产surfactin枯草芽胞杆菌168重组菌株构建及其对黄瓜枯萎病的控制作用

发布时间：2018-04-05 07:42

  本文选题：同源重组　切入点：sfp基因　出处：《中国农业科学院》2015年硕士论文

【摘要】：黄瓜枯萎病是由尖孢镰刀菌黄瓜专化型Fusarium oxysporum f.sp.cucumerinum(Foc)引起的危害严重的土传病害。防治黄瓜枯萎病的高效菌株枯草芽胞杆菌B006可产生脂肽类抗生素表面活性素surfactin和芬枯草菌素fengycin。前人研究证明脂肽类表面活性素surfactin通过促进芽胞杆菌生物膜形成以及在植物根部的定殖、诱导植物产生系统抗性等发挥生防作用。但对于surfactin是否能够抑制根际病原菌的侵染和在植株内的蔓延来控制病害的发生和发展尚缺乏依据。本研究首先从生防芽胞杆菌B006菌株中克隆了编码4’-磷酸泛酰巯基乙胺转移酶Sfp的sfp基因并连接到载体pDG1730上,并通过同源重组的方法将该基因整合到不产生surfactin的模式菌株B.subtilis 168(B168)基因组中。转化子于1%淀粉平板和5%血平板初步筛选,获得不水解淀粉并产生溶血圈的重组菌株B168S。采用HPLC-ESI-MS对B168S在牛肉膏蛋白胨培养液中培养48 h后的surfactin进行检测,证明B168S可产生surfactin,sfp基因重组菌株构建成功。对重组菌株B168S和B168的生物学特性的测定表明:B168和B168S在黄瓜根系分泌物培养基上菌落形态略有差异,B168S的菌落更厚。两菌株接种于LB培养基,37℃静止培养24h后,B168S在液面处产生的生物膜较B168厚。平板对峙实验表明B168S在CRE培养基上对Foc菌丝生长有一定的抑制作用,抑制率R2/R1为1.22,而B168菌株无抑制作用。采用组织印迹法对重组菌株B168S和原始菌株B168在黄瓜根部的定殖和分布状态测定结果表明,B168S在黄瓜根中部和根尖处菌量明显大于B168。采用平板菌落计数法对B168S和B168在黄瓜根各部位的定殖量测定结果表明,菌悬液(106 cfu·mL-1)浸泡90分钟的黄瓜种子播种于无菌石英砂3天后,B168S在黄瓜根基部和根尖的定殖量分别为B168的2~3倍和9~10倍。温室盆栽实验表明,用浓度为106 cfu·mL-1的B168S和B168菌悬液蘸根后,移栽到接种Foc孢子(105个·g-1)的病土中,移栽后第3周B168S和B168处理的防效分别为21.1%和17.9%;对不同高度黄瓜茎中Foc的组织分离结果表明,B168S处理后Foc在黄瓜茎内的相对高度低于B168和Foc处理,蔓延速度减慢。以柠檬酸、苹果酸、琥珀酸作为单一有机酸的根分泌物培养液培养芽胞杆菌B006,HPLC方法检测各培养液中B006菌株产生的surfactin。结果表明,三种有机酸可改变surfactin的产量,并改变surfactin各同系物的比例。外源添加surfactin粗提物能够促进芽胞杆菌生物膜的形成,从葡萄糖和琥珀酸培养液中获得的surfactin粗提物A和B分别在50μM和10μM以上对芽胞杆菌B168和B168S生物膜的形成具有明显促进作用。上述结果进一步明确了surfactin可明显促进芽胞杆菌在黄瓜根部的定殖,并通过抑制枯萎病菌的侵染和在黄瓜茎中的蔓延而增强对黄瓜枯萎病的防治效果。研究结果将有助于深入了解芽胞杆菌代谢产物surfactin在植物根际的功能,为芽胞杆菌的应用提供理论基础。
[Abstract]:Fusarium wilt is a serious soil-borne disease caused by Fusarium oxysporum Fusarium oxysporum F. sp. cucumerinum focus.Bacillus subtilis B006, a highly effective strain of Fusarium subtilis, produced surfactant surfactin and fengycinin for lipopeptide antibiotics.Previous studies have proved that lipopeptide surfactant surfactin plays a role in biological control by promoting the formation of biofilm of Bacillus spp., colonizing in plant roots and inducing systemic resistance in plants.However, there is no basis for whether surfactin can inhibit the infection and spread of rhizosphere pathogens to control the occurrence and development of the disease.In this study, the sfp gene encoding Sfp was cloned from Bacillus biocontrol strain B006 and ligated to the vector pDG1730.The gene was integrated into the genome of the model strain B.subtilis 168 B168 without surfactin production by homologous recombination.The recombinant strain B168Swas obtained by screening on 1% starch plate and 5% blood plate, which could not hydrolyze starch and produce hemolytic circle.HPLC-ESI-MS was used to detect the surfactin of B168S cultured in beef extract peptone medium for 48 h. It was proved that B168S could produce a recombinant strain with SFP gene.The biological characteristics of the recombinant strains B168S and B168 showed that the colony morphology of B168S and B168S on cucumber root exudate medium was slightly different from that of B168S, and the colony of B168S was thicker than that of B168S.The biofilm of B168S was thicker than that of B168 after 24 hours of static culture at 37 鈩，

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