本文選題：施氏假單胞菌　切入點：碳代謝物抑制　出處：《中國農(nóng)業(yè)大學(xué)》2015年博士論文

【摘要】：碳代謝物抑制(CCR)是指當(dāng)環(huán)境中存在多種碳源時,微生物優(yōu)先利用優(yōu)勢碳源而抑制非優(yōu)勢碳源的利用從而達(dá)到最佳生長的現(xiàn)象。假單胞菌中的碳代謝物抑制調(diào)控系統(tǒng)由雙組份系統(tǒng)CbrAB、碳代謝物抑制蛋白Crc以及非編碼RNA (CrcZ、CrcY和CrcX)組成,非編碼RNA在碳代謝物抑制過程中發(fā)揮了重要的作用,但目前固氮基因表達(dá)是否受碳代謝物抑制調(diào)控尚未見報道。固氮施氏假單胞菌A1501分離自南方水稻根際土壤,具有較強的與水稻聯(lián)合固氮的能力。全基因組序列分析表明,A1501攜帶一套與其他假單胞菌類似的碳代謝物抑制調(diào)控系統(tǒng),包括兩個非編碼RNA CrcZ和CrcY,但其調(diào)控機制尚不清楚。本文對非編碼RNA CrcZ和CrcY在固氮施氏假單胞菌A1501中碳代謝物抑制的調(diào)控作用進(jìn)行了研究,同時對兩個非編碼RNA在固氮酶活的調(diào)控功能進(jìn)行了探索,取得了如下結(jié)果： 1.在4mM琥珀酸與15mM葡萄糖作為混合碳源的條件下,A1501表現(xiàn)出了二次生長現(xiàn)象,A1501優(yōu)先利用琥珀酸作為碳源,抑制葡萄糖的代謝,當(dāng)琥珀酸消耗完畢后,開始利用葡萄糖作為碳源繼續(xù)生長。結(jié)果表明,A1501具有典型的碳代謝物抑制現(xiàn)象,乳酸鈉和琥珀酸是A1501生長的優(yōu)勢碳源,而葡萄糖和苯甲酸是非優(yōu)勢碳源。 2.A1501中具有兩個非編碼RNA,其中CrcZ與銅綠假單胞菌中的CrcZ同源性達(dá)到了70%,CrcY與惡臭假單胞菌中CrcY同源性達(dá)到了65%,二者之間的同源性則達(dá)到了60%。Northern blot與Real-time PCR分析結(jié)果表明,在cbrB或rpoN單基因突變株中,兩個非編碼RNA的表達(dá)量與野生型相比明顯下調(diào)。5'RACE分析確定了兩個非編碼RNA各自的轉(zhuǎn)錄起始位點及轉(zhuǎn)錄方向。在兩個非編碼RNA的上游,我們發(fā)現(xiàn)二者均具有雙組份系統(tǒng)CbrAB和σ54(RpoN)保守的結(jié)合位點,凝膠阻滯試驗進(jìn)一步證明CbrB能夠與兩個非編碼RNA的上游啟動子序列結(jié)合,表明兩個非編碼RNA的表達(dá)受到了上述兩個調(diào)控因子的調(diào)控。在乳酸鈉或琥珀酸優(yōu)勢碳源存在時,雙組份系統(tǒng)cbrAB和兩個非編碼RNA的表達(dá)量顯著低于非優(yōu)勢碳源,表明其表達(dá)受環(huán)境中碳源變化的誘導(dǎo),進(jìn)而參與碳代謝物抑制。 3.為進(jìn)一步研究非編碼RNA CrcZ和CrcY的功能,分別構(gòu)建了crcZ、crcY單突變株和crcZ/Y雙突變株。結(jié)果表明,兩個非編碼RNA單獨突變菌株在優(yōu)勢碳源(乳酸鈉、琥珀酸)或非優(yōu)勢碳源(葡萄糖、苯甲酸)存在時,其生長與野生型類似。相反,crcZ/Y雙突變株在非優(yōu)勢碳源中無法生長,也喪失了在混合碳源中所表現(xiàn)出的二次生長現(xiàn)象。Real-time PCR結(jié)果表明,在兩個單獨缺失突變菌株中,未被突變的非編碼RNA的表達(dá)量顯著上調(diào)。由此推測兩個非編碼RNA在碳代謝物抑制調(diào)控的功能上存在互補性。 4.以乳酸鈉為碳源crcZ／Y雙突變株的固氮酶活與野生型相比下降了1／3,而之前的研究表明cbrA、cbrB和crc突變株的固氮酶活都下降了40%左右。Real-time PCR結(jié)果表明,與野生型相比crcZ/Y雙突變株中的固氮酶結(jié)構(gòu)基因和固氮相關(guān)調(diào)控基因表達(dá)全部下調(diào),這說明A1501中碳代謝物抑制系統(tǒng)不但影響了碳代謝調(diào)控而且也影響了固氮酶活。 綜上所述,非編碼RNA CrcZ和CrcY是固氮施氏假單胞菌A1501碳代謝物抑制中非常重要的組分,它們能夠使A1501在多碳源復(fù)雜環(huán)境中對碳源進(jìn)行高效利用,并確保其穩(wěn)定生長和高效固氮。根據(jù)研究,我們提出了A1501中CrcZ和CrcY介導(dǎo)的碳代謝物抑制與固氮調(diào)控的模型,為進(jìn)一步研究A1501碳氮代謝全局調(diào)控過程奠定了基礎(chǔ)。
[Abstract]:Carbon repressio (CCR) means that when there are many carbon sources in the environment, the use of microbial carbon source and inhibit the advantage of priority non dominant carbon source so as to achieve the best growth phenomenon. Carbon metabolites of Pseudomonas in the regulation system by the two-component system CbrAB, carbon metabolites inhibiting protein Crc and encoding RNA (CrcZ, CrcY and CrcX), non encoding RNA played an important role in carbon repressio process, but the nitrogen fixation gene expression is affected by the carbon repressio regulation has not been reported. The nitrogen fixing Pseudomonas stutzeri A1501 isolated from rhizosphere soil of southern rice, has a strong ability of nitrogen fixing and rice whole genome. Sequence analysis showed that A1501 carry a similar to other Pseudomonas repressio carbon regulation system, including two non RNA encoding CrcZ and CrcY, but the regulation mechanism is unclear. The non The regulation role of RNA CrcZ and CrcY in the inhibition of carbon metabolite in Pseudomonas Azotobacter A1501 was studied. Meanwhile, the function of two non coding RNA in nitrogenase activity was explored, and the following results were obtained.
