發(fā)布時(shí)間：2018-03-09 20:23

  本文選題：沙雷氏菌　切入點(diǎn)：基因組　出處：《南京農(nóng)業(yè)大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：粘質(zhì)沙雷氏菌FS14是本實(shí)驗(yàn)室分離自白術(shù)根腐病害樣品的一株產(chǎn)紅色色素的腸桿菌。在平板對(duì)峙實(shí)驗(yàn)中,FS14表現(xiàn)出對(duì)植物病害真菌如尖孢鐮刀菌和核盤菌等具有很強(qiáng)的拮抗作用,另外FS14對(duì)番茄病原細(xì)菌青枯雷爾氏菌也具有明顯的抑制作用。雖然粘質(zhì)沙雷氏菌的幾丁質(zhì)酶很早就被用于防治植物真菌病害,也有報(bào)道用粘質(zhì)沙雷氏菌防治昆蟲病害,但是對(duì)粘質(zhì)沙雷氏菌拮抗植物病原的分子機(jī)制還不清楚。為了研究粘質(zhì)沙雷氏菌FS14生物防治植物病害的機(jī)制,使其能穩(wěn)定的用于農(nóng)業(yè)生產(chǎn),本研究對(duì)該菌株的全基因組進(jìn)行了測(cè)序并做了比較基因組學(xué)分析,從基因水平了解沙雷氏菌的基本遺傳特征,分析了拮抗相關(guān)基因,并表達(dá)和結(jié)晶了在FS14拮抗植物病原細(xì)菌中可能起關(guān)鍵作用的Ⅵ型分泌系統(tǒng)的翻譯后調(diào)控蛋白和免疫蛋白。具體研究結(jié)果包括:1.本研究利用454焦磷酸測(cè)序?qū)闒S14進(jìn)行了全基因組測(cè)序。FS14的基因組由一個(gè)閉合環(huán)狀染色體組成,基因組全長(zhǎng)5,249,875 bp,G+C含量為59.46%(GenBank序列號(hào):CP005927)。結(jié)合Glimmer和GeneMark預(yù)測(cè)得到4,761個(gè)編碼序列,編碼區(qū)域總長(zhǎng)度為4,505,070bp,覆蓋了FS14全基因組序列的85.8%。結(jié)合其他9株已公布的沙雷氏菌全基因組對(duì)該屬進(jìn)行了全面的比較基因組學(xué)分析,以FS14為參考菌株進(jìn)行泛基因組分析,發(fā)現(xiàn)沙雷氏菌泛基因組的大小隨著基因組數(shù)目的增加而增加,屬于開放型泛基因組。10株沙雷氏菌的泛基因組含有10,837個(gè)編碼基因,大約是單個(gè)基因組大小的2倍,表明沙雷氏菌不同菌株間存在豐富的遺傳多樣性;核心基因組包含1,964個(gè)編碼基因(約是FS14基因數(shù)目的41%),其中大部分基因與細(xì)菌的基本功能相關(guān);菌株特異性序列主要包括原噬菌體和假想蛋白,在這些特異性序列中還包括Ⅰ型限制修飾系統(tǒng)、Ⅲ型分泌系統(tǒng)、Ⅵ型分泌系統(tǒng)和碲抗性基因等,這些基因只出現(xiàn)在部分沙雷氏菌的基因組中。2.進(jìn)一步分析發(fā)現(xiàn),菌株FS14的基因組擁有多種與它的拮抗特性相關(guān)的基因,如靈菌紅素、細(xì)菌素、嗜鐵素、多重耐藥性、表面活性劑、幾丁質(zhì)酶、蛋白酶、脂肪酶和磷脂酶等的編碼基因。值得注意的是,在FS14的基因組中還發(fā)現(xiàn)了兩個(gè)獨(dú)立的Ⅵ型分泌系統(tǒng)(T6SS),其中一個(gè)與具有拮抗細(xì)菌功能的T6SS同源。用生物信息學(xué)方法找到已測(cè)序的沙雷氏菌的所有T6 S S,對(duì)其編碼基因簇的組成結(jié)構(gòu)進(jìn)行比較分析,并利用130個(gè)T6SS保守蛋白ClpV的序列構(gòu)建分子進(jìn)化樹,發(fā)現(xiàn)沙雷氏菌的T6SS分成四個(gè)分類群:T6SS-a,T6SS-b,T6SS-c和T6SS-d,其中T6SS-a保守存在于所有包含T6SS的沙雷氏菌中。菌株FS14的基因組包含T6SS-a和T6SS-b。這是目前為止,首次在沙雷氏菌的一個(gè)基因組中發(fā)現(xiàn)兩種T6SS。3.沙雷氏菌Db10的T6SS-a被證明用于攻擊同一環(huán)境中的其它細(xì)菌,因此菌株FS14的T6SS-a可能在其拮抗青枯雷爾氏菌中起關(guān)鍵作用。而T6SS的激活依賴絲氨酸/蘇氦酸激酶(PpkA)和絲氨酸/蘇氨酸磷酸酶(PppA)的翻譯后調(diào)控。PpkA使T6SS中的Fha蛋白蘇氨酸磷酸化,繼而激活T6SS分泌復(fù)合物的組裝和Hcp蛋白的分泌。PppA的作用與PpkA相反,使Fha去磷酸化。Fha是PpkA和PppA的共同作用底物,是T6S分泌裝置的關(guān)鍵組分。翻譯后調(diào)控使得T6SS能快速應(yīng)對(duì)環(huán)境變化,對(duì)細(xì)菌適應(yīng)環(huán)境并保持競(jìng)爭(zhēng)優(yōu)勢(shì)起重要作用。將粘質(zhì)沙雷氏菌T6SS-a的PpkA的N-端激酶結(jié)構(gòu)域(PpkN)及C-端結(jié)構(gòu)域(PpkAC)、PppA和Fha的基因通過(guò)PCR方法擴(kuò)增并用大腸桿菌C43(DE3)表達(dá),經(jīng)親和層析和凝膠過(guò)濾層析對(duì)重組蛋白進(jìn)行分離純化。體外表達(dá)并純化的PpkAC有很強(qiáng)的降解DNA和RNA的核酸酶活性。純化得到的PpkAN、PppA和Fha蛋白用坐滴氣相擴(kuò)散法進(jìn)行蛋白質(zhì)結(jié)晶條件的篩選和條件優(yōu)化。PpkAN的初始結(jié)晶條件為:①0.1M NaCl,0.1M HEPES pH7.5,1.6M(NH4)2SO4;②0.1M HEPES-Na pH 7.5,1.5M Li2SO4;PppA的結(jié)晶條件為0.1M HEPES-Na pH7.5,0.75M Li2SO4;Fha蛋白較好的兩個(gè)結(jié)晶條件為:①0.2M MgCl2,0.1M BIS-TRIS pH 5.5,25%PEG3350;②0.2M MgCl2,0.1M Tris-HCl,pH 8.5,30%PEG4000。通過(guò)對(duì)這些晶體進(jìn)行初步X-光射線衍射分析發(fā)現(xiàn)這些晶體的分辨率并不理想。正在對(duì)這些結(jié)晶條件進(jìn)一步優(yōu)化,以便能夠得到衍射能力良好的晶體并解析結(jié)構(gòu),為研究T6SS的翻譯后調(diào)控機(jī)制提供結(jié)構(gòu)基礎(chǔ)。