本文關(guān)鍵詞： 遼寧慢生根瘤菌 Tn5-pRL1063a 銅抗性 操縱子　出處：《西北農(nóng)林科技大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：土壤的銅污染會給其中微生物造成一定的選擇壓力,根瘤菌要保證自身的存活并且與宿主植物形成有效的共生固氮體系,必須采取一定的保護(hù)機(jī)制來抵抗高銅濃度造成的脅迫。分離自陜西寧強(qiáng)代家壩銅鋅礦中的遼寧慢生根瘤菌Bradyrhizobium liaoningense CCNWSX0360對銅的最大耐受濃度為2.0 mM。本文利用轉(zhuǎn)座子Tn5-pRL1063a對B.liaoningense CCNWSX0360進(jìn)行隨機(jī)突變,建立了庫容為17247的突變體庫。銅敏感性篩選獲得6株突變株Bln-d、Bln-163、Bln-c、Bln-29、Bln-32和Bln-54。采用質(zhì)粒挽救法確定Tn5的插入位點,結(jié)果顯示Bln-d和Bln-163中Tn5插入到同一個基因的不同位置,相似性比對鑒定為編碼重金屬轉(zhuǎn)運(yùn)P1B-type ATPase的基因,命名為cueA,其余4個突變體中Tn5分別插入到基因copA(編碼多銅氧化酶)、ctpA(編碼羧基末端處理酶)、tolC(編碼TolC家族蛋白)和lptE(編碼脂多糖轉(zhuǎn)運(yùn)分子伴侶)內(nèi)部。重金屬耐受性分析結(jié)果顯示:Bln-d、Bln-163、Bln-c專一對銅敏感,說明cueA和cop A特異地介導(dǎo)菌株對銅的抗性,在保護(hù)細(xì)菌免受銅的毒害中起關(guān)鍵作用;除Cu以外,Bln-32、Bln-54和Bln-29不同程度地對其它重金屬敏感,說明ctpA、tolC和lptE通過間接途徑賦予根瘤菌銅抗性。根據(jù)上述基因的功能注釋得出:在B.liaoningense CCNWSX0360中至少存在3種抗銅機(jī)制:跨膜轉(zhuǎn)運(yùn)機(jī)制(cueA和tolC)、氧化/還原機(jī)制(copA)和膜屏障保護(hù)機(jī)制(ctpA和lptE),共同賦予細(xì)菌抵抗銅脅迫的能力。根據(jù)跨膜金屬結(jié)合基序及標(biāo)簽序列信息本研究將cueA編碼的蛋白CueA歸為Cu+/Ag+轉(zhuǎn)運(yùn)P1B-type ATPase。CueA的N端的金屬結(jié)合基序為兩段His-rich區(qū),而不是典型的GXXCXXC基序。利用同源重組構(gòu)建了突變體ΔcsoR和ΔcueA,銅耐受性測試結(jié)果顯示:ΔcueA和cueA::Tn5的銅敏感程度相同,對銅的最大耐受濃度(MTC)為0.8mM,顯著低于野生型;互補(bǔ)cueA基因能夠回復(fù)突變體到野生型菌株的抗性水平。qRT-PCR和lacZ融合表達(dá)分析結(jié)果表明:Cu2+顯著誘導(dǎo)cueA的表達(dá)且表達(dá)水平依賴于Cu2+的濃度;一價銅離子螯合劑BCS能夠消除(減小)Cu2+對cueA的誘導(dǎo)和對細(xì)菌的毒害。結(jié)合銅耐受性測試、qRT-PCR以及l(fā)acZ融合表達(dá)分析結(jié)果證實了cueA通過向細(xì)胞周質(zhì)空間轉(zhuǎn)運(yùn)一價Cu+來維持根瘤菌的銅抗性。運(yùn)用qRT-PCR和lacZ融合表達(dá)分析技術(shù)檢測了野生型、突變體ΔcsoR以及互補(bǔ)菌株ΔcsoR(csoR)中csoR和cueA的表達(dá)水平,結(jié)果表明:缺失csoR引起基因cueA和csoR高水平組成型表達(dá);互補(bǔ)csoR恢復(fù)依賴Cu2+的誘導(dǎo)表達(dá),證明了CsoR利用去阻遏機(jī)制負(fù)調(diào)節(jié)csoR和cueA。實驗結(jié)果還表明:突變csoR顯著增加了菌株對Cu、Zn和Cd的抗性,并且這3種金屬離子均能誘導(dǎo)基因csoR和cueA的表達(dá);在Escherichia coli GG48中表達(dá)CueA能夠提高該菌對Zn和Cd抗性,說明cueA不僅能夠介導(dǎo)Cu+而且介導(dǎo)Zn2+和Cd2+的抗性。此外,N端截短實驗發(fā)現(xiàn):缺失N端的His-rich區(qū)喪失CueA的部分銅抗性功能,完全喪失在CueA的Zn/Cd抗性功能,表明N端的His-rich區(qū)在CueA的抗銅功能中發(fā)揮重要作用但不是必須的,而在抗Zn和Cd的功能中是絕對需要的,具體作用還不清楚,可能通過金屬離子結(jié)合位點來緩沖細(xì)胞內(nèi)的金屬離子濃度。通過共接菌實驗比較野生菌和突變體?cueA的競爭結(jié)瘤能力,得出根瘤菌的銅抗性能夠賦予其在銅脅迫下的結(jié)瘤能力�；蛲蛔円约般~耐受性分析結(jié)果顯示:cop操縱子中6個基因均專一對銅敏感,其中copA和copB是整個系統(tǒng)的核心組分,二者需要同時存在才能發(fā)揮作用;新基因2212和2213能夠賦予cop操縱子的完全的銅抗性;copC和cus F則對菌株的耐受性貢獻(xiàn)不大,推測這6個基因編碼的蛋白可能相互作用形成介導(dǎo)銅抗性的符合體。這6個基因在不同銅濃度脅迫下具有相同的表達(dá)趨勢,即在較低Cu2+濃度下,隨著Cu2+濃度的升高各個基因的轉(zhuǎn)錄水平逐漸增加,在0.625 mM Cu2+濃度下表達(dá)量均達(dá)到最大,在高濃度下則轉(zhuǎn)錄水平下降。綜合共轉(zhuǎn)錄驗證實驗及5’RACE實驗得出這6個基因為一個轉(zhuǎn)錄單元,受同一個啟動子啟動轉(zhuǎn)錄,轉(zhuǎn)錄起始點為基因2213起始密碼子上游第26個堿基“G”。啟動子活性分析以及l(fā)acZ融合表達(dá)分析結(jié)果表明2213與2212之間的間隔序列在調(diào)節(jié)操縱子的表達(dá)起著關(guān)鍵作用,但具體調(diào)控機(jī)制還未研究不清楚。生物信息學(xué)分析數(shù)據(jù)顯示:cop操縱子可能為一個進(jìn)化上較為古老的銅抗性操縱子。與單獨敲除cueA或copA相比,同時敲除這兩個基因菌株更為敏感,說明cueA和cop操縱子兩個獨立的系統(tǒng),共同保護(hù)細(xì)胞免受銅離子的毒害。