In 1. 4mM succinic acid with 15mM glucose as the sole carbon source under the condition of A1501 showed a two growth phenomenon, A1501 preferred using succinic acid as carbon source, inhibition of glucose metabolism, when succinate consumed, using glucose as a carbon source to continue growth. The results show that A1501 has the typical carbon metabolites inhibition phenomenon. Sodium lactate and succinate is the advantage of carbon source A1501 growth, while glucose and benzoic acid is a non dominant carbon source.
With two non RNA encoding 2.A1501, CrcZ and Pseudomonas aeruginosa CrcZ homology reached 70%, CrcY and Pseudomonas putida CrcY homology reached 65%, two homology between blot and Real-time reached 60%.Northern PCR analysis results showed that in the cbrB or rpoN single gene mutation strain, expression of two non encoding RNA compared with the wild type significantly reduced.5'RACE analysis identified their transcription initiation site and direction of transcription two non RNA encoding. In the upper reaches of the two non RNA encoding, we found that two have the two-component system CbrAB and 54 sigma (RpoN) conserved binding sites the gel retardation experiments show that CbrB can further upstream and two non encoding RNA promoter sequence with two RNA showed that the expression of non encoding by regulation of the two regulation factors. In sodium lactate or carbon storage in advantage of succinic acid, The expression of cbrAB and two non coding RNA in two component system was significantly lower than that in non dominant carbon sources, indicating that their expression was induced by the change of carbon source in the environment and then involved in the inhibition of carbon metabolites.
3. for the further study of non RNA CrcZ encoding and CrcY function, we constructed crcZ, crcY single mutant crcZ/Y and double mutant. The results showed that two non RNA encoding separate mutant strains in advantage of carbon source (sodium lactate, succinate) or non dominant carbon sources (glucose, benzoic acid) is present, the the growth is similar to the wild type. On the contrary, crcZ/Y double mutant cannot grow in the non dominant carbon source, but also the loss shown in the mixed carbon source in the two growth phenomenon of.Real-time PCR showed that in two separate mutant strains, the expression is not mutation of non coding RNA was significantly raised. Therefore there are complementary to two non encoding RNA in carbon repressio control function.
4. using sodium lactate as carbon source crcZ / Y double mutant strains of nitrogenase activity compared with wild type decreased by 1 / 3, while the previous research showed that cbrA, cbrB and CRC mutant strains of nitrogenase activity were decreased by about 40%.Real-time PCR. The results showed that compared with the wild type crcZ/Y double mutant nitrogen fixation enzyme structure genes and genes involved in the regulation of nitrogen fixation lines was all down, indicating that the system not only affects the carbon repressio carbon metabolism and A1501 also affected the nitrogenase activity.
In summary, RNA CrcZ and CrcY encoding non nitrogen fixing Pseudomonas stutzeri A1501 carbon metabolites very important in inhibition group, they can make the A1501 in the carbon source in the complex environment of carbon source utilization, and to ensure its stable growth and efficient nitrogen fixation. According to the research, we put forward the carbon and nitrogen fixing repressio the regulation of A1501 in CrcZ and CrcY mediated model, laid a foundation for the further study of A1501 global carbon and nitrogen metabolism regulation.

【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：Q935

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