灵芝多糖合成途径关键酶基因过表达的研究

发布时间：2018-06-16 20:47

本文选题：灵芝多糖 + 磷酸葡萄糖变位酶　；参考：《江南大学》2017年硕士论文

【摘要】：灵芝(Ganoderma lucidum)是名贵的食药用真菌,灵芝多糖(polysaccharides)是灵芝主要的活性成分之一,众多研究表明,灵芝多糖具有调节免疫、抗肿瘤、抗氧化、抗纤维化、降血压等生物活性。灵芝多糖主要从发酵液、菌丝体及子实体中获得。目前关于灵芝多糖的研究大多集中在通过发酵调控技术增加其产量,而通过分子水平操作来调控多糖合成代谢的研究却很欠缺。本课题通过构建重组质粒,采用农杆菌转化法(ATMT)使得灵芝多糖代谢途径中的关键酶磷酸葡萄糖变位酶(α-PGM)、磷酸甘露糖异构酶(PMI)的基因在灵芝菌体内获得过表达,通过qRT-PCR法检测基因在转录水平的相对表达量,并对灵芝多糖产量及其单糖组成、及灵芝多糖合成途径中相关酶的酶活进行研究。此外,对野生型及重组型的灵芝菌株采用扫描电镜(SEM)与透射电镜(TEM)技术,观察其菌体形态和菌体细胞内超微结构的区别。研究结果主要有:1.由NCBI查找灵芝菌株中编码α-PGM、PMI蛋白酶的基因序列,使用DNAMAN软件设计引物,以所提灵芝总DNA为模板,经PCR扩增目的基因片段,核酸凝胶电泳检测并对其测序,结果显示基因序列一致。以pCAMBIA1301为骨架,采用同源重组的方法构建重组质粒PJW-PGM、PJW-PMI,根癌农杆菌介导转化灵芝原生质体,并从含有相应抗生素的平板上筛选出重组菌株。2.以野生型灵芝菌株为对照,研究发现灵芝重组菌株的菌体生物量与野生型相比有一定程度的提高,且其还原糖消耗速率更快。重组型菌株与野生型灵芝胞外多糖(EPS)和胞内多糖(IPS)的产量在整个发酵过程中的产量明显高于野生型灵芝。对发酵过程中多糖组成及比例的变化进行研究,结果表明,由于pgm、pmi基因的过表达,葡萄糖分别流向不同的合成代谢分支,影响灵芝多糖中的单糖组分及比例。利用RT-PCR技术从转录水平对参与糖核苷酸供体的重要酶进行验证,结果显示pgm、pmi基因的过表达,不仅使其自身在mRNA水平的相对表达量高于野生型菌株,也能促进其上下游基因的表达。以野生型灵芝菌株为对照,对参与灵芝多糖合成途径中糖核苷酸供体合成的相关酶的比酶活变化规律进行研究,结果发现重组型灵芝中不仅PGM、PMI的比酶活高于野生型,也促进多糖合成途径中磷酸葡萄糖异构酶(PGI)、UDP-葡萄糖焦磷酸化酶(UGPG)两种酶比酶活的提高。3.以野生型灵芝菌株为对照,对重组型灵芝菌株采用SEM与TEM技术,观察其菌体形态和菌体细胞内超微结构的区别。结果显示,SEM下重组型的菌体表面覆盖的灵芝多糖较野生型多,TEM下重组型与野生型的细胞内超微结构整体无明显差异。
[Abstract]:Ganoderma lucidum is a valuable medicinal fungus, and Ganoderma lucidum polysaccharide (polysaccharides) is one of the main active ingredients of Ganoderma lucidum. Many studies have shown that Ganoderma lucidum polysaccharide has biological activities such as regulating immunity, anti-tumor, antioxidation, anti fibrosis and lowering blood pressure. The polysaccharide of Ganoderma lucidum is mainly obtained from fermentation broth, mycelium and fruiting body. The study of Ganoderma lucidum polysaccharides is mostly focused on increasing its yield through fermentation technology, but the research on the regulation of polysaccharide synthesis and metabolism through molecular level operation is very short. By constructing recombinant plasmid and using ATMT to make the key enzyme glucose mutant enzyme (alpha -PGM) in the metabolite of Ganoderma lucidum polysaccharides. The gene of phosphoric mannose isomerase (PMI) was overexpressed in Ganoderma lucidum. The relative expression of gene at the transcriptional level was detected by qRT-PCR method, and the enzyme activity of the polysaccharide yield and monosaccharide composition of Ganoderma lucidum, and the enzyme activities in the polysaccharide synthesis pathway of Ganoderma lucidum were studied. Microscopy (SEM) and transmission electron microscopy (TEM) were used to observe the difference between the morphology of the mycelium and the ultrastructure in the cells of the mycelium. The main results were as follows: 1. the sequence of the gene encoding alpha -PGM, PMI protease in Ganoderma lucidum was found by NCBI, and the primers were designed by DNAMAN software, and the total DNA of Ganoderma lucidum was used as a template. The target gene fragment was amplified by PCR, and the nucleic acid gel electricity was amplified by PCR. The result showed that the gene sequence was consistent. The recombinant plasmid PJW-PGM, PJW-PMI, PJW-PMI, Agrobacterium tumefaciens mediated transformation of Ganoderma protoplasts were constructed by the method of homologous recombination, and the recombinant strain.2. was selected from the plate containing the corresponding antibiotics and the wild Ganoderma lucidum strain was used as the control. The biomass of the recombinant strain increased to a certain extent compared with the wild type, and the rate of reducing sugar consumption was faster. The yield of recombinant strain and wild Ganoderma lucidum (EPS) and intracellular polysaccharide (IPS) in the whole fermentation process was obviously higher than that of wild Ganoderma lucidum. The results show that, due to the overexpression of PGM, PMI gene, glucose flows to different anabolic branches, affecting the monosaccharide components and proportions of ganoderma polysaccharide respectively. RT-PCR technology is used to verify the important enzymes involved in the sugar nucleotide donor from the transcriptional level. The results show that the overexpression of PGM, PMI gene, not only makes its own in mR. The relative expression of NA level is higher than that of the wild type strain, and it can also promote the expression of the upstream and downstream genes. With the wild Ganoderma lucidum as the control, the specific enzyme activity of the enzymes involved in the glyconucleotides donor synthesis in the polysaccharide synthesis pathway of Ganoderma lucidum is studied. The results show that the recombinant Ganoderma lucidum is not only PGM, but the specific enzyme activity of PMI is higher than that in the wild. The two enzymes of glucose phosphate isomerase (PGI) and UDP- glucose pyrophosphorylase (UGPG) were also promoted in the polysaccharide synthesis pathway. Compared with the increase of the enzyme activity, the wild Ganoderma lucidum was compared with the enzyme activity. The recombinant type Ganoderma lucidum strain was treated with SEM and TEM technology to observe the difference between the morphology of the mycelium and the ultrastructure in the cell. The results showed that the recombinant type of recombinant Ganoderma was reorganized under SEM. Ganoderma lucidum polysaccharides on the surface of bacteria were more than those in wild type. There was no significant difference in cell ultrastructure between recombinant and wild type under TEM.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ929

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