[Abstract]:The soil copper contamination caused by microorganisms which will give certain selection pressure, rhizobia to ensure their own survival and the formation of host plant symbiotic nitrogen fixation system effectively, must take protective mechanism to stress resistance caused by high copper concentration. Isolated from Shaanxi Ningqiang daijiaba copper zinc ore in Liaoning Bradyrhizobium Bradyrhizobium liaoningense CCNWSX0360 maximum tolerated concentration of copper is 2 mM. by using Tn5-pRL1063a transposon random mutation of B.liaoningense CCNWSX0360, a capacity of 17247 of the mutant library. Copper sensitivity screening 6 strains Bln-d, Bln-163, Bln-c, Bln-29, Bln-32 and Bln-54. using plasmid rescue method to determine the Tn5 insertion site, the results showed Bln-d and Bln-163 Tn5 into different positions of the same gene, similarity comparison identified as encoding P1B-type heavy metal transporter ATPase The gene, named cueA, the remaining 4 mutants in Tn5 were inserted into the copA gene (encoding multicopper oxidase), ctpA (encoding the carboxyl terminal processing enzyme (tolC), encoding TolC protein family) and lptE (encoding transporter lipopolysaccharide molecular chaperone). Heavy metal tolerance analysis showed: Bln-d, Bln-163, Bln-c specific the copper sensitive, cueA and cop A specifically mediate resistance to copper, plays a key role in the protection of the bacteria from copper toxicity; except Cu, Bln-32, Bln-54 and Bln-29 in different degrees of other heavy metal sensitive, that ctpA, tolC and lptE by indirect way gives rhizobia copper resistance. According to the functional annotation of these genes showed that: there are at least 3 kinds of copper resistance mechanism in B.liaoningense CCNWSX0360: transmembrane transport mechanism (cueA and tolC), oxidation / reduction mechanism (copA) and membrane barrier protection mechanisms (ctpA and lptE), to give bacterial resistance to copper Stress. According to the transmembrane metal binding motif and tag sequence information on the cueA encoding protein CueA belongs to the Cu+/Ag+ ATPase.CueA transfer P1B-type N end of the metal binding motif for the two His-rich area, rather than the typical GXXCXXC motif. We constructed a mutant csoR and cueA homologous recombination. The test results showed that copper tolerance of Delta cueA and cueA: copper Tn5 sensitivity, the maximum tolerated concentration of copper (MTC) 0.8mM, was significantly lower than that of the wild type; complementary cueA gene can recover the mutant to wild type strain resistance level of.QRT-PCR and lacZ fusion expression analysis results showed that the concentration of cueA and the expression depends on the level of Cu2+ was significantly induced by Cu2+; copper ion chelating agent BCS can eliminate (decrease) induced by Cu2+ on cueA and on bacterial toxicity. Combined with copper tolerance test, qRT-PCR and lacZ fusion expression analysis results Confirmed through cueA to the periplasmic space transport of monovalent Cu+ to maintain the rhizobia of copper resistance. Using qRT-PCR and lacZ fusion analysis technique was used to detect the expression of the wild type and mutant and complementary strain Delta csoR Delta csoR (csoR) expression in csoR and cueA. The results showed that the