【作者】 何鹏飞；

【导师】 侯明生；

【作者基本信息】 华中农业大学 ， 植物病理学， 2014， 博士

【摘要】 植物促生根围细菌是一类生活于植物根围土壤,并能促进植物生长的一类细菌,是农业生物防治的重要组成部分。B9601-Y2（以下简称Y2）是实验室从小麦根际土壤中分离并保存的一株解淀粉芽孢杆菌,隶属于植生亚种。离体条件下,Y2对多种植物病原真菌和细菌具有很强的拮抗活性；在大田试验中,能促进多种农作物的生长和增产。为了研究其促生防病机制,本研究对该菌株的全基因组进行了测序和解析,并以基质辅助激光解吸电离飞行时间质谱（matrix-assisted laser desorption/ionization-time of flight-mass spectrometry, MALDI-TOF-MS）证实其分泌抗生素类物质；构建了绿色荧光（green fluorescent protein,GFP）表达载体,研究其在玉米根际土壤及玉米体内定殖动态；通过基因工程方法,恢复了该菌株形成自然感受态的能力。取得了如下研究结果：1.Y2的基因组大小为4238624bp, GC含量占45.76%,在NCBI数据库中的登录号为NC₁7912.1,它含有4159个CDS,编码区总长度为3704557bp,约占全基因组的87.4%；它共有91个tRNA基因、10个rDNA拷贝、69个串联重复区域、73个转座子和24个插入序列元件。2.通过基因组注释,发现Y2具有多种生存策略,能够忍受极端的环境条件。它含有芽孢杆菌DNA损伤修复的保守核心基因,如切除修复系统所需的alkA （MUS₄255）、ung（MUS₄184）、mutM（MUS₃183）等基因成员；错配修复系统mutS （MUS₁860）、mutL（MUS₁861）、XseA （MUS₂727）以及XseB （MUS₂726）;非同源末端接合以及同源修复系统所需的关键基因等。Y2含有利用纤维素、半纤维素、果胶以及淀粉类的基因成员,如MUS₂161、P（MUS₄265）、bglS（MUS₄304）、 pelB（MUS₄319）以及amyE （MUS₀286）等,能够降解利用土壤中有限的大分子物质并快速壮大自己种群数量；同时还存有包括脱乙酰几丁质酶csn（MUS₃567）等在内的编码基因降解几丁质。Y2拥有与植物寄主相容全套基因簇和调控元件。3.Y2基因组中含有多种抗生素的合成基因簇,MALDI-TOF-MS也证实Y2除了分泌如芬荠素、伊枯草菌素、嗜铁素、溶杆菌素、杆菌烯、大环内酯以及地非西丁在内的一些脂肽类和聚酮类抗生物质外,还能合成羊毛硫抗生素化合物摩萨西丁,拮抗多种植物病原菌,减少植物病害的发生。基因组中还有杀线虫相关的碱性丝氨酸蛋白酶基因及促生长化合物2,3-丁二醇、3-羟基-2-丁酮、生长素（IAA）的合成代谢途径,从而为Y2菌株促生长、防治真菌性病害和根结线虫病害提供了分子依据。4.Y2缺失表面活性素的合成基因簇srfA的第二个成员srfAB,导致不能合成表面活性素（surfactin）。srfAB缺失的间接后果导致了与感受态形成相关的必需基因comS丢失,致使Y2不能形成自然感受态。comS的上游调控基因comX也发生了缺失现象。Y2具有编码Ⅰ型限制修复系统的基因簇hsdRMS （MUS₃105～MUS₃103）,导致Y2的后续分子操作困难。5.以芽孢杆菌表达载体pHT01为载体骨架,构建了不同类型的大肠杆菌-芽孢杆菌GFP表达穿梭载体。通过自然转化,将5种GFP表达载体导入“保姆”菌株枯草芽孢杆菌168和解淀粉芽孢杆菌FZB42,提取质粒并电击转化Y2感受态细胞,成功地获得了3株稳定性好、具GFP标记的Y2菌株,分别命名为Y2-pHAPII、 Y2-pGFP4412以及Y2-P43GFPmut3a。6.选用GFP标记菌Y2-P43GFPmut3a为接种材料,初步研究了Y2在玉米根系及根际土壤的定殖动态和内生性。Y2在玉米的根内、根表及根际土壤有良好的定殖,在接近2个月的期间内,菌浇数量分别维持在103～104、106-107以及105～106cfu/g（鲜根或土）,种群数量变化不大。Y2也能在玉米的茎、叶组织中定殖,定殖密度为102cfu/g鲜组织。利用激光共聚焦扫描显微镜观察组织压片和切片,证明在接种2天后,Y2就在玉米根毛、根的伸长区和成熟区定殖,接种第5天和第9天,就能在玉米茎和叶组织中找到定殖菌体。组织观察与菌落回收的结果相吻合。7.以pHT01为出发载体,构建了诱导表达感受态形成关键因子ComK的载体pHT01-comK并导入Y2感受态细胞。通过诱导表达ComK, Y2的自然感受特性得到了恢复。当以解淀粉芽孢杆菌自然转化生长培养基GCHE培养Y2至OD600达0.5～0.6,加入终浓度为lmM IPTG诱导1h,再将质粒与Y2混合后复苏3h,Y2的转化子可达3.06±0.22×104cfu/μg质粒DNA。在世界上首次将自然转化缺陷型野生解淀粉芽孢杆菌恢复了自然转化能力,为这类菌株的基因操作提供了新的技术策略。更多还原

