【作者】 刘慧芹；

【导师】 温祥珍； 赵廷昌；

【作者基本信息】 山西农业大学 ， 蔬菜学， 2014， 博士

【摘要】 铁、锌等金属离子是细菌生长的必要微量元素,细菌通过调控铁、锌吸收系统和排放系统来调节细胞内的离子平衡。铁吸收调节蛋白(Ferric uptake regulator, Fur)和锌吸收调节蛋白(Zinc uptake regulator, Zur)是Fur金属蛋白家族两个重要成员,在许多G细菌中负调控高亲和性的铁、锌吸收系统。由番茄-辣椒疮痂病菌(Xanthomonas campestris pv. vesicatoria)引起的番茄、辣椒细菌性疮痂病是番茄和辣椒的一种世界性病害。本研究采用同源重组方法对X. campestris pv. vesicatori中fur、zur基因进行了克隆,得到了两个基因的突变体(fur-m、zur-m),对两个基因的功能进行了较为全面的分析；同时研究了Fe在寄主——病原物互作中的作用；筛选出一株拮抗作用较强的内生细菌X8,研究了X8及fur突变体在番茄疮痂病防治过程中所具有的潜能。获得了以下研究结果：1.fur基因分析 以NCBI登录的番茄疮痂病菌X. campestris pv. vesicatoria 85-10标准菌株的全基因组序列为参考,fur由136个氨基酸组成,与其它几种黄单胞菌fur相比较,氨基酸同源性均为90%以上。系统发育树分析表明,Xcv中的fur与其它黄单胞杆菌相比其同源性高,尤其是和Xac(Xanthomonas axonopodis)、Xoo(Xanthomonas. oryzae pv. oryzae)、Xcr (Xanthomonas campestris pv. raphani)、Xcc (Xanthomonas campestris pv. campestris)同源性在90%以上,说明fur在黄单胞杆菌中高度保守。2.fur基因功能表达fur突变后(fur-m)其表型发生了显著的变化。突变株可以产生铁载体,增加了对铁的吸收；几乎不能产生生物膜；产生群体感应的能力明显减弱；在运动性测定中,半固体培养基中的突变体游动直径中有8.57mm,为野生型的39.5%,运动性减弱；胞外多糖(EPS)含量只有0.52 mg/mL,仅为野生型的40.2%；fur-m对链黑菌素(SNG)的敏感性明显增加,其生存数只有Ig7.46 cfu/mL,而野生型有1g11.85cfu/mL;对过氧化氢(H202)的抗性显著降低,突变体抑菌圈直径为18.12mm,生存数为lg7.68 cfu/mL,野生型直径为14.59mm,生存数为突变的104倍；菌株的生长曲线基本上呈现“S”型曲线,但fur-m的生长速度明显低于野生型。fur-m对番茄的致病性明显减弱,症状出现较晚,且不典型,病情指数由野生型的20.5降为3.2,差异显著；fur-m不能在烟草上激发产生过敏性坏死反应。fur-m在低铁浓度下吸收的铁最多,达2172.2g/g；低浓度的铁可以促进fur-m的生长,不同的金属离子对fur-m 6勺生长均有明显影响,除Mn2+之外,其它七种离子(Zn2+、Fe3+、Pb2+、Cu2+、Co2+、Ni2+、Cd2+)均显著抑制突变体的生长。对突变体进行互补后(fur-c),以上这些特性均得到了回补,各项表现接近与野生型。说明fur基因在Xcv中是一个全局性调节因子。3.zur基因分析及功能表达Xcv的ZUr基因的同源性在各黄单胞杆菌中较高,尤其是与Xac相比同源性达100%,Xcc-8004达89%,说明zur在黄单胞杆菌中也高度保守。zur突变后(zur-m)某些表型也发生了变化。在铁载体的产生、生物膜生成、产生群体感应的能力均与野生型相近。zur-m运动性明显减弱,其游动直径为9.60mm,是野生型的44.3%；胞外多糖(EPS)含量仅有0.70 mg/mL,为野生型的61.8%；zur-m对链黑菌素(SNG)的敏感性增加,其生存数为lg10.70 cfu/mL,约为野生型的十分之一(lg11.85cfu/mL);对过氧化氢(H202)的抗性显著降低,抑菌圈直径为17.50mm,生存数为1g9.25个/mL,而野生型直径为14.59mm,生存数为突变的102倍；菌株的生长曲线基本上呈现“S”型曲线,zur-m 6勺生长速度减缓。3种接种方法均表明zur-m对番茄的致病性减弱,不能在烟草上激发过敏性坏死反应；七种金属离子(Zn2+、Pb2+、Cu2+、 Co2+、Ni2+、Cd2+、Mn2+等)均对zur-m的生长有明显的抑制作用,且金属浓度越高其抑制作用越大。对突变体进行互补后(zur-c),部分缺失的表型可以得到了回补,接近与野生型。4. Fur、zur对其它相关基因表达的影响fur显著负调控bfd.Xcv935基因,对copB. Xcv1654、katE、zur、sufC、hrpG基因是呈现一定的负调控；显著正调控bfr基因,对fliM、pilU、sodB、sufD基因表现为一定的正调控。zur对多个基因呈现出负调控,尤其是bfd、Xcv935、sufD、sufC、pilU、Xcv1654、copB、hrpG,其中bfr和fliM表达量减少最明显。对bfr、fliM、katE、sodB基因表现为正调控。5.Fe及Xcv对番茄的影响zt (zur突变,zur-m)和ft (fur哭变,fur-m)对种子的附着能力完全不同：野生型附着量为1.9×108,而zt和ft分别为3.29x 108和1.10∏104,ft附着能力显著下降。不同浓度的铁和细菌均对番茄种子的萌发和生长有一定的作用。其中低Fe和ft有促进作用,而高铁和Y有抑制作用。不同铁浓度下,接种两种菌后番茄的POD活性均明显的升高。接种两菌株后,番茄CAT活性升高,整体活性表现为中铁下活性最高,在低铁下活性最小。各铁浓度下,三个处理的叶片SOD活性变化均在6d时活性升高,形成峰值,之后变化平缓。低铁下,ft接种后番茄PAL活性基本高于Y接种的；而高铁下则正相反。不同铁浓度下,三个处理间的MDA含量基本趋于上升,对于接种ft和Y的番茄其MDA含量顺序基本为低铁>高铁>中铁。低铁下,接种Y和ft后番茄SS含量是先升后降。中高铁下,3个处理的变化均为先降再升再降,处理基本为Y>对照>ft。中铁条件下,ft接种后的番茄Pro含量较其它浓度要高,Y的最低。高铁下,各菌株Pro含量基本变化为先降后升。各铁浓度下,三个处理的叶绿素总量变化相似,都在9d时达到最低,呈现先降后升的趋势。各处理下,胡萝卜素浓度变化较一致,9d时明显升高,12d时又明显下降。中铁下的各处理在株高、总鲜重、茎粗、叶面积均最高,接种ft的各生物量在各处理中也基本是最高的,而且ft有利于番茄叶片对铁的吸收。在高铁下接种Y后,番茄病害发生减轻,病情指数明显下降。6.生防菌X8的鉴定与生防作用 在十几种果蔬中分离到6株拮抗作用较强的细菌菌株,其中X8菌株对多种病原真菌和细菌具有较强拮抗作用。经16S rDNA序列分析,X8菌株与枯草芽孢杆菌Bacillus subtilis的16S rDNA序列的同源性达99%。综合形态特征、生理生化特性,将X8鉴定为B. subtilis,在GenBank中序列登录号为HQ647257。利用X8和ft防治疮痂病。在保护作用中,X8的防效在21d时为42.9%,在ft防效为50.4%；在治疗作用中,X8的防效在21d时为25.3%,ft则没有防效；综合防治中,以上各处理配合上铁均能取得更好的效果,接近于药剂的防效。更多还原

