本文選題：溶磷菌 + 溶磷特性　； 參考：《吉林農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：溶磷微生物能將土壤中難溶性磷酸鹽轉(zhuǎn)化為能被植物吸收利用的可溶性磷形態(tài),從而提高土壤磷素利用效率,在農(nóng)業(yè)磷素循環(huán)利用方面有著重要作用。本論文針對(duì)大豆根際土壤中溶無(wú)機(jī)磷細(xì)菌溶磷特性進(jìn)行一系列的研究,主要取得以下成果:本試驗(yàn)從東北大豆根際土壤中分離和篩選出4株具有較強(qiáng)溶磷能力的細(xì)菌,其最大溶磷量分別為558μg/mL、478μg/mL、596μg/mL和586μg/mL。經(jīng)過(guò)Vitek2生理生化系統(tǒng)分析和16S rDNA序列測(cè)定,鑒定4株菌株分別為假單胞菌、腸桿菌、蒼白桿菌、克雷伯氏菌,并命名為wj1、wj3、wj5和wj6。本文以NBRIP分離培養(yǎng)基為基礎(chǔ),利用L25(53)正交設(shè)計(jì)試驗(yàn),從碳源、氮源及pH值三個(gè)方面對(duì)菌株溶磷培養(yǎng)基進(jìn)行優(yōu)化。結(jié)果表明:最佳碳源為葡萄糖,氮源為蛋白胨,初始pH值為8。對(duì)wj6溶解Ca3(PO4)2、FePO4和AlPO4特性的研究發(fā)現(xiàn),該菌株能不同程度的降解Ca3(PO4)2、FePO4和AlPO4三種磷酸鹽,最高溶磷量分別為409.28μg/mL、60.59μg/mL、32.90μg/mL。但是,在溶解FePO4和AlPO4時(shí)受培養(yǎng)環(huán)境pH的影響較大,將wj6接種到pH 8的溶磷培養(yǎng)基中,該菌株能溶解FePO4和AlPO4,在培養(yǎng)基原液(pH 3)中不具有溶磷能力。從溶磷菌產(chǎn)酸角度對(duì)溶磷機(jī)制進(jìn)行了研究。在以磷酸鈣為唯一磷源的條件下,菌株溶磷過(guò)程中伴隨著pH值的降低。采用GC-MS對(duì)菌株在溶磷過(guò)程中產(chǎn)生的有機(jī)酸分析:4株溶磷菌主要產(chǎn)生的有機(jī)酸有:2,2-二甲氧基丙酸、α-酮戊二酸、4-hexadienedioicacid、3-hydroxydecanoic Acid、蘋(píng)果酸、hepta-2,4-dienoic acid、乙酸、琥珀酸、檸檬酸等。酶活性檢測(cè)結(jié)果表明,在溶磷過(guò)程中菌液磷酸酶活性顯著增強(qiáng)。從wj1、wj3、wj5和wj6溶磷菌中克隆GDH基因,分別獲得大小為2007bp、2066bp、1727bp、2391bp的基因片段。序列分別與GenBank中Pseudomonas brassicacearum subsp.brassicacearum NFM421、Enterobacter asburiae L1、Ochrobactrum anthropi ATCC 49188、Klebsiella pneumoniae subsp.pneumoniae KP5-1中的GDH基因相似性達(dá)到92.30%、96.51%、85.12%、99.29%。綜合12h和48h時(shí)間下wj1和wj3GDH基因在不同磷源以及不同pH值的NBRIP培養(yǎng)基中GDH基因表達(dá)量的變化結(jié)果表明,菌株在溶磷過(guò)程中GDH基因都有表達(dá),同種菌在不同磷源的NBRIP培養(yǎng)基中表達(dá)量差異較大,且隨著培養(yǎng)時(shí)間的延長(zhǎng)表達(dá)量也在變化。培養(yǎng)相同時(shí)間,wj1和wj3兩種菌株GDH基因在pH值為5~9的NBRIP培養(yǎng)基中表達(dá)量變化趨勢(shì)一致。12h時(shí),在pH 5的培養(yǎng)基中表達(dá)量最大,48h時(shí)在pH 9的培養(yǎng)基中表達(dá)量最大。綜上所述,本研究可以推測(cè)4株溶磷菌在溶磷過(guò)程中pH值下降以及產(chǎn)酸都是在適宜的環(huán)境下誘導(dǎo)出來(lái)的。本試驗(yàn)成功克隆了產(chǎn)葡萄糖酸的GDH基因,GDH基因的表達(dá)量也受磷源和菌液pH值的影響。
[Abstract]:Phosphorus soluble microorganisms can transform insoluble phosphate in soil into soluble phosphorus forms which can be absorbed and utilized by plants, thus improving the utilization efficiency of phosphorus in soil, which plays an important role in the recycling of phosphorus in agriculture. In this paper, a series of studies were carried out on the phosphorus dissolving characteristics of inorganic phosphorus bacteria in soybean rhizosphere soil. The main results were as follows: in this experiment, four strains of bacteria with strong phosphorus solubilizing ability were isolated and screened from soybean rhizosphere soil in Northeast China. The maximum phosphorus solubilization was 558 渭 g / mL and 586 渭 g / mL, respectively, and 596 渭 g / mL and 586 渭 g / mL, respectively. The four strains were identified as Pseudomonas pseudomonas, Enterobacter pallidus, Klebsiella klebsiella, and named wj1wj3wj5 and wj6 by physiological and biochemical analysis of Vitek2 and 16s rDNA sequencing. On the basis of NBRIP separation medium, using L25 (53) orthogonal design experiment, the phosphorus solubilizing medium of the strain was optimized from three aspects: carbon source, nitrogen source and pH value. The results showed that the optimum carbon source was glucose, the nitrogen source was peptone, and the initial pH was 8. The characteristics of dissolving Ca _ 3 (PO _ 4) _ 2 FePO _ 4 and AlPO _ 4 by wj6 were studied. It was found that the strain could degrade three kinds of phosphate, Ca _ 3 (PO _ 4) _ 2 FePO _ 4 and AlPO _ 4 to varying degrees, and the highest dissolved phosphorus content was 409.28 渭 g 路mL ~ (-1) ~ 60.59 渭 g 路mL ~ (-1) ~ (32. 