【Abstract】 Plant growth-promoting rhizobacteria （PGPR）, is one kind of bacteria living in the plant rhizospheric soil and could promote crop growth and control plant diseases. Bacillus amyloliquefaciens B9601-Y2（Y2）, belonging to plantarum group, was isolated from rhizospheric soil of wheat and stored in our laboratory. In vitro, Y2has strong antagonistic activity against many common phytopathogenic fungi and bacteria, and could promote a number of crops growth and increase yield in the field. To understand its mechanisms, Y2genome was sequenced and analyzed. Y2’s ability to produce antibiotic metabolites and colonize in maize and its rhizospheric soil was confirmed by MALDI-TOF-MS and through GFP-tagged technology, respectively. Y2natural competence was recovered with gene-engineering method. The results obtained in this study were as follows:1. The genome size of Y2is4238624bp（base pairs） with accession number NC17912.1in NCBI database. It was predicted that Y2had45.76%GC content and4159CDS with the overall length3704557bp, accounting for87.4%of the whole genome. The number of tRNA gene, rDNA operon, tandem repeated region, transposon and inserted sequence element are91,10,69,73and24, respectively.2. Many living strategies were found in Y2through genomic annotation, which indicate that Y2could tolerate harsh environments and survive. Y2has conserved core genes related to the DNA repair system in Bacillus, such as alkA （MUS₄255）, ung（MUS₄184）,mutM（MUS₃183） and other genes acquired in excision repair system; mutS（MUS₁860）, mutL（MUS₁861）, XseA（MUS₂727） and XseB （MUS₂726） in mismatch repair system and some essential genes in non-homologous end joining and homologous repair system. Members of gene utilizing polysaccharides such as cellulose, hemicellulose, pectin and starch were discovered in Y2genome, such as MUS₂161, ydhP（MUS₄265）, bglS （MUS₄304）, pelB （MUS₄319） and amyE （MUS₀286）, suggesting that Y2could make use of limit nutrient in the soil without appropriate plant hosts to increase its own population rapidly. Meanwhile, some genes encoding the chitin-degrading enzymes were also found, partially explaining why Y2has so strong antagonism against some pathogenic fungi. Y2owns the synthetic clusters and regulatory genes of biofilm, which shows Y2has a good mutual benefit relationship with plant host. 3. Many antibiotic biosynthetic clusters were found in Y2genome, whose products were detected by MALDT-TOF-MS. Besides the production of some common lipopeptides fengycin, iturin, bacillibactin, bacilysin and polyketides bacillaene, macrolactin and difficidin, Y2could secrete bacteriocins such as mersacidin to inhibit the growth of phytopathogenic fungi and reduce the occurrence of plant disease. The alkaline serine protease gene and metabolic pathways of some plant growth-promoting volatile compound2,3-butanediol and acetoin discovered in Y2genome provide molecular evidence for Y2to control diseases caused by fungi and root-knot nematode and promote plant growth.4. An amazing phenomenon we discovered is that Y2is a surfactin-deficient strain because of the deletion of srfAB, the second gene member in surfactin operon srfA. The indirect result of srfAB deletion leads to the loss of comS, an essential gene in natural competence, which makes Y2deficient in natural transformation. comX, an upstream regulatory gene of comS, was also absent. At the same time, Y2contains type I restriction-modification system hsdRMS （MUS₃105～MUS₃103）. All of that leads to difficulty in molecular manipulation of Y2.5. Several types of Escherichia coli-Bacillus shuttle vector in GFP expression had been constructed based on the backbone of Bacillus expression plasmid pHT01. These vectors were transformed into transient hosts B. subtilis168and B. amyloliquefaciens FZB42by natural transformation. Y2electro-competent cells were electroporated with the plasmid DNA extracted from B168and FZB42.3GFP-tagged Y2strains were obtained and named as Y2-pHAPII, Y2-pGFP4412and Y2-P43GFPmut3a, respectively.6. The colonization dynamics and endophytic characteristic of Y2in maize root and rhizo spheric soil were studied primarily, with the GFP-tagged strain Y2-P43GFPmut3a as inoculant. The results showed that Y2had a good colonization in maize roots, root surface and rhizospheric soil, whose population number has been10～104,106～107and105～106cfu/g （root or soil）, respectively, and hasn’t changed sharply during two months. Furthermore, Y2could colonize in maize stem and leaf although the number was only102cfu/g （fresh tissue）. The tissue slices were observed under the confocal laser scanning microscopy. And the results showed2days after inoculation （DAI）, Y2-P43GFPmut3a could colonize the root hair, elongation region and matured zone of maize root and in maize stem and leaf tissue at5DAI and9DAI, respectively. The phenomenon was proved by fluorescence observation. 7. Inducible expression vector pHT01-comK was constructed, based on pHT01skeleton. The natural competence of Y2was recovered through the inducible expression of ComK, a key factor in Bacillus natural competence. The condition of natural competence formation was optimized primarily, and resulted in the highest transformation efficiencies3.06±0.22×104cfu/μg DNA when using GCHE medium as growth medium, and inducibly expressing1h with a final concentration of lmM IPTG with0.5-0.6OD6oo, recovering3h after the addition of exogenous plasmid DNA. So far, it is the first report to recover the natural transformation of undomesticated B. amyloliquefaciens deficient in natural competence, which could provide a technological strategy for gene manipulation in these strains.更多还原