expression of cueA and csoR caused by the high level group the lack of forming csoR; complementary induced expression of csoR dependent Cu2+ recovery, proved by CsoR derepression mechanism of negative regulation of csoR and cueA., the experiment results also indicated that the mutation of csoR significantly increased the resistance of Zn strains to Cu, and Cd, and the expression of the 3 kinds of metal ions were induced by csoR gene and cueA CueA can be improved; the bacteria of Zn and Cd expression in Escherichia coli GG48 resistance, cueA resistance and can not only mediate Cu+ mediated Zn2+ and Cd2+. In addition, found N truncated experiment: part of the copper loss of N end His-rich CueA anti loss zone Complete loss of function, function in Zn/Cd resistant CueA, showed that the N side of the His-rich area in CueA copper resistance function plays an important role but not necessary, and in the anti Zn and Cd function is absolutely necessary, the specific function is not clear, the concentration of metal ions may buffer cells via metal ion binding sites. By CO inoculation experiments comparing wild strains and mutants? Competitive nodulation ability of cueA, the rhizobia can give its Cu resistance under the stress of copper nodulation ability. Gene mutation and copper tolerance analysis results show that the cop operon in 6 genes specific for copper sensitive copA and copB is the core component of the whole system, the two need to exist at the same time can play a role; completely new copper resistance gene 2212 and 2213 to give the cop operon and cus F copC; the strain tolerance contributed little to push the 6 test The gene encoding protein may be formed with copper resistance mediated interactions. These 6 genes have the same expression trend in different concentrations of copper stress, even at a low concentration of Cu2+, with elevated levels of transcription of each gene Cu2+ concentration increased gradually, reached the maximum amount of expression at 0.625 mM concentration of Cu2+ then, the transcription level decreased at high concentration. The comprehensive cotranscription validation experiment and 5 RACE experiment shows that the 6 gene is a transcription unit, by the same promoter transcription, transcription start point for 2213 genes upstream of the start codon twenty-sixth nucleotides G. The promoter activity of lacZ and fusion expression analysis showed that the interval between 2213 and 2212 sequence plays a key role in regulating the expression of the operon, but the specific regulatory mechanism has not been studied clearly. Bioinformatics analysis data show that: the cop operon may be a A more ancient copper resistance operon is more sensitive than knocking out cueA or copA individually. It is more sensitive to knock out the two genes, indicating that two independent systems of cueA and cop operon protect the cells from copper toxicity.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q939.114
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本文編號：1456964