4.T6SS的效應(yīng)蛋白被注射到缺少免疫蛋白的靶細(xì)胞中抑制靶細(xì)胞的生長(zhǎng),而相應(yīng)的免疫蛋白能中和效應(yīng)蛋白使自身細(xì)胞免受攻擊。在FS14的T6SS-a中發(fā)現(xiàn)3個(gè)免疫蛋白的編碼基因(L085_13755、L085_13760和L085_13765),其中L085_13755和L085_13760分別與粘質(zhì)沙雷氏菌Db10的Rap2b和Rap2a有很高的同源性(90%),而L085_13765在菌株Db10中沒(méi)有找到對(duì)應(yīng)序列,但是在PDB中比對(duì)發(fā)現(xiàn)其與菌株Db10的T6SS免疫蛋白R(shí)ap2a具有32%的同源性,推測(cè)L085_13765編碼FS14 T6SS-a的第三個(gè)免疫蛋白,命名為Rap2c。本研究用大腸桿菌C43(DE3)誘導(dǎo)表達(dá)Rap2c蛋白,經(jīng)親和層析和凝膠過(guò)濾層析對(duì)重組蛋白進(jìn)行分離純化,得到了高純度且聚合狀態(tài)單一的目的蛋白。將純化得到的Rap2c蛋白濃縮至適當(dāng)濃度后,用坐滴氣相擴(kuò)散法進(jìn)行蛋白質(zhì)結(jié)晶條件的篩選和條件優(yōu)化。對(duì)在這些條件下生長(zhǎng)得到的晶體進(jìn)行初步X-光射線衍射分析發(fā)現(xiàn)這些晶體的分辨率約為7A。通過(guò)對(duì)Rap2c的二級(jí)結(jié)構(gòu)和三級(jí)結(jié)構(gòu)的預(yù)測(cè),發(fā)現(xiàn)Rap2c同Tsi家族的免疫蛋白一樣由5個(gè)α螺旋(α1-α5)折疊成束狀物,Cys31-Cys95可以形成二硫鍵將α2和α4連接,α3螺旋可能是結(jié)合效應(yīng)蛋白的關(guān)鍵部位,而α3-α4 loop可能在阻斷效應(yīng)蛋白的活性時(shí)起主要作用。
[Abstract]:Serratia marcescens FS14 isolated from Atractylodes root rot disease samples of a strain producing red pigment of Enterobacteriaceae in our laboratory. In plate confrontation test, FS14 has shown strong antagonism against plant disease fungi such as Fusarium oxysporum and Sclerotinia sclerotiorum, in addition FS14 on tomato pathogen Ralstonia ray Yersinia also has obvious inhibitory effects. Although Serratia marcescens chitinase has long been used for prevention and treatment of fungal diseases of plants, have also been reported with Serratia marcescens insect disease, but the molecular mechanism of Serratia marcescens against plant pathogens is not clear. In order to study the mechanism of Serratia marcescens FS14 biological control of plant diseases, which can be used to stabilize the agricultural production, the Research on the whole genome of the strain was sequenced and the comparative genomics analysis, from gene level to understand the basic genetics of Serratia Transmission characteristics, analysis of the antagonistic related genes, and the expression and regulation of protein crystallization and immune protein of type VI may play a key role in FS14 against plant pathogenic bacteria in the secretory system of translation. The research results include: 1. the study of the strain FS14 were whole genome sequencing the genome of.FS14 consists of a closed ring chromosome using 454 pyrosequencing, full-length 5249875 BP, the content of G+C is 59.46% (GenBank serial number: CP005927). The combination of Glimmer and GeneMark predicted 4761 encoding sequence, the total length of encoding region was 4505070bp, covering the whole genome sequence of FS14 85.8%. with other 9 strains have been published on the whole genome of Serratia sp. a comprehensive study of comparative genomic analysis, using FS14 as the reference strains of Pan genome