【作者】 余贤美；

【导师】 张修国；

【作者基本信息】 山东农业大学 ， 植物病理学， 2009， 博士

【摘要】 铁是一切生命体不可或缺的必需元素,尽管其含量丰富,但由于在中性pH氧化条件下铁离子可溶性很低,常常成为限制生命体生长的营养元素。因此,许多微生物合成具有螯合铁离子能力的嗜铁素以获取所需要的铁离子。嗜铁微生物可通过分泌嗜铁素提高环境中铁的生物有效性,从而促进植物的生长,还可通过与病原微生物竞争环境中有限的铁离子,达到控制植物病害的目的。在缺铁条件下,枯草芽孢杆菌可分泌儿茶酚型嗜铁素bacillibactin(BB)以获取所需要的铁离子。本研究首次通过CAS(Chrome Azurol Sulphonate)检测平板法从海南岛橡胶树根际土壤分离获得一批嗜铁细菌,并以在CAS检测平板上产生较大橘黄色晕圈的拮抗细菌枯草芽孢杆菌CAS15为研究对象,克隆了B.subtilis嗜铁素基因dhbC并实现了在大肠杆菌中的表达,并通过同源重组法构建dhbC基因缺失突变株和回复株,验证了dhbC基因的功能,证明了dhbC基因在B.subtilis嗜铁素生物合成中发挥重要作用。该论文的研究结果为嗜铁微生物的筛选利用及其生防机理的研究奠定了基础,为植物病害的生物防治提供了新的思路。主要研究结果和结论如下:1.根据菌株的形态特征和生理生化特性,将CAS15初步鉴定为芽孢杆菌属(Bacillus);16S rDNA序列分析显示,CAS15的16S rDNA与枯草芽孢杆菌Bacillus subtilis 16S rDNA序列具有99.4%的同源性,在所构建的系统进化树上,CAS15与枯草芽孢杆菌(登录号为DQ207730和EU047884)及芽孢杆菌(登录号为AB188212)的遗传距离最近,处在同一分支,因此,将CAS15鉴定为枯草芽孢杆菌(Bacillus subtilis)。2.通过电喷雾离子化质谱分析(Electrospray Ionization Mass Spectrometry,ESI-MS)研究了CAS15分泌的嗜铁素,结果表明,CAS15提取物在m/z 881.2([M-H]1-)处的质谱峰值与3H2O(DHB-Gly-Thr)3的峰值一致,其准确分子质量为881.25;通过DHB(G)分析,研究了铁离子对CAS15嗜铁素产量的影响,发现铁离子抑制嗜铁素的产生,而且,培养基中FeCl3浓度越高,对嗜铁素产生的抑制作用越强。当培养基中不含FeCl3时,其OD510/OD600值为0.10～0.39;当培养基中含有5μmol/L FeCl3时,其OD510/OD600值为0.005～0.04;而当培养基中含有50μmol/L FeCl3时,其OD510/OD600值仅为0.001～0.01。3.对CAS15嗜铁素分泌影响因素进行了研究,结果表明,改良MM培养基为CAS15嗜铁素分泌的最适培养基,以葡萄糖为碳源,以色氨酸为外源氨基酸,pH7.2,37℃条件下培养,有利于CAS15分泌嗜铁素,可获得较高产量的嗜铁素。4.通过室内平板对峙试验研究了CAS15对15个常见病原菌的拮抗作用,结果显示,CAS15对这15个常见病原菌有较强的拮抗作用,拮抗带宽带为6～10 mm;并研究了CAS15菌液过滤液对15个病原菌的抑制作用,结果显示,抑制率为20.18%～94.07%,表明,CAS15具有较强较广泛的拮抗谱。5.黄瓜种子发芽试验结果表明,100倍液和1000倍液能有效促进芽的生长,其中100倍液效果最好。离体叶片接种试验结果显示,CAS15菌悬液对芒果炭疽病具有较强的抑制作用,病情指数下降了53.34%,防效达到78.95%,表明CAS15具有较好的促进植物生长及生物防治潜能。6.根据已报道的枯草芽孢杆菌基因组序列,设计特异性引物,通过PCR扩增,获得了CAS15 dhbC基因片段,该片段长1197 bp,预期编码398个氨基酸残基的多肽。BLASTn搜索结果显示,该基因片段与B.subtilis subsp. subtilis str.168和Bacillus subtilis subsp. subtilis str. NCIB 3610的dhbC基因序列( accession No.Z99120.2和NZ_ABQL01000005.1)分别具有99.74%和99.58%同源性,在核苷酸序列上,分别只有4个和5个碱基不同,在氨基酸序列上,分别有2个和3个氨基酸残基不同。7.通过网络工具http://www.expasy.org/对CAS15 dhbC基因编码产物DhbC进行结构分析与预测,结果显示:CAS15 dhbC基因编码产物的等电点为5.30,为酸性蛋白质,稳定系数为53.00,表明该蛋白质性质不稳定;总平均疏水指数(Grand average of hydropathicity, GRAVY)为-0.303;DhbC蛋白含有带负电荷的氨基酸残基(Asp+Glu)57个,带正电荷的氨基酸残基(Arg+Lys)39个,总原子数为6098,其分子式为C1910H3039N545O592S12;DhbC蛋白含有5个蛋白激酶C-磷酸化位点;含有6个酪蛋白激酶Ⅱ磷酸化位点,1个酪氨酸激酶磷酸化位点及2个N-豆蔻酰化位点;由于其蛋白结构的不稳定性,DhbC有可能由氨基酸14-394,122-397,69-393各形成一种三级结构模型。8.将CAS15 dhbC基因片段连接到表达载体pET-30a(+),并经氯化钙转化法导入大肠杆菌BL21(DE3),获得工程菌E.coli BL21(DE3)/pET-30a-dhbC,以1 mmol/L的IPTG进行诱导,经30℃诱导4 h后实现了高效表达,获得了48.8 kDa的融合蛋白,表达产物主要以可溶形式存在,重组蛋白的表达量约占菌体总蛋白的58%。由于所采用的pET-30a(+)表达系统所表达的蛋白质带有6个连续的组氨酸,可以通过Ni2+金属亲和层析进行重组蛋白的纯化并通过Western blot进行分析,结果表明,重组蛋白可与兔抗His-tag多克隆抗体发生特异性反应。9.通过同源重组法,将CAS15基因组中dhbC基因敲除,获得了dhbC基因缺失突变株,并将dhbC基因重新导入CAS15 dhbC基因缺失突变株,获得CAS15 dhbC基因回复株,经CAS检测平板检测,回复株能够和原CAS15菌株一样,在检测平板上产生明显的橘黄色晕圈,而CAS15 dhbC基因缺失突变株不能,表明dhbC基因与嗜铁素的产生密切相关,验证了dhbC基因在B.subtilis isochorismate生物合成过程中的重要性,说明dhbC基因在CAS15嗜铁素生物合成中发挥重要作用。更多还原

【Abstract】 Iron is essential to the growth of virtually all organisms. Despite its abundance in nature, iron is often a growth limiting nutrient because of the low solubility of ferric iron under aerobic conditions at neutral pH. Consequently, many bacteria produce ferric iron chelating compounds known as siderophores to gain access to various iron sources. Siderophore producing microorganisms can promote plant growth by increasing the bioavailability of iron via secreting siderophore and control plant diseases by antagonizing against plant pathogens via competing