【Abstract】 Iron, zinc and other metal ions are essential trace elements in bacteria growth. Bacteria adjust themselves ion balance by regulating intracellular iron absorption system and exhaust system. Fur (ferric uptake regulator) and Zur (Zinc uptake regulator) are two important members of the Fur family protein, it can regulate many high-affinity iron and zinc absorption systems in G-negative bacteria. Tomatoes-peppers scab caused by X. campestris pv vesicatoria is a worldwide disease. Fur and zur gene in X. campestris pv. vesicatoriwas were cloned and obtained two mutant (fur-m, zur-m) with method of homologous recombination in this study. The functions of the two genes were more comprehensively analyzed, simultaneously, the role of Fe in the host-pathogen interaction and potentiality of fur-m preventing bacteria disease were studied. A strong antagonistic endophytic bacteria X8 was screened, control potential of X8 and fur mutant on tomato scab was studied. Obtained the following results:1. fur gene analysis According genome sequence of X. campestris pv. vesicatoria 85-10, standard strains in NCBI, as a reference, fur was composed of 136 amino acids, compared with fur in several other Xanthomonas, it amino acid homology are more than 90%. Phylogenetic analysis showed fur in Xcv as has high homology compared to other Xanthomonas spp., especially to the Xac (X.axonopodis Xac29-1), Xoo (X. oryzae pv. Oryzae), Xcr (X. campestris pv. raphani) and Xcc (X. campestris pv. campestris), homology is above 90%, which indicates that fur is highly conserved in Xanthomonas spp.2. function expression of fur phenotypes fur mutant (fur-m) exhibit abnormalities in many ways. fur-m can produce siderophores, increasing the absorption of iron. It almost can not produce biofilms. The ability to produce quorum sensing is significantly weakened. Swimming diameter in fur-m was 8.57 mm, and only was 39.5% of wild type in semi-solid medium on swimming motility essay. Swimming ability became weak. Extracellular polysaccharide content (EPS) in fur-m was 0.52 mg/mL, and only was 40.2% of wild-type. Sensitivity of fur-m to streptonigrin (SNG) significantly increased the survival number of bacteria was only Ig7.46 cfu/mL, and only was one ten thousandth of the wild type (lg11.85 cfu/mL). Resistance of mutant to hydrogen peroxide (H2O2) was significantly reduced. Inhibit diameter was 18.12 mm and survival number was Ig7.68 cfu/mL, whereas Inhibit diameter in wildtype was 14.59 mm and survival number was 104 times