90) 渭 g 路mL ~ (-1), respectively. However, the pH of the culture environment affected the dissolution of FePO4 and AlPO4. The strain could dissolve FePO4 and AlPO4 by inoculating wj6 into pH 8 medium, but had no ability to dissolve phosphorus in the medium solution (pH 3). The mechanism of phosphorus dissolving was studied from the point of acid production of phosphorus-soluble bacteria. Under the condition of calcium phosphate as the sole phosphorus source, the pH value of the strain decreased in the process of phosphorus dissolution. GC-MS Analysis of Organic acids produced in the process of phosphorus dissolution by GC-MS the main organic acids produced by the four strains were: 1: 2 (2-dimethoxypropionic acid), 4-hexadienedioicacididine (4-hexadienedioic acid) 3-hydroxydecanoic Acid-malic acid hepta-24-dienoic acidic acid, acetic acid, succinic acid, citric acid and so on. The results of enzyme activity test showed that phosphatase activity of bacteria liquid increased significantly in the process of phosphorus dissolving. The GDH gene was cloned from wj1wj3wj5 and wj6, and the gene fragment of 2007bpn2066bp1 1727bpmc2391bp was obtained. The sequence was similar to that of Pseudomonas brassicacearum subsp.brassicacearum NFM421 Enterobacter asburiae L1 Ochrobactrum anthropi ATCC 49188 and Klebsiella pneumoniae subsp.pneumoniae KP5-1, respectively. The similarity of the GDH gene in Pseudomonas brassicacearum subsp.brassicacearum NFM421 and Klebsiella pneumoniae subsp.pneumoniae KP5-1 was 92.3096.51and 85.120.99.29. The expression of wj1 gene and wj3GDH gene in different phosphorous sources and different pH value NBRIP medium under 12h and 48h time were analyzed. The results showed that the GDH gene was expressed in the process of phosphorus solubilization. The expression of the same strain was different in NBRIP medium with different phosphorus sources, and the expression was also changed with the extension of culture time. At the same time, the expression of GDH gene was the same in NBRIP medium with pH 5 ~ 9, and the highest in pH 5 medium for 48 h, and the maximum expression level in pH 5 medium for 48 h. The expression level of GDH gene was the same in NBRIP medium with pH 5 and pH 5 at 48 h. The expression of GDH gene was the highest in pH 9 medium. To sum up, it can be inferred that the pH value decreased and the acid production induced by the four phosphorus soluble bacteria in the suitable environment. The expression of GDH gene was also influenced by phosphorus source and pH value.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S565.1;S154.3

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 張愛(ài)華,楊啟銀,陳育如,虞光華;解磷細(xì)菌突變株P(guān)S-01P菌株的選育及發(fā)酵條件的研究[J];安徽農(nóng)業(yè)大學(xué)學(xué)報(bào);2004年03期

2 趙小蓉,林啟美;微生物解磷的研究進(jìn)展[J];土壤肥料;2001年03期

3 劉文干;何園球;張坤;樊建波;曹慧;;一株紅壤溶磷菌的分離、鑒定及溶磷特性[J];微生物學(xué)報(bào);2012年03期

，

本文編號(hào)：2056983