analysis, found that Serratia pan genome size with genome number Increases, belong to the open type pan genome of.10 strain Shaleishi bacteria pan genome contains 10837 genes encoding, is about 2 times that of a single genome size, showed that Serratia marcescens isolates existed abundant genetic diversity; the core genome contains 1964 encoding gene (FS14 gene number is about 41%). One of the most basic functions of the related gene and bacterial strains; specific sequences including the original phage and hypothetical protein, also included in these specific sequences of type I restriction modification system, type III secretion system, the type VI secretion system and tellurium resistance genes, these genes appear only in part of the genome of Serratia.2. further analysis showed that the genome of strain FS14 has a variety of characteristics associated with its antagonistic genes, such as prodigiosin, bacteriocin, siderophore, multidrug resistance, surfactant, chitinase, The gene encoding protease, lipase and phospholipase etc.. It is worth noting that in the genome of FS14 was found in two independent type VI secretion system (T6SS), which has a homology with antagonistic bacteria T6SS. Sarre's function of bacteria by Bioinformatics Method to find sequenced all T6 S S and compare the composition of its encoding gene cluster, and constructed a phylogenetic tree using a sequence of 130 conserved T6SS protein ClpV, found that Serratia T6SS divided into four taxa: T6SS-a, T6SS-b, T6SS-c and T6SS-d, the conservative T6SS-a exists in all T6SS containing Serratia strain FS14. The genome contains T6SS-a and T6SS-b. which is now in a genome of Serratia found two T6SS.3. Serratia Db10 T6SS-a proved for other bacterial attack in the same environment for the first time, so strain FS14 T6S S-a may play a key role in the antagonism of Ralstonia solanacearum. Activation of T6SS dependent serine / threonine kinase (PpkA) and helium acid serine / threonine phosphatase (PppA) post-translational regulation of.PpkA Fha protein threonine phosphorylation of T6SS, PpkA and.PppA secretion function and activation of Hcp protein and assembly the secretion of T6SS complexes of dephosphorylated Fha on the contrary,.Fha is the combined effect of PpkA and PppA substrate, T6S group is the key device. The secretion of post-translational regulation makes T6SS can quickly respond to environmental changes, to adapt to the environment of bacteria and maintain the competitive advantage. It will play an important role in Serratia marcescens T6SS-a PpkA the N- terminal kinase domain (PpkN) and C- domain (PpkAC), PppA and Fha genes were amplified by PCR and Escherichia coli C43 (DE3) expression of recombinant protein was purified by affinity chromatography and gel filtration chromatography in