of mutant. Growth curves of strains essentially show "S" curve, but the growth rate in fur-m was less than half of that of wildtype. Pathogenicity of the fur-m to tomatoes reduced was significant, fur-m can not stimulate hypersensitive response (HR) in the tobacco. fur-m can absorb most iron under low iron concentration,up to 2172.2 μg/g. Iron can promote the growth of fur-m under low concentration. Different metal ions affected significantly the growth of the fur-m. The other seven ions (Zn2+、Pb2+、Cu2+、Co2+、Ni2、Cd2+、Mn2+) significantly inhibited the growth of fur-m except Mn2+. After fur-m was complemented (fur-c), above phenotypes obtain recover, being lose to the performance of the wild type. It is showed fur gene is a global regulator in Xcv.3. Function expression and genetic analysis of zur homologous genes of zur in Xcv and other Xanthomonas were higher, especially when compared with Xac and Xcc-8004, homology is up to 100% and 89%, especially, it indicates zur is also highly conserved in Xanthomonas.Some phenotypes of zur mutant (zur-m) had also changed. Siderophores produce, biofilm formation, capable of producing quorum sensing in mutant were similar compared with the wild type. Movement significantly weakened, its swimming diameter was 9.60 mm,and only was 44.3% of wild-type. Extracellular polysaccharides (EPS) had 0.70 mg/mL, only was 61.8% of the wild-type. Sensitivity of mutant to streptonigrin (SNG) is increased, the survival bacteria are lg10.70 cfu/mL, only about one-tenth of wild-type (lg 11.85 cfu/mL). Resistance to hydrogen peroxide (H2O2) was significantly reduced, inhibit diameter was 17.50 mm and survival number was Ig9.25 cfu/mL, however inhibit diameter was 14.59 mm and survival number was ten times of mutant. Growth curve also showed "S" curve, growth rate of mutants was lowed. Pathogenicity of zur mutant to tomatoes was greatly reduced with three inoculating methods. Mutants can hardly stimulate the hypersensitive response in the tobacco. Seven different metal ions (Zn2+、 Pb2+、Cu2+、Co2+、Ni+、Cd2+、Mn2+) significantly inhibited the growth of mutant, and higher the metal concentration was, the stronger the inhibition was. After the mutant was complemented (zur-c), some phenotypes can recover, be close to the wild type.4. Impact of fur, zur to the expression of other genes related fur negatively regulates bfd and Xcv935 significantly, negatively regulates copB, Xcv 1654, katE, zur, sufC, hrpG slightly, fur significantly positively regulates bfr gene, slightly positively regulates fliM, pilU, sodB, sufD gene, zur negatively regulates many genes, especially bfd and Xcv935, sufD, sufC, pilU, Xcv 1654, copB, hrpG. bfr and fliM reduce most obviously. It positively regulates bfr, fliM, katE and sodB.5. Effect of Fe and Xcv on the germination and growth of tomato Adhesion amount of zt