本文選題：同源重組　切入點(diǎn)：sfp基因　出處：《中國(guó)農(nóng)業(yè)科學(xué)院》2015年碩士論文

【摘要】：黃瓜枯萎病是由尖孢鐮刀菌黃瓜專(zhuān)化型Fusarium oxysporum f.sp.cucumerinum(Foc)引起的危害嚴(yán)重的土傳病害。防治黃瓜枯萎病的高效菌株枯草芽胞桿菌B006可產(chǎn)生脂肽類(lèi)抗生素表面活性素surfactin和芬枯草菌素fengycin。前人研究證明脂肽類(lèi)表面活性素surfactin通過(guò)促進(jìn)芽胞桿菌生物膜形成以及在植物根部的定殖、誘導(dǎo)植物產(chǎn)生系統(tǒng)抗性等發(fā)揮生防作用。但對(duì)于surfactin是否能夠抑制根際病原菌的侵染和在植株內(nèi)的蔓延來(lái)控制病害的發(fā)生和發(fā)展尚缺乏依據(jù)。本研究首先從生防芽胞桿菌B006菌株中克隆了編碼4’-磷酸泛酰巰基乙胺轉(zhuǎn)移酶Sfp的sfp基因并連接到載體pDG1730上,并通過(guò)同源重組的方法將該基因整合到不產(chǎn)生surfactin的模式菌株B.subtilis 168(B168)基因組中。轉(zhuǎn)化子于1%淀粉平板和5%血平板初步篩選,獲得不水解淀粉并產(chǎn)生溶血圈的重組菌株B168S。采用HPLC-ESI-MS對(duì)B168S在牛肉膏蛋白胨培養(yǎng)液中培養(yǎng)48 h后的surfactin進(jìn)行檢測(cè),證明B168S可產(chǎn)生surfactin,sfp基因重組菌株構(gòu)建成功。對(duì)重組菌株B168S和B168的生物學(xué)特性的測(cè)定表明:B168和B168S在黃瓜根系分泌物培養(yǎng)基上菌落形態(tài)略有差異,B168S的菌落更厚。兩菌株接種于LB培養(yǎng)基,37℃靜止培養(yǎng)24h后,B168S在液面處產(chǎn)生的生物膜較B168厚。平板對(duì)峙實(shí)驗(yàn)表明B168S在CRE培養(yǎng)基上對(duì)Foc菌絲生長(zhǎng)有一定的抑制作用,抑制率R2/R1為1.22,而B(niǎo)168菌株無(wú)抑制作用。采用組織印跡法對(duì)重組菌株B168S和原始菌株B168在黃瓜根部的定殖和分布狀態(tài)測(cè)定結(jié)果表明,B168S在黃瓜根中部和根尖處菌量明顯大于B168。采用平板菌落計(jì)數(shù)法對(duì)B168S和B168在黃瓜根各部位的定殖量測(cè)定結(jié)果表明,菌懸液(106 cfu·mL-1)浸泡90分鐘的黃瓜種子播種于無(wú)菌石英砂3天后,B168S在黃瓜根基部和根尖的定殖量分別為B168的2~3倍和9~10倍。溫室盆栽實(shí)驗(yàn)表明,用濃度為106 cfu·mL-1的B168S和B168菌懸液蘸根后,移栽到接種Foc孢子(105個(gè)·g-1)的病土中,移栽后第3周B168S和B168處理的防效分別為21.1%和17.9%;對(duì)不同高度黃瓜莖中Foc的組織分離結(jié)果表明,B168S處理后Foc在黃瓜莖內(nèi)的相對(duì)高度低于B168和Foc處理,蔓延速度減慢。以檸檬酸、蘋(píng)果酸、琥珀酸作為單一有機(jī)酸的根分泌物培養(yǎng)液培養(yǎng)芽胞桿菌B006,HPLC方法檢測(cè)各培養(yǎng)液中B006菌株產(chǎn)生的surfactin。結(jié)果表明,三種有機(jī)酸可改變surfactin的產(chǎn)量,并改變surfactin各同系物的比例。外源添加surfactin粗提物能夠促進(jìn)芽胞桿菌生物膜的形成,從葡萄糖和琥珀酸培養(yǎng)液中獲得的surfactin粗提物A和B分別在50μM和10μM以上對(duì)芽胞桿菌B168和B168S生物膜的形成具有明顯促進(jìn)作用。上述結(jié)果進(jìn)一步明確了surfactin可明顯促進(jìn)芽胞桿菌在黃瓜根部的定殖,并通過(guò)抑制枯萎病菌的侵染和在黃瓜莖中的蔓延而增強(qiáng)對(duì)黃瓜枯萎病的防治效果。研究結(jié)果將有助于深入了解芽胞桿菌代謝產(chǎn)物surfactin在植物根際的功能,為芽胞桿菌的應(yīng)用提供理論基礎(chǔ)。
[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，

本文編號(hào)：1713822