本文選題：靈芝多糖 + 磷酸葡萄糖變位酶�。� 參考：《江南大學(xué)》2017年碩士論文

【摘要】：靈芝(Ganoderma lucidum)是名貴的食藥用真菌,靈芝多糖(polysaccharides)是靈芝主要的活性成分之一,眾多研究表明,靈芝多糖具有調(diào)節(jié)免疫、抗腫瘤、抗氧化、抗纖維化、降血壓等生物活性。靈芝多糖主要從發(fā)酵液、菌絲體及子實(shí)體中獲得。目前關(guān)于靈芝多糖的研究大多集中在通過(guò)發(fā)酵調(diào)控技術(shù)增加其產(chǎn)量,而通過(guò)分子水平操作來(lái)調(diào)控多糖合成代謝的研究卻很欠缺。本課題通過(guò)構(gòu)建重組質(zhì)粒,采用農(nóng)桿菌轉(zhuǎn)化法(ATMT)使得靈芝多糖代謝途徑中的關(guān)鍵酶磷酸葡萄糖變位酶(α-PGM)、磷酸甘露糖異構(gòu)酶(PMI)的基因在靈芝菌體內(nèi)獲得過(guò)表達(dá),通過(guò)qRT-PCR法檢測(cè)基因在轉(zhuǎn)錄水平的相對(duì)表達(dá)量,并對(duì)靈芝多糖產(chǎn)量及其單糖組成、及靈芝多糖合成途徑中相關(guān)酶的酶活進(jìn)行研究。此外,對(duì)野生型及重組型的靈芝菌株采用掃描電鏡(SEM)與透射電鏡(TEM)技術(shù),觀(guān)察其菌體形態(tài)和菌體細(xì)胞內(nèi)超微結(jié)構(gòu)的區(qū)別。研究結(jié)果主要有:1.由NCBI查找靈芝菌株中編碼α-PGM、PMI蛋白酶的基因序列,使用DNAMAN軟件設(shè)計(jì)引物,以所提靈芝總DNA為模板,經(jīng)PCR擴(kuò)增目的基因片段,核酸凝膠電泳檢測(cè)并對(duì)其測(cè)序,結(jié)果顯示基因序列一致。以pCAMBIA1301為骨架,采用同源重組的方法構(gòu)建重組質(zhì)粒PJW-PGM、PJW-PMI,根癌農(nóng)桿菌介導(dǎo)轉(zhuǎn)化靈芝原生質(zhì)體,并從含有相應(yīng)抗生素的平板上篩選出重組菌株。2.以野生型靈芝菌株為對(duì)照,研究發(fā)現(xiàn)靈芝重組菌株的菌體生物量與野生型相比有一定程度的提高,且其還原糖消耗速率更快。重組型菌株與野生型靈芝胞外多糖(EPS)和胞內(nèi)多糖(IPS)的產(chǎn)量在整個(gè)發(fā)酵過(guò)程中的產(chǎn)量明顯高于野生型靈芝。對(duì)發(fā)酵過(guò)程中多糖組成及比例的變化進(jìn)行研究,結(jié)果表明,由于pgm、pmi基因的過(guò)表達(dá),葡萄糖分別流向不同的合成代謝分支,影響靈芝多糖中的單糖組分及比例。利用RT-PCR技術(shù)從轉(zhuǎn)錄水平對(duì)參與糖核苷酸供體的重要酶進(jìn)行驗(yàn)證,結(jié)果顯示pgm、pmi基因的過(guò)表達(dá),不僅使其自身在mRNA水平的相對(duì)表達(dá)量高于野生型菌株,也能促進(jìn)其上下游基因的表達(dá)。以野生型靈芝菌株為對(duì)照,對(duì)參與靈芝多糖合成途徑中糖核苷酸供體合成的相關(guān)酶的比酶活變化規(guī)律進(jìn)行研究,結(jié)果發(fā)現(xiàn)重組型靈芝中不僅PGM、PMI的比酶活高于野生型,也促進(jìn)多糖合成途徑中磷酸葡萄糖異構(gòu)酶(PGI)、UDP-葡萄糖焦磷酸化酶(UGPG)兩種酶比酶活的提高。3.以野生型靈芝菌株為對(duì)照,對(duì)重組型靈芝菌株采用SEM與TEM技術(shù),觀(guān)察其菌體形態(tài)和菌體細(xì)胞內(nèi)超微結(jié)構(gòu)的區(qū)別。結(jié)果顯示,SEM下重組型的菌體表面覆蓋的靈芝多糖較野生型多,TEM下重組型與野生型的細(xì)胞內(nèi)超微結(jié)構(gòu)整體無(wú)明顯差異。
[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.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：TQ929

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本文編號(hào)：2028054