and ft on seeds was different. Adhesion amount of Y (widetype) was 1.9x 108 strains/mL,while zt (zur mutant, zur-m) and ft (fur mutant, fur-m) were 3.29×108 and 1.10×104 strains/mL respectively. This ability of ft decreased remarked. ft and zt had some promotion on seed germination, but Y had significant inhibition. Different iron concentrations and bacteria could effect seed germination and growth of tomato, ft and low iron concentration had promotion and high concentration and Y had inhibition. strainsUnder different iron concentrations, POD activity of tomatoes was significantly increased. CAT activity of tomatoes increased inoculated by to mutant. CAT activity was highest under middle iron, was lowest low-iron. SOD activity was increased to peak at sixth day under each concentration, then changed smoothly. Under low-iron, PAL activity of tomatoes inoculated by ft was higher than that by Y. It was contrary in high-iron. Under different concentrations, MDA content of each treatments tend to rise. Tomatoes inoculated by ft and Y, its basic sequence of MDA content was low iron> CK> high iron. Under low-iron, SS content of tomatoes inoculated by Y and ft increased then decreased, the Changes of three treatments were down-up-down under middle and high iron; treatments were basically Y> CK> ft. Pro content of tomatoes inoculated by ft was higher under middle-iron, while that of Y was lower. Pro change in each treatment was drop then rise. Under each concentration, total chlorophyll changes was similar in three treatments. It was the lower at 9 d and first drop then rise. The change of carotenoid concentration was consistent, rise at 9 d and sown at 12 d significantly. Height, total fresh weight, stem diameter and leaf area were the most in middle iron concentration. All biomass of tomatoes inoculated by ft also were most. Ft promoted tomato leaves to absorb more iron. Tomato bacteria scab mitigate and disease index decreased when inoculated by ft in high iron concentration.6. Identification of biocontrol bacteria X8 and control of tomato scab Six antagonistic bacteria strains were isolated from mangy vegetables and fruit. Strains X8 has stronger antagonism to many pathogens. With the 16S rDNA sequence analysis, strain X8 shared 99% homology with Bacillus subtilis. According to morphology, physiological and biochemical characters as well as 16S rDNA sequences, strain X8 was identified as B. subtilis (GenBank accession number HQ647257). Control of X8 and ft to scab.7. Control of tomato scab In protection, control effect of X8 to tomato scab was 42.90% at 21th day, while that of ft was 50.4%%. In therapy, effect of X8 was 25.32% at 21th day, oppositely, ft has no effect. In integrated control, control effects above treatment combined with Fe were better, and those effects were closed to chemical agents’.更多还原