧肽霉素的生物合成途径具有重要意义。更多还原

【Abstract】 Cytosinpeptidemycin is a newly reported antiphytoviral agricultural antibiotic produced by Streptomyces ahygroscopicus var.Liaoningensis,which owns high control effect to some plant viral and fungal diseases.To study the biosynthesis of cytosinpeptidemycin in the view of molecular biology,the gene transformation system of Streptomyces ahygroscopicus was developed,some biosynthetic gene was cloned and analyzed in the study.The results are as fellows:1.Wild strain of Streptomcyes ahygroscopicus was mutagenized by UV irradiation.A matant,UV2136,blocked in the biosynthesis of cytosinpeptidemycin was used to clone cytosinpeptidemycin biosynthetic genes.Meanwhile,a high-producing strain was induced, which can be used in the cytosinpeptidemycin production.2.Targeted with UV2136,the mutant of Streptomyces ahygroscopicus var.Liaoningensis blocked in cytosinpeptidemycin biosynthesis,the optimal conditions for mycelium growth, protoplasts formation and regeneration were developed.Exquisite and abundant mycelium was typically grown in 40ml of adopted YEME medium containing 15%of glucose and 0.5% of glycine.The optimal lysozyme system,including lysozyme concentration,temperature and lysis time,was studied by orthogonal design analysis,and the results indicated that the highest formation ratio of 99%was achieved when the lysozyme solution was 2mg/ml, incubated at 28℃for 90min.The protoplasts were plated on dry plates of R₂YE by spreading with a glass spreader,and the regeneration ratio was over 48%when cultured at 25.5℃.3.The gene transformation system of mutant UV2136 was developed by protoplasts transformation in the study.UV2136,the mutant blocked in cytosinpeptidemycin biosynthesis,was highly sensitive to thiostrepton,plasmid pIJ702 and pWHM3 containing thiostrepton resistance gene,tsr,were used to transform UV2136.Without endogenous plasmid,UV2136 owns strong restriction and modification systems which prevent the transformation of foreign plasmid.Many possible solutions were adopted,including heat attenuation,heat treatment,chill treatment,EDTA treatment and plasmid denaturation,no transformant was found when pIJ702 was used.Non-methylated pWHM3 derived from E.coli ET12567 also can not be transformed into UV2136,while the pWHM3 from methylatng host,E.coli DH5α,can be introduced at a low frequency,4 transformants perμg pWHM3,and the pWHM3 modified by UV2136 itself can be transformed at a higher frequency,10⁴ transformants perμg pWHM3.4.Conjugation was used to develop the transformation system of wild strain of cytosinpeptidemycin producing 04-2-2.To construct pKC1139-tsr,pIJ702 was digested by BelⅠ,the fragment of 1,055bp containing tsr gene was recovered and inserted into the pKC1139/BglⅡwhich was dephosphorylated by CIAP.To construct pSET152-tsr,tsr gene was cloned by PCR,digested by ApaI and NheI and ligated with pSET152/ApaI/NheI.By conjugation,pKC1139-tsr can be transformed into the wild strain 04-2-2,while pSET152-tsr cannot be introduced,probably because there is no attP site which is necessary for the replication of pSET152-tsr. 5.By complementation of UV2136,three genes involved in the biosynthesis of cytosinpeptidemycin were cloned and function analyzed.Total DNA of the wild strain 04-2-2 was partially digested by EcoRI and HindⅢindividually and 3-15kb fragments were recovered,ligated with pWHM3/EcoRI and pWHM3/HindⅢwhich was dephosphorylated by CIAP.The ligated product was introduced into UV2136 by protoplast transformation system and the cytosinpeptidemycin activity of thiostrepton resistant strains was determained. A transformant,UV2136-R,can produce cytosinpeptidemycin,and the insert was analyzed. There are three ORFs designated cytoA,cytoB and cytoC.cytoA,consisted of 1464bp, encodes a protein with 487 amion acid resdues.Database searching showed that CytoA belongs to HATPase_c superfamily,is homologous to sensor kianse from Streptomyces pristinaespiralis ATCC 25486 with 66%identities,suggesting that the expressed product of cytoA maybe histidine kinase.cytoB,consisted of 570bp,encodes a protein with 189 amino acid residues.Database Blasting showed that CytoB belongs to REC superfamily,is highy homologous to response regulator from Streptomyces griseus subsp.griseus NBRC 13350 with 86%identities,illuminating that CytoB is response regulator,consisting of two-competent system together with CytoA,and gene accession No.was GQ149124. Disruptant of cytoA/B could produce cytosinpeptidemycin,which indicated that cytoA/B was not related to the production of cytosinpeptidemycin directly,but was regulatory genes.cytoC, consisted of 783bp,encodes a protein with 260 amino acid residues.Database searing showed that CytoC belongs to DUF899 superfamily,is high homologous to conserved hypothetical protein which is thioredoxin-like from Frankia sp.ccI3 and Streptomyces pristinaespiralis ATCC 25486 with 85%identities.Disrupant of cytoC losing the ability to produce cytosinpeptidemycin,suggested that cytoC is an important gene involved in the biosynthesis of cytosinpeptidemycin.6.By homologous cloning,a biosynthetic gene named cytoD encoding glycosyltransferase was cloned and function analyzed.There are cytosinine in both cytosinpeptidemycin and Blasticidin S,it can be induced that there are similar genes involved in the biosynthesis.Using partial sequence of BlsD as probe,total digested genome DNA of 04-2-2 was Southern blotted,and a 2.1kb signal fragment was found.By construction of gene sub-library and colony in situ hybridization,a colony containing signal fragment was achieved and the plasimid was named pUC18-CGA.There is 1 ORF named cytoD,which is 981bp and the expressed product is a protein with 321 amino acid residues.Database blasting showed that CytoD is high homologous to glycosyltransferase from Streptomyces griseus subsp.griseus NBRC 13350 with 85%identities,suggesting that CytoD maybe glycosyltransferase.Disruptant of cytoD lose the ability to produce cytosinpeptidemycin, which illustrated that cytoD is an important gene involved in the biosynthesis of cytosinpeptidemycin.All the results were essential for the cloning of cytosinpeptidemycin biosynthetic genes and illustrating the total biosynthesis.更多还原