【作者】 韩冬梅；

【导师】 孙明；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 本论文共包括两部分工作,一部分是对枯草芽胞杆菌(Bacillus subtilis)BSn5中新型抑菌蛋白APn5进行了鉴定及性质研究;另一部分是利用构建质粒物理图谱的方法,对苏云金芽胞杆菌YBT-1520菌株全基因组测序拼接中的pBMB69和pBMB95两个质粒进行了验证。1.枯草芽胞杆菌BSn5菌株是从被细菌污染的花魔芋愈伤组织中分离得到的对魔芋软腐病具有抑制作用的魔芋内生菌。利用饱和度为30%的硫酸铵从BSn5菌株的发酵上清液中沉淀胞外蛋白,利用不同截留量的超滤管进行超滤离心去除小分子物质,经SDS-PAGE检测,仅可见一条大小约为31.6 kDa的蛋白带,命名为APn5蛋白,对胡萝卜软腐欧文氏菌胡萝卜软腐亚种(Erwinia carotovora subsp.carotovora)SCG1具有较好的抑菌活性。APn5蛋白抑菌谱较窄,对所检测的真菌性植物病原菌具有微弱的抑菌活性,而在所检测的细菌性植物病原菌中,仅对胡萝卜软腐欧文氏菌胡萝卜软腐亚种和带化红球菌(Rhodococcus fascians)具有较好的抑制作用。该蛋白对高温敏感,对蛋白酶E敏感,对蛋白酶K和胰蛋白酶部分敏感;在酸性环境下其抑菌活性稳定,碱性环境下活性不稳定。在菌株BSn5的生长周期中,该蛋白的抑菌活性不能稳定存在,其发酵上清液在对数生长期抑菌活性逐渐升高,进入稳定期后抑菌活性开始下降,直至完全消失。经调查,枯草芽胞杆菌B47也能产生与APn5分子量大小和抑菌活性均相似的抑菌蛋白,命名为APn5-like。利用APn5蛋白制备抗体,Western blot检测APn5-like蛋白,可获得阳性杂交信号。MALDI-TOF-MS和Q-TOF分析,未发现APn5蛋白和APn5-like蛋白与Matrix Science数据库中己知的蛋白具有高度相关性,它们具有两段完全一致的肽段,均与枯草芽胞杆菌鞭毛蛋白N端序列具有相似性,此外还具有功能相似的两个肽段,分别与DnaJ和金属结合蛋白具有相似性。综合理化性质和质谱分析结果,APn5蛋白和APn5-like蛋白为同一类抑菌蛋白,它们很可能是一种新功能的抗菌蛋白,具有自己的抑菌特点。2.苏云金芽胞杆菌菌株YBT-1520是从土壤中分离筛选,对棉铃虫、小菜蛾等鳞翅目害虫具有高毒力的专利菌株。本实验室利用全基因组鸟枪法测定了该菌株的全基因组序列,进行了初步分析,结果表明该菌株具有10个质粒。在质粒的测序拼接过程中,由于插入因子和重复序列的大量存在,导致在质粒的前后拼接结果有所不同。尤其是pBMB69和pBMB95是否是两个不同的质粒还是一个质粒上存在着很大分歧。在上述研究基础上,本研究利用构建质粒物理图谱的方法,对于苏云金芽胞杆菌YBT-1520测序拼接中前后拼接结果不同的质粒pBMB69和pBMB95以及plasmid137进行了验证。根据在质粒pBMB69和pBMB95的复制子ori44和ori60以及在这两个质粒上均存在的cry1Ac10基因序列设计引物,利用PCR的方法筛选已构建好的苏云金芽胞杆菌YBT-1520全基因组BAC文库。在筛选文库的过程中,共获得了9个可以同时扩增出ori44和cry1Ac10基因的转化子,1个可以同时扩增出ori60和cry1Ac10基因的转化子。利用NotⅠ对这些转化子进行酶切验证,从中筛选出装载的外源片段最大的转化子EMB2H05(含有pBMB69片段)和EMB2D10(含有pBMB95片段)。根据测序结果,选择合适的限制性内切酶进行酶切分析,利用琼脂糖凝胶普通电泳和脉冲电泳分析酶切片段并估计大小,最终构建物理图谱。本研究绘制出了质粒pBMB69的全部序列和pBMB95上重复区域的物理图谱。与测序拼接结果比较分析,pBMB69上有一段40 kb左右的片段发生了颠转,其余部分与拼接结果一致;pBMB95重复区域与测序拼接结果一致。结果表明,pBMB69与pBMB95为两个独立质粒。更多还原

【Abstract】 The research includs two parts:one is identification and characterization the novel antimicrobial protein APn5 isolated from Bacillus subtilis strain BSn5;another is to construct the restriction maps of plasmids pBMB69 and pBMB95 in Bacillus thuringiensis strain YBT-1520.1.Bacillus subtilis strain BSn5 was isolated from Amorphophallus konjac calli contaminated by bacteria.BSn5 could inhibit Erwinia carotovora subsp.carotovora strain SCG1,which causes Amorphophallus soft rot disease and affects Amorphophallus industry development.The extracellular proteins was isolated from the BSn5 culture by ammonium sulfate precipitation with 30%relative saturation and further purified by ultrafiltration.There was only a 31.6 kDa band could be detected by SDS-PAGE,which was named as APn5.APn5 showed a narrow inhibition spectrum,showing low inhibition activity to plant pathogenic fungus and strong inhibition activity only to Erwinia carotovora