iron nutrient with them. In response to iron deprivation, B.subtilis secretes the catecholic siderophore bacillibactin (BB) to acquire iron.In this study, we isolated a series of siderophore producing bacteria from the rhizosphere soil of the rubber trees in Hainan Island by CAS agar plate assay, and CAS15, which produced distinct orange halo at CAS agar plate and had strong antagonism to common plant pathogens, was selected for the following studies. We amplified the siderophore producing gene dhbC and expressed it in E.coli, and then verified the function of dhbC by delecting it and retransforming it into to the dhbC- mutants via the homologous recombination method. The results of this study will provide the basis for the exploitation and utilization of the siderophore producing microorganisms and the study of the mechanism of their biological control, and provide a new way for the biological control of the plant diseases. The main results and conclusions are as follows.According to the characteristics of morphological, cultural, physiological and biochemical, CAS15 was identified as Bacilllus primarily. 16S rDNA sequence analysis result showed that the 16S rDNA sequence of CAS15 shared 99.4% identity with that of Bacillus subtilis. The genetic distance were closest between the 16S rDNA of CAS15 and that of Bacillus subtilis (accession No. DQ207730 and EU047884) and Bacillus sp.(accession No. AB188212), they are located at the same branch of the constructed phylogenetic tree, thus, CAS15 was identified as Bacillus subtilis.The type of siderophore produced by CAS15 and the siderophore productivity were analyzed by ESI-MS assay and DHB(G) assay respectively. The results showed that the siderophore produced by CAS15 was catecholic siderophore 2,3-dihydroxybenzoate-glycine- threonine trimeric ester bacillibactin, with the calculated mass of 881.25, and the siderophore productivity was significantly inhibited by iron. The inhibition was stronger with the higher concentration of iron. When there was no FeCl3, the OD510/OD600 value is 0.10～0.39, when there was 5μmol/L FeCl3 in the medium, the OD510/OD600 value is 0.005～0.04, while when there was 50μmol/L FeCl3 in the medium, the OD510/OD600 value is 0.001～0.01.The influencing factors for the siderophore production of CAS15 were studied, the results showed that modified minimal medium (MM) was the optimal medium for CAS15 siderophore production, and when cultured at pH7.2 and 37℃with glucose as the carbon source and tryptophan as the exogenous nitrogen source, a high yield of siderophore was obtained from the culture broth of CAS15, which indicated that the up-mentioned culture condition was benefit for CAS15 siderophore production.The antagonism of CAS15 against the common plant fungal pathogens was studied by the duel-culture test, the result showed that CAS15 had strong antagonism against the 15 selected fungal pathogens, with the inhibition width of 6～10 mm. Then the inhibiton of the cell-free supernatant of CAS15 was studied, the result showed that the cell-free supernatant of CAS15 significantly inhibited the growth of the 15 pathogens significantly, with the inhibiting rate of 20.18%～94.07%, which indicated that CAS15 had wide range of antagonistivity against plant fungal pathogens.Then we study the promotion of CAS15 cell-free supernatant on the seed germination of