本文關(guān)鍵詞： 南極酵母 Pseudozyma sp.JCC207 海藻糖 TPS1基因 TPS2基因 提取優(yōu)化 功能分析　出處：《山東大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：海藻糖是生物體中一種重要的非還原糖,對生物體組織和生物大分子具有非特異性保護作用,逐漸成為備受關(guān)注的天然物質(zhì)。海藻糖具有在凍結(jié)、干燥、高滲透壓等這類嚴酷的環(huán)境下,保護生物體膜、膜蛋白、DNA等的功效,使生物在極端環(huán)境中更容易存活,可見,海藻糖是生物體中重要的功能物質(zhì)。南極酵母是指生活在南極海冰、海冰邊緣或海水等極端環(huán)境中一大類酵母的總稱,是南極主要的真菌生物。南極酵母長期在低溫、高鹽環(huán)境中生長和繁殖,海藻糖基因及海藻糖發(fā)揮了重要功能。南極酵母海藻糖合成酶指導(dǎo)合成海藻糖是其適應(yīng)南極極端環(huán)境的重要生存機制,因此從分子水平上研究南極酵母的海藻糖產(chǎn)生機制是非常有必要的。南極酵母Pseudozyma sp. JCC207是南極酵母里具有代表性的一株菌,對它海藻糖形成機制研究可以為南極生境和其他物種的研究提供理論基礎(chǔ)。本論文主要進行了以下研究內(nèi)容：1)南極酵母Pseudozyma sp. JCC207的全基因組測序；2)普通PCR法克隆獲得南極酵母Pseudozyma sp. JCC207基因rPSl并對TPS1、TPS2進行了生物信息學(xué)分析；3)應(yīng)用實時定量PCR技術(shù)對南極酵母Pseudozyma sp. JCC207 TPS1的轉(zhuǎn)錄調(diào)控機制進行了研究；4)通過單因素實驗法優(yōu)化了南極酵母Pseudozyma sp. JCC207的培養(yǎng)條件；5)通過單因素實驗、Box-Behnkens設(shè)計和響應(yīng)面分析法對三氯乙酸法提取海藻糖的提取條件進行了優(yōu)化,建立了影響因素的二次回歸方程；6)通過添加不同濃度海藻糖與甘油、二甲基亞砜保種試劑對比,探究海藻糖對南極酵母Pseudozyma sp. JCC207保種的作用。全基因組測序得到了南極酵母Pseudozyma sp. JCC207全部基因,其中包含了本實驗所需的TPS1、TPS2基因；克隆得到南極酵母Pseudozyma sp. JCC207 TPS1基因ORF全長1827 bp,編碼蛋白608個氨基酸,預(yù)測蛋白相對分子質(zhì)量為64.46 Da；測序得到TPS2基因ORF全長3963 bp,編碼蛋白1321個氨基酸,預(yù)測蛋白相對分子質(zhì)量為144.47 KDa; qRT-PCR結(jié)果顯示,在高鹽和低溫條件下TPS1表達量高,說明,TPS1對高鹽和低溫敏感；單因素試驗優(yōu)化得到南極酵母最適培養(yǎng)溫度為25℃,pH為5-9范圍內(nèi)酵母生長情況差別不大；響應(yīng)面分析得到三氯乙酸法提取海藻糖的最佳條件為三氯乙酸濃度06 mol/L、體積15.16mL、提取時間2976 min,經(jīng)驗證,優(yōu)化條件下的海藻糖提取量與理論預(yù)測值基本吻合；海藻糖保種實驗中,功能試驗表明,與甘油(20%)和二甲基亞砜(10%)等傳統(tǒng)保種試劑相比,0.5%海藻糖溶液在短期內(nèi)(5 d)對酵母具有更好的保種能力。
[Abstract]:Trehalose is an important non-reducing sugar in organism, which has nonspecific protective effect on organism tissue and biological macromolecule, and has gradually become a natural substance of much concern. Trehalose has been frozen and dried. Such harsh environments as high osmotic pressure protect the effectiveness of biological membranes, membrane proteins, DNA, and so on, so that organisms in extreme environments easier to survive, can be seen. Trehalose is an important functional substance in organisms. Antarctic yeast refers to a large category of yeast living in extreme environments such as Antarctic sea ice, sea ice edge or sea water. Antarctic yeast grows and reproduces in low temperature and high salt environment for a long time. Trehalose genes and trehalose play an important role in Antarctic yeast trehalose biosynthesis which is an important survival mechanism to adapt to Antarctic extreme environment. Therefore, it is necessary to study the mechanism of trehalose production in Antarctic yeast at molecular level. JCC207 is a representative strain of Antarctic yeast. The study on the mechanism of trehalose formation can provide a theoretical basis for the study of Antarctic habitats and other species. Genome sequencing of Antarctic yeast Pseudozyma sp. JCC207; 2) the Pseudozyma sp. JCC207 gene rPSl of Antarctic yeast was cloned by ordinary PCR method and the bioinformatics analysis of TPS1 and TPS2 was carried out. 3) the mechanism of transcriptional regulation of Pseudozyma sp. JCC207 TPS1 in Antarctic yeast was studied by real-time quantitative PCR. 4) the culture conditions of Antarctic yeast Pseudozyma sp. JCC207 were optimized by single factor experiment. 5) the extraction conditions of trehalose by trichloroacetic acid were optimized by Box-Behnkens design and response surface analysis, and the quadratic regression equation of influencing factors was established. 6) by adding different concentrations of trehalose and glycerol, dimethyl sulfoxide preservative reagent contrast. The effect of trehalose on species conservation of Antarctic yeast Pseudozyma sp. JCC207 was studied. All JCC207 genes. It contains the TPS1 / TPS2 gene needed in this experiment. The total length of ORF of Antarctic yeast Pseudozyma sp. JCC207 TPS1 was 1 827 BP, encoding 608 amino acids. The relative molecular weight of predicted protein was 64.46 Da. The total length of TPS2 gene ORF was 3963 BP, encoding 1321 amino acids, and the relative molecular weight of predicted protein was 144.47 KDa. The results of qRT-PCR showed that the expression of TPS1 was high under high salt and low temperature, indicating that TPS1 was sensitive to high salt and low temperature. The results of single factor experiment showed that the optimum culture temperature of Antarctic yeast was 25 鈩，

本文編號：1488501