节杆菌HW08降解苦马豆素相关酶的筛选与乙醇脱氢酶的表达

发布时间：2018-03-22 01:05

本文选题：苦马豆素　切入点：节杆菌　出处：《西北农林科技大学》2015年硕士论文　论文类型：学位论文

【摘要】：疯草是指黄芪属(Astragalus spp.)和棘豆属(Oxytropis spp.)有毒植物,广泛分布在我国西北等地草原地区,其毒性成分苦马豆素(swainsonine,SW)能导致动物以神经障碍和繁殖障碍为特征的中毒,严重危害我国草原牧业的发展,目前尚缺乏有效的防治方法。微生物降解SW可作为解决动物疯草中毒的一种新的思路,本实验室已经分离出了一株能高效降解SW的菌株-节杆菌属HW08(Arthrobacter sp.HW08)。为了确定节杆菌HW08中降解SW相关的酶,本研究使用液相色谱串联质谱技术(LC-MS/MS)对节杆菌HW08进行差异蛋白质组学研究,筛选降解SW的相关酶,实时荧光定量PCR(Real-time quantitative PCR,qRT-PCR)检测降解酶基因的相对表达量;对依赖NADP乙醇脱氢酶A1R6C3基因进行克隆及原核表达,并检测表达产物降解SW的能力,取得的结果如下:(1)采用Lable-free定量方法,对经SW诱导与未经SW诱导的节杆菌HW08进行差异蛋白组学分析。通过比较不同条件下差异蛋白的相对表达量,结合Gene Ontology和KEGG代谢通路分析,筛选出8个与降解SW相关的酶,分别为磷酸糖类异构酶A1R5X7和A1R5X8、依赖NADP的乙醇脱氢酶A1R6C3、未知蛋白A1R872、NAD(P)转氢酶β亚基A1RBS8、冷休克DNA结合蛋白家族A0JY59、乙酰CoA乙酰基转移酶A0JZ95和冷休克蛋白J7LTJ9。采用实时荧光定量PCR法,对这8个蛋白质从转录组水平检测其基因的相对表达,结果试验组基因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)相对对照组显著增加(p0.05),与蛋白质相对表达量结果一致;而AAur-2719(0.81)和AAur-4018(0.61)相对对照组基因差异不显著。(2)对依赖NADP的乙醇脱氢酶A1R6C3基因进行克隆表达与纯化,检测纯化酶降解SW的能力。重组菌E.coli BL21-pET32a-AAur 2040表达出56 kDa大小的蛋白,与预期目的蛋白的大小一致;对IPTG诱导条件进行优化,当浓度为1.0 mmol/L,诱导3 h蛋白表达量即达到最高;用带His标签的Ni柱对目标蛋白进行亲和层析纯化,经Western blot鉴定正确;纯化后的乙醇脱氢酶(蛋白浓度为0.5 mg/mL)经气相色谱检测,1 h能降解约18.52μg的SW,降解率为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%.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.87

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