cucumber, the result showed that 100×and 1000×dilution promoted the growth of cucumber seedling significantly, and 100×dilution had the best effect. The result of inoculation in vitro showed that the cultured broth of CAS15 inhibited the mango anthracnose effectively by decreasing 53.34% of the disease index than the control, and the prevention rate was 78.95%, which indicated that CAS15 had great potential in plant growth promotion and biological control.A pair of specific primers were designed based on the reported genomic sequence of Bacillus subtilis, and then a 1197 bp fragment of CAS15 dhbC was amplified by PCR. CAS15 dhbC was predicted to encode a 43.8 kDa polypeptide with 398 amino acid residues. The BLASTn result showed that the nucleotide acids of dhbC gene (accession No.FJ194456) of CAS15 shared 99.74% and 99.58% identity with that of dhbC gene of B.subtilis subsp. subtilis str.168 (accession No. Z99120.2) and Bacillus subtilis subsp. subtilis str. NCIB 3610 (accession No. NZ_ABQL01000005.1) repectively. There were only 4- and 5-base difference in nucleotide sequence and 2- and 3-residue difference in amino acid sequence with that of Bacillus subtilis strain 168 and NCIB 3610 respectively.The structure analysis and prediction of DhbC encoded by CAS15 dhbC was studied by network (http://www.expasy.org), the results showed that the protein encoded by CAS15 dhbC was a acidic polypeptide, with the theoretical PI of 5.30. The instability index of DhbC was computed to be 53.00, which classified the protein as unstable. The Grand average of hydropathicity (GRAVY) is -0.303. The total number of negatively charged residues (Asp + Glu) and positively charged residues (Arg + Lys) of DhbC was 57 and 39 respectively, and its formula was C1910H3039N545O592S12, with a total number of 6098 atoms. DhbC contains 5 protein kinase C phosphorylation sites, 6 casein kinaseⅡphosphoralylation sites, 1 tyrosine kinase phosphorylation site and 2 N-myristorylation sites. According to its unstability, DhbC could form 3 3D structures by 14-394, 122-397 and 69-393 amino acids.The gene fragment of CAS15 dhbC was ligated to expression vector pET-30a(+) and then transformed into E.coli BL21(DE3) by calcium chloride transformation method, and the recombinant E.coli BL21(DE3)/pET-30a-dhbC was obtained. The recombinant DhbC of 48.8 kDa was exressed mainly in soluble form via 1 mmol/L IPTG induction in E.coli, and the amount reached highest at 30℃for 4 h. According to the N-terminal fusion 6 His-tag, the recombinant protein was purified by Ni2+ metal affinity chromatography and then identified by western blot, the result showed that the recombinant DhbC had the antigenicity to rabbit anti-his-tag polyclonal antibody.Finally, CAS15 dhbC gene was deleted by gene knockout and dhbC gene-delected mutant (CAS15 dhbC-del) was obtained. Then the dhbC gene was retransformed into the CAS15 dhbC-del via homologus recombination, and dhbC gene-comlemented mutant (CAS15 dhbC-com) was obtained. The function of siderophore production was tested at CAS agar plate, the result showed that CAS15 dhbC-com could produce the distinct orange halo at CAS agar plate as CAS15, while CAS15 dhbC-del could not, which indicated that dhbC gene was directly related to the siderophore production and it was confirmed to play an important role in the siderophore biosynthesis of CAS15.更多还原