本文選題：發(fā)菜　切入點：糖基轉移酶　出處：《陜西科技大學》2017年碩士論文　論文類型：學位論文

【摘要】：發(fā)菜,學名發(fā)狀念珠藻(Nostoc flagelliforme),是一種具有較高食用及藥用價值的光能自養(yǎng)型藍藻。在生長代謝過程中,發(fā)菜細胞向胞外分泌大量的發(fā)菜多糖,對細胞起到保護作用,同時研究發(fā)現(xiàn)其亦具有抗氧化、抗病毒、抗腫瘤以及提高免疫等生物活性,有一定的藥用價值。在鹽脅迫條件下,發(fā)菜多糖分泌量增加。前期對鹽脅迫及正常培養(yǎng)條件下的發(fā)菜樣品進行轉錄組測序分析,挖掘參與發(fā)菜響應高鹽脅迫的相關基因。本課題在轉錄組測序結果的基礎上,篩選出部分可能參與鹽脅迫下發(fā)菜多糖代謝相關的差異表達基因,以發(fā)菜基因組為模板,對其進行克隆與表達,分析差異表達基因的基本特征,豐富發(fā)菜的基因數據,為進一步研究發(fā)菜多糖的代謝調控機制提供理論基礎。對發(fā)菜中的三個糖基轉移酶基因(GT1、GT2、GT3)、GDP-甘露糖4,6-脫水酶基因(GMD)、多糖輸出蛋白基因(PEP)進行克隆,得到基因序列,分別對其進行生物信息學分析,結果如下:GT1、GT2、GT3、GMD、PEP基因序列大小分別為1290 bp、1173 bp、948 bp、1080 bp、1467 bp,核酸序列分析均具有較高的保守性。GT1、GT2、GT3、GMD、PEP蛋白分子量大小分別為47.54 k Da、43.16 k Da、35.99 k Da、41.08 k Da、51.28 k Da,理論等電點分別為9.33、7.64、6.34、5.73、5.50。GT1、GT2、GT3、GMD、PEP均為親水性蛋白,不具有跨膜活性。GT1、GT2、GT3的二級結構主要為隨機卷曲和α螺旋,GMD和PEP的二級結構為隨機卷曲、α螺旋和折疊。將GT1、GT2、GT3、GMD、PEP進行擴增,與載體p ET28a連接,IPTG誘導其在大腸桿菌BL21中表達,分別以IPTG濃度、加入IPTG時菌液OD值、培養(yǎng)溫度為變量進行單因素實驗,SDS-PAGE蛋白電泳比較目的蛋白的表達效果,確定目的蛋白表達的適宜條件。結果表明,蛋白表達的條件為:IPTG終濃度為1 m M,加入IPTG時菌液OD值為0.8,加入IPTG后培養(yǎng)溫度為16℃,在該條件下,四種蛋白均表達得到了與預期大小一致的重組蛋白。研究結果為進一步研究GT1、GT2、GT3、GMD的結構、功能及在發(fā)菜多糖代謝調控中的作用奠定了基礎,為研究發(fā)菜多糖的代謝調控機制提供了理論依據和實驗思路。
[Abstract]:Nostoc flagelliforme.Nostoc flagelliforme.Nostoc flagelliforme.Nostoc flagelliforme.It is a photoautotrophic cyanobacterium with high edible and medicinal value. At the same time, the study found that it also has antioxidant, anti-virus, anti-tumor and enhance immune biological activities, and has certain medicinal value. The exudation of polysaccharides was increased. In the early stage, the samples under salt stress and normal culture were analyzed by transcriptome sequencing, and the related genes involved in the response to high salt stress were excavated. This study was based on the results of transcriptome sequencing. Some differentially expressed genes which may be involved in the metabolism of polysaccharides were screened out and cloned and expressed using the genome as template to analyze the basic characteristics of differentially expressed genes and to enrich the gene data. In order to provide a theoretical basis for further study on the metabolic regulation mechanism of caraway polysaccharides, three glycosyltransferase genes (GT1, GT2 and GT3), GDP-mannose 6-dehydrase gene (GMDN) and Polysaccharide exportation protein gene (PEP), were cloned and sequenced. The bioinformatics analysis was carried out respectively, The results showed that the PEP gene size of the GMDP gene was 1290 bp1, 1173 BP, 948 BP, 1080 bp1, 1467 BP, and the molecular weight of GMDPEP protein was 47.54 k, 43.16 k, 43.99 k, 41.08 k, respectively, and the theoretical isoelectric point was 9.33 / 7.66.66.36.36.35.732 / 0.GT1 / GT2GMDPEP was hydrophilic, respectively, and the molecular weight of GMDPEP was 47.54 k, and the molecular weight of GMDPEP was 43.16 k / d = 35.99 k, respectively, and the theoretical isoelectric point was 9.33 / 76.66.36.36.36.73nb / 55.50.GT1 / GT2GMDPEP was a hydrophilic protein, and the molecular weight of GMDPEP / GMDPEP was 47.54 / kg / kg, respectively. The secondary structures of GT1 GMD and PEP were random crimp, 偽 helix, 偽 helix and folding. The GMD-PEP was amplified and ligated with the vector p ET28a to induce its expression in Escherichia coli BL21. Single factor experiment was carried out to compare the expression effect of the target protein by SDS-PAGE protein electrophoresis with the concentration of IPTG, the OD value of the bacteria solution and the culture temperature as variables, respectively, and the suitable conditions for the expression of the target protein were determined, the results showed that the optimal conditions for the expression of the target protein were obtained. The conditions of protein expression were as follows: the final concentration of IPTG was 1 mm, the OD value of bacteria solution was 0.8 when IPTG was added, and the culture temperature was 16 鈩，

本文編號：1585612