vitro expression. Nuclease activity and purification of PpkAC has a strong degradation of DNA and RNA. The purified PpkAN, initial crystallization condition screening and optimization of.PpkAN PppA and Fha protein using the sitting drop vapor diffusion method for protein crystallization conditions as follows: 0.1M NaCl, 0.1M HEPES pH7.5,1.6M (NH4) 2SO4; 0.1M HEPES-Na pH 7.5,1.5M Li2SO4; PppA 0.1M HEPES-Na as the crystallization conditions of pH7.5,0.75M Li2SO4; two Fha protein crystallization conditions better: 1 0.2M MgCl2,0.1M BIS-TRIS pH 5.5,25%PEG3350; MgCl2,0.1M Tris-HCl pH 8.5,30%PEG4000. 0.2M, through a preliminary X- ray diffraction analysis shows that these crystals are not ideal for the resolution of these crystals. These are further optimization of crystallization conditions and in order to be able to get good crystal diffraction ability and analytical structure, provide the structural basis of.4.T6SS regulation effect and mechanism of T6SS after translation The protein was injected into the target cells lack immune protein in inhibiting cell growth, while the corresponding immune protein can neutralize effector proteins to make their own cells from attack. The genes encoding the 3 immune protein found in FS14 T6SS-a (L085_13755, L085_13760 and L085_13765), the L085_13755 and L085_13760 respectively with Serratia marcescens Db10 Rap2b and Rap2a have very high homology (90%), and L085_13765 in strain Db10 was not found in the corresponding sequence, but found that the strain Db10 and T6SS immune protein Rap2a has 32% homology in the PDB ratio of third, suggesting that L085_13765 encoding FS14 T6SS-a protein, named Rap2c. in this study Escherichia coli C43 (DE3) induced the expression of Rap2c protein, the recombinant protein was purified by affinity chromatography and gel filtration chromatography, obtained high purity and aggregation of single target protein will be pure. The obtained Rap2c protein concentration to the appropriate concentration, screening and optimization of protein crystallization conditions using the sitting drop vapor diffusion method. A preliminary X- ray diffraction analysis showed that the crystal resolution is about 7A. by predicting the two level structure of Rap2c and the three stage structure on the growth obtained in these conditions crystal, found that the immune protein Rap2c with Tsi as the family consists of 5 alpha helix (alpha 1- alpha 5) folded into clusters, Cys31-Cys95 can form two disulfide bonds in the alpha 2 and alpha 4 alpha 3 screw connection, may be the key site binding protein, alpha 3- alpha 4 loop may play a major role in effect of blocking the activity of the protein.

【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：S432.4

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本文編號(hào)：1590108