本文選題：苦馬豆素　切入點(diǎn)：節(jié)桿菌　出處：《西北農(nóng)林科技大學(xué)》2015年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：瘋草是指黃芪屬(Astragalus spp.)和棘豆屬(Oxytropis spp.)有毒植物,廣泛分布在我國(guó)西北等地草原地區(qū),其毒性成分苦馬豆素(swainsonine,SW)能導(dǎo)致動(dòng)物以神經(jīng)障礙和繁殖障礙為特征的中毒,嚴(yán)重危害我國(guó)草原牧業(yè)的發(fā)展,目前尚缺乏有效的防治方法。微生物降解SW可作為解決動(dòng)物瘋草中毒的一種新的思路,本實(shí)驗(yàn)室已經(jīng)分離出了一株能高效降解SW的菌株-節(jié)桿菌屬HW08(Arthrobacter sp.HW08)。為了確定節(jié)桿菌HW08中降解SW相關(guān)的酶,本研究使用液相色譜串聯(lián)質(zhì)譜技術(shù)(LC-MS/MS)對(duì)節(jié)桿菌HW08進(jìn)行差異蛋白質(zhì)組學(xué)研究,篩選降解SW的相關(guān)酶,實(shí)時(shí)熒光定量PCR(Real-time quantitative PCR,qRT-PCR)檢測(cè)降解酶基因的相對(duì)表達(dá)量;對(duì)依賴NADP乙醇脫氫酶A1R6C3基因進(jìn)行克隆及原核表達(dá),并檢測(cè)表達(dá)產(chǎn)物降解SW的能力,取得的結(jié)果如下:(1)采用Lable-free定量方法,對(duì)經(jīng)SW誘導(dǎo)與未經(jīng)SW誘導(dǎo)的節(jié)桿菌HW08進(jìn)行差異蛋白組學(xué)分析。通過(guò)比較不同條件下差異蛋白的相對(duì)表達(dá)量,結(jié)合Gene Ontology和KEGG代謝通路分析,篩選出8個(gè)與降解SW相關(guān)的酶,分別為磷酸糖類(lèi)異構(gòu)酶A1R5X7和A1R5X8、依賴NADP的乙醇脫氫酶A1R6C3、未知蛋白A1R872、NAD(P)轉(zhuǎn)氫酶β亞基A1RBS8、冷休克DNA結(jié)合蛋白家族A0JY59、乙酰CoA乙�；D(zhuǎn)移酶A0JZ95和冷休克蛋白J7LTJ9。采用實(shí)時(shí)熒光定量PCR法,對(duì)這8個(gè)蛋白質(zhì)從轉(zhuǎn)錄組水平檢測(cè)其基因的相對(duì)表達(dá),結(jié)果試驗(yàn)組基因AAur-1890(3.41)、AAur-1891(2.27)、AAur-2040(2.12)、Arth-2600(2.27)、Arth-2986(3.73)和ARUE-c09510(11.4)相對(duì)對(duì)照組顯著增加(p0.05),與蛋白質(zhì)相對(duì)表達(dá)量結(jié)果一致;而AAur-2719(0.81)和AAur-4018(0.61)相對(duì)對(duì)照組基因差異不顯著。(2)對(duì)依賴NADP的乙醇脫氫酶A1R6C3基因進(jìn)行克隆表達(dá)與純化,檢測(cè)純化酶降解SW的能力。重組菌E.coli BL21-pET32a-AAur 2040表達(dá)出56 kDa大小的蛋白,與預(yù)期目的蛋白的大小一致;對(duì)IPTG誘導(dǎo)條件進(jìn)行優(yōu)化,當(dāng)濃度為1.0 mmol/L,誘導(dǎo)3 h蛋白表達(dá)量即達(dá)到最高;用帶His標(biāo)簽的Ni柱對(duì)目標(biāo)蛋白進(jìn)行親和層析純化,經(jīng)Western blot鑒定正確;純化后的乙醇脫氫酶(蛋白濃度為0.5 mg/mL)經(jīng)氣相色譜檢測(cè),1 h能降解約18.52μg的SW,降解率為46.3%。
[Abstract]:The poisonous plants of Astragalus spp.and Oxytropis spp.are widely distributed in the grasslands of northwest China. The toxic component of the poisonous plant, Astragalus spp.and Oxytropis spp., can lead to the poisoning of animals characterized by neurological and reproductive disorders. It is a serious harm to the development of grassland animal husbandry in China. At present, there is a lack of effective control methods. Microbial degradation of SW can be used as a new way to solve the poisoning of animal wild grass. A strain of the genus HW08(Arthrobacter sp. HW08, which can efficiently degrade SW, has been isolated in our laboratory. In order to determine the SW-related enzymes in HW08, In this study, LC-MS / MS was used to study the differential proteomics of Arthrobacter HW08, to screen the SW-degrading enzyme, and to detect the relative expression of the degrading enzyme gene by real-time quantitative PCR(Real-time quantitative PCR-qRT-PCR. The A1R6C3 gene of ethanol dehydrogenase dependent on NADP was cloned and expressed in prokaryotic cells, and the ability of the expressed product to degrade SW was detected. The results obtained were as follows: 1) Lable-free quantitative method was used. The differential proteomic analysis of SW-induced and non-SW-induced HW08 was carried out. By comparing the relative expression of differentially expressed proteins under different conditions and combining with the analysis of Gene Ontology and KEGG metabolic pathways, eight enzymes related to SW degradation were screened out. NADP dependent alcohol dehydrogenase A1R6C3, unknown protein A1R872 (NADS-8) transhydrogenase 尾 subunit A1RBS8, cold shock DNA binding protein family A0JY59, acetyl CoA acetyltransferase A0JZ95 and cold shock protein J7LTJ9 were used. The relative expression of the eight proteins was detected at the transcriptional level. The results showed that the relative expression of AAur-1890 (3.41C) and ARUE-c0951011.4) in the test group was significantly higher than that in the control group, and the results were consistent with the results of the relative protein expression. However, there was no significant difference between AAur-2719P 0.81) and AAur-40180.61) compared with the control group, the A1R6C3 gene of ethanol dehydrogenase was cloned, expressed and purified. The recombinant strain E.coli BL21-pET32a-AAur 2040 expressed 56 kDa protein, which was the size of 56 kDa. The result showed that the protein expression reached the highest level at 1.0 mmol / L for 3 h, and the target protein was purified by affinity chromatography with Ni column with His label, and identified correctly by Western blot. The purified ethanol dehydrogenase (0.5 mg 路mL ~ (-1)) could be reduced to 18.52 渭 g SWD by gas chromatography for 1 h, and the degradation rate was 46.3%.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S859.87

