盐胁迫下发菜多糖代谢相关差异表达基因的克隆与原核表达

发布时间：2018-03-08 21:20

  本文选题：发菜　切入点：糖基转移酶　出处：《陕西科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：发菜,学名发状念珠藻(Nostoc flagelliforme),是一种具有较高食用及药用价值的光能自养型蓝藻。在生长代谢过程中,发菜细胞向胞外分泌大量的发菜多糖,对细胞起到保护作用,同时研究发现其亦具有抗氧化、抗病毒、抗肿瘤以及提高免疫等生物活性,有一定的药用价值。在盐胁迫条件下,发菜多糖分泌量增加。前期对盐胁迫及正常培养条件下的发菜样品进行转录组测序分析,挖掘参与发菜响应高盐胁迫的相关基因。本课题在转录组测序结果的基础上,筛选出部分可能参与盐胁迫下发菜多糖代谢相关的差异表达基因,以发菜基因组为模板,对其进行克隆与表达,分析差异表达基因的基本特征,丰富发菜的基因数据,为进一步研究发菜多糖的代谢调控机制提供理论基础。对发菜中的三个糖基转移酶基因(GT1、GT2、GT3)、GDP-甘露糖4,6-脱水酶基因(GMD)、多糖输出蛋白基因(PEP)进行克隆,得到基因序列,分别对其进行生物信息学分析,结果如下:GT1、GT2、GT3、GMD、PEP基因序列大小分别为1290 bp、1173 bp、948 bp、1080 bp、1467 bp,核酸序列分析均具有较高的保守性。GT1、GT2、GT3、GMD、PEP蛋白分子量大小分别为47.54 k Da、43.16 k Da、35.99 k Da、41.08 k Da、51.28 k Da,理论等电点分别为9.33、7.64、6.34、5.73、5.50。GT1、GT2、GT3、GMD、PEP均为亲水性蛋白,不具有跨膜活性。GT1、GT2、GT3的二级结构主要为随机卷曲和α螺旋,GMD和PEP的二级结构为随机卷曲、α螺旋和折叠。将GT1、GT2、GT3、GMD、PEP进行扩增,与载体p ET28a连接,IPTG诱导其在大肠杆菌BL21中表达,分别以IPTG浓度、加入IPTG时菌液OD值、培养温度为变量进行单因素实验,SDS-PAGE蛋白电泳比较目的蛋白的表达效果,确定目的蛋白表达的适宜条件。结果表明,蛋白表达的条件为:IPTG终浓度为1 m M,加入IPTG时菌液OD值为0.8,加入IPTG后培养温度为16℃,在该条件下,四种蛋白均表达得到了与预期大小一致的重组蛋白。研究结果为进一步研究GT1、GT2、GT3、GMD的结构、功能及在发菜多糖代谢调控中的作用奠定了基础,为研究发菜多糖的代谢调控机制提供了理论依据和实验思路。
[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, 伪 helix, 伪 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，

本文编号：1585612