固氮施氏假单胞菌非编码RNA CrcZ和CrcY介导的碳代谢物抑制调控

发布时间：2018-04-03 21:54

本文选题：施氏假单胞菌　切入点：碳代谢物抑制　出处：《中国农业大学》2015年博士论文

【摘要】：碳代谢物抑制(CCR)是指当环境中存在多种碳源时,微生物优先利用优势碳源而抑制非优势碳源的利用从而达到最佳生长的现象。假单胞菌中的碳代谢物抑制调控系统由双组份系统CbrAB、碳代谢物抑制蛋白Crc以及非编码RNA (CrcZ、CrcY和CrcX)组成,非编码RNA在碳代谢物抑制过程中发挥了重要的作用,但目前固氮基因表达是否受碳代谢物抑制调控尚未见报道。固氮施氏假单胞菌A1501分离自南方水稻根际土壤,具有较强的与水稻联合固氮的能力。全基因组序列分析表明,A1501携带一套与其他假单胞菌类似的碳代谢物抑制调控系统,包括两个非编码RNA CrcZ和CrcY,但其调控机制尚不清楚。本文对非编码RNA CrcZ和CrcY在固氮施氏假单胞菌A1501中碳代谢物抑制的调控作用进行了研究,同时对两个非编码RNA在固氮酶活的调控功能进行了探索,取得了如下结果： 1.在4mM琥珀酸与15mM葡萄糖作为混合碳源的条件下,A1501表现出了二次生长现象,A1501优先利用琥珀酸作为碳源,抑制葡萄糖的代谢,当琥珀酸消耗完毕后,开始利用葡萄糖作为碳源继续生长。结果表明,A1501具有典型的碳代谢物抑制现象,乳酸钠和琥珀酸是A1501生长的优势碳源,而葡萄糖和苯甲酸是非优势碳源。 2.A1501中具有两个非编码RNA,其中CrcZ与铜绿假单胞菌中的CrcZ同源性达到了70%,CrcY与恶臭假单胞菌中CrcY同源性达到了65%,二者之间的同源性则达到了60%。Northern blot与Real-time PCR分析结果表明,在cbrB或rpoN单基因突变株中,两个非编码RNA的表达量与野生型相比明显下调。5'RACE分析确定了两个非编码RNA各自的转录起始位点及转录方向。在两个非编码RNA的上游,我们发现二者均具有双组份系统CbrAB和σ54(RpoN)保守的结合位点,凝胶阻滞试验进一步证明CbrB能够与两个非编码RNA的上游启动子序列结合,表明两个非编码RNA的表达受到了上述两个调控因子的调控。在乳酸钠或琥珀酸优势碳源存在时,双组份系统cbrAB和两个非编码RNA的表达量显著低于非优势碳源,表明其表达受环境中碳源变化的诱导,进而参与碳代谢物抑制。 3.为进一步研究非编码RNA CrcZ和CrcY的功能,分别构建了crcZ、crcY单突变株和crcZ/Y双突变株。结果表明,两个非编码RNA单独突变菌株在优势碳源(乳酸钠、琥珀酸)或非优势碳源(葡萄糖、苯甲酸)存在时,其生长与野生型类似。相反,crcZ/Y双突变株在非优势碳源中无法生长,也丧失了在混合碳源中所表现出的二次生长现象。Real-time PCR结果表明,在两个单独缺失突变菌株中,未被突变的非编码RNA的表达量显著上调。由此推测两个非编码RNA在碳代谢物抑制调控的功能上存在互补性。 4.以乳酸钠为碳源crcZ／Y双突变株的固氮酶活与野生型相比下降了1／3,而之前的研究表明cbrA、cbrB和crc突变株的固氮酶活都下降了40%左右。Real-time PCR结果表明,与野生型相比crcZ/Y双突变株中的固氮酶结构基因和固氮相关调控基因表达全部下调,这说明A1501中碳代谢物抑制系统不但影响了碳代谢调控而且也影响了固氮酶活。综上所述,非编码RNA CrcZ和CrcY是固氮施氏假单胞菌A1501碳代谢物抑制中非常重要的组分,它们能够使A1501在多碳源复杂环境中对碳源进行高效利用,并确保其稳定生长和高效固氮。根据研究,我们提出了A1501中CrcZ和CrcY介导的碳代谢物抑制与固氮调控的模型,为进一步研究A1501碳氮代谢全局调控过程奠定了基础。
[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.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：Q935

【相似文献】