【相似文獻(xiàn)】

相關(guān)期刊論文 前6條

1 趙更峰,馬曉航,賈小明,王園園;節(jié)桿菌(Arthrobacter)42-1菌株的分離、鑒定及產(chǎn)肌氨酸氧化酶條件研究[J];浙江大學(xué)學(xué)報(bào)(農(nóng)業(yè)與生命科學(xué)版);2005年01期

2 李娜;雷麗萍;夏振遠(yuǎn);吳德喜;;節(jié)桿菌K15發(fā)酵條件的研究[J];云南農(nóng)業(yè)大學(xué)學(xué)報(bào)(自然科學(xué)版);2010年06期

3 楊美英;蔣春玲;李文明;曲振環(huán);王乾欽;;節(jié)桿菌PJ3降解咔唑和聯(lián)苯[J];生態(tài)學(xué)雜志;2010年07期

4 張磊;談曙明;繆鑫昕;訾小利;胡南;;阿氏節(jié)桿菌菌株DA-1的厭氧反硝化研究[J];湖北農(nóng)業(yè)科學(xué);2013年01期

5 ;生物防治因子[J];生物技術(shù)通報(bào);1986年12期

6 ;[J];;年期

相關(guān)博士學(xué)位論文 前4條

1 堯宇翔;細(xì)菌分解代謝氮雜環(huán)污染物尼古丁的分子機(jī)制研究[D];上海交通大學(xué);2014年

2 陳熙明;節(jié)桿菌中的滲透壓脅迫感受器調(diào)節(jié)細(xì)胞分裂以及細(xì)胞壁發(fā)育[D];蘭州大學(xué);2011年

3 張海紅;節(jié)桿菌電轉(zhuǎn)化方法的優(yōu)化及6-磷酸海藻糖酯酶基因的功能研究[D];蘭州大學(xué);2011年

4 姜穎;低溫下節(jié)桿菌海藻糖-6-磷酸合成酶催化效率及表達(dá)調(diào)控[D];蘭州大學(xué);2010年

相關(guān)碩士學(xué)位論文 前4條

1 韓秀芳;土壤放線菌分離菌株的分類(lèi)鑒定與拮抗性篩選[D];北京理工大學(xué);2015年

2 翟阿官;節(jié)桿菌HW08降解苦馬豆素相關(guān)酶的篩選與乙醇脫氫酶的表達(dá)[D];西北農(nóng)林科技大學(xué);2015年

3 姜穎;節(jié)桿菌和大腸桿菌海藻糖-6-磷酸合成酶在低溫下的催化效率比較[D];蘭州大學(xué);2007年

4 歐陽(yáng)小珍;重金屬轉(zhuǎn)化菌的分離篩選及轉(zhuǎn)化機(jī)理初探[D];河北大學(xué);2013年

，

本文編號(hào)：1646355