subsp.carotovora and Rhodococcus fascians.APn5 was sensitive to high temperature and alkaline pH,pronase E,and partial sensitive to trypsin and proteinase K. During strain BSn5 growth phase,the inhibition activity was unstable,which would gradually lose on stationary phase and disappeared finally.After investigating all Bacillus subtilis strains stored in our lab,only B47 produced a protein with the similar molecular weight and inhibition activity to APn5,and the protein was named as APn5-like.APn5-like showed positive signal during the Western blot using the antibody prepared by APn5.MALDI-TOF-MS and Q-TOF analysis showed that there was no high relative to known proteins from Matrix Science database.There were two pairs of identical peptides in APn5 and APn5-like,all of that had similarity to N-terminal sequence of Bacillus subtilis flagellin;in addition,two peptides in the APn5 and APn5-like show functional similarity,respectively being similar to DnaJ and metal-binding protein.On the basis of the difference on inhibition spectrum and characteristics,APn5 and APn5-like are allied to a novel antimicrobial protein. 2.Bacillus thuringiensis strain YBT-1520,which is highly toxic to lepidopteran pests, was isolated from the soil and protected by patent.The whole genome of YBT-1520 was sequenced by shot-gun sequencing,and the preliminary analysis of the sequence revealed that there were 10 plasmids harbored in YBT-1520.Because of the insertion sequence and repeat sequence,there was discrepancy between the different sequence splice results in whether pBMB69 and pBMB95 were two different plasmids or they should be joined together as one plasmid.This work tried to clarify this discrepancy by constructing the restriction maps of plasmids pBMB69 and pBMB95.The BAC library of YBT-1520 total DNA was screened by PCR using the primers designed on the basis of ori44,ori60 and cry1Ac10 sequence. Finally,there were 9 clones containing ori44 and cry1Ac10,and only 1 clones harboring ori60 and cry1Ac10.The clones EMB2H05(contain pBMB69 fragment) and EMB2D10 (contain pBMB95 fragment) with the largest inserts were selected and digested with the appropriate restriction enzymes.We estimated the size of the restriction fragments separated by pulsed field gel electrophoresis and agarose gel electrophoresis,and at last, constructed the restriction maps of the whole pBMB69 and the repeat region of pBMB95. Compared with the sequence splice results,there was a 40 kb reversal fragment in pBMB69;the repeat region in pBMB95 was in accordance with the sequence splice results.According to the restriction maps,pBMB69 and pBMB95 were two independent plasmids.更多还原