企鹅珍珠贝葡萄糖转运蛋白1同源异构型基因的克隆及对葡萄糖应激的表达响应分析

发布时间：2018-02-24 02:24

  本文关键词： 企鹅贝 GLUT1 基因克隆 葡萄糖　出处：《上海海洋大学》2016年硕士论文　论文类型：学位论文

【摘要】：葡萄糖转运蛋白(GLUTs)是能量代谢的关键因子,用于血液和组织间的葡萄糖转运,维持机体能量代谢,为了探索GLUT1(glucose transporter type1)在企鹅珍珠贝中的功能,本文开展了企鹅珍珠贝GLUT1的克隆与功能研究。主要结果如下:1.利用企鹅珍珠贝转录组序列数据,筛选得到两条GLUT1基因EST序列,利用RACE技术分别克隆出两条企鹅贝GLUT1基因cDNA全长,一条蛋白分子量为57kDa,并命名为PpGLUT1,另一条蛋白分子量为58kDa。GLUT1(57kDa)基因cDNA全长为2 068bp,ORF为1 569bp,5’UTR为216bp,3’UTR为276 bp。该基因编码522个氨基酸,包含一个SP功能结构域,12个跨膜螺旋区,N端和C端都在胞质侧,有多个磷酸化位点,分子量为57.2264kDa,理论等电点为5.17。该基因序列与其他物种GLUT1序列一致性为57%-72%。不同组织荧光定量结果表明,该基因在闭壳肌、肝胰腺、肠、性腺、足、外套膜和鳃中都有表达,在肠和外套膜中表达量最高,闭壳肌中的表达量显著低于其他组织。50%的高浓度葡萄糖注射闭壳肌结果表明,在注射后30min内,肠PpGLUT1表达量升高,在30min之后,表达量逐渐降低,注射2h时表达量最低,2h之后表达量略有上升。不同浓度葡萄糖注射实验表明,肠道中的PpGLUT1表达量高于对照组的表达量,当葡萄糖浓度为10%-40%时,PpGLUT1表达量逐渐上升,浓度为40%时,PpGLUT1表达量最高,40%之后,表达量逐渐降低。肠道组织的原位杂交显色结果表明PpGLUT1在肠道组织中表达量很丰富且多集中在固有层和粘膜下层(基底膜附近)等处,肌层中表达量极少。2.GLUT1(58kDa)基因cDNA cDNA全长为2 237 bp,开放阅读框为1 584bp,5’非编码区(5’UTR)为494 bp,3’UTR为159 bp,包含有29个poly A尾巴,该基因编码527个氨基酸,分子量预测为58.9388 kDa,理论等电点pI为8.16,亲水性(GRAVY)总评均值为0.542,因此该蛋白为疏水性蛋白。该蛋白共有14个Ser、1个Thr和4个Tyr磷酸化位点,5个糖基化位点(NYTF,NKTI,NKTS,NSTM和NISL),不存在信号肽序列。跨膜结构域预测表明,该基因存在12个跨膜螺旋结构域,N端和C端都在胞质侧。功能结构域分析表明,该基因氨基酸序列的84aa-500aa处具有SP(sugar porter family)特征结构域,并且还具有xylE同源结构域和AraJ(arabinose efflux permease)透膜酶结构域。多重序列比对分析表明,PpGLUT1有3个特征性保守序列(XXQQXSG,EXFXQXPR and PETKXXTFXEI),分别位于309-316aa,430-437aa and 490-500aa的氨基酸序列处,其中XXQQXSG位于跨膜螺旋区上,EXFXQXPR and PETKXXTFXEI位于胞质侧。同源性比对发现,与长牡蛎(Crassostrea gigas)的序列一致性为75%,与海蜗牛(Aplysia californica)的相似性达到83%,一致性为56%,与其他物种的比对结果表明,相似性都在36-50%,序列一致性多为58-70%,50%的高浓度葡萄糖注射实验结果显示,在注射后30 min内,GLUT1(58kDa)表达量升高;在30 min之后,表达量逐渐降低;当注射时间为2 h时,GLUT1(58kDa)表达量最低;2 h之后,表达量逐渐上升,在4h时,表达水平稳定。葡萄糖注射实验结果表明,企鹅贝在注射不同浓度葡萄糖后,肠组织中的GLUT1(58kDa)表达量都比注射前增加,当葡萄糖浓度小于0.2g/mL时,表达量依次升高;当浓度为0.2g/mL时,GLUT1(58kDa)表达量达到最高,当浓度大于0.2g/mL时,表达量逐渐降低上述结果表明,企鹅珍珠贝GLUT1(57kDa)和GLUT1(58kDa)主要在肠和外套膜表达,对葡萄糖注射有表达响应,表达量变化幅度相似,两个葡萄糖转运蛋白可能存在相互作用。
[Abstract]:The glucose transporter (GLUTs) is the key factor for energy metabolism, glucose transport between blood and tissues, maintain energy metabolism, in order to explore the GLUT1 (glucose transporter type1) in Pteria penguin in function, this paper carried out the cloning and functional study of Pteria penguin GLUT1. The main results are as follows: 1. the transcriptome of Pteria penguin sequence data were obtained two GLUT1 gene EST sequence were cloned using RACE two GLUT1 gene cDNA from Pteria penguin, a protein with a molecular weight of 57kDa, and was named PpGLUT1, another protein with a molecular weight of 58kDa.GLUT1 (57kDa) gene cDNA was 2 068bp, ORF 1 569bp, 5 UTR '216bp, 3' UTR 276 bp. of the gene encoding 522 amino acids, containing a SP domain, 12 transmembrane helices N and C ends at the cytoplasmic side, there are multiple phosphorylation sites, a molecular weight of 5 7.2264kDa, the isoelectric point of 5.17. of the gene sequence with other species GLUT1 sequence consistency in different tissues of 57%-72%. fluorescence quantitative results showed that the gene in adductor muscle, hepatopancreas, intestine, gonads, foot, mantle and gill are expressed in the intestine and the mantle of the highest expression level, the expression of shell. Muscle was significantly lower than the high glucose injection of adductor muscle tissue.50% results showed that after the injection of 30min, intestinal PpGLUT1 expression increased after 30min, the expression decreased gradually after 2H injection the lowest expression, 2h expression increased slightly. Experiments showed that glucose injection of different concentrations in the intestinal tract PpGLUT1 expression was higher than the control group, when the glucose concentration was 10%-40%, PpGLUT1 expression gradually increased, the concentration is 40%, then the expression of PpGLUT1 was highest, 40%, the expression decreased gradually. In situ hybridization of intestinal tissue color 缁撴灉琛ㄦ槑PpGLUT1鍦ㄨ偁閬撶粍缁囦腑琛ㄨ揪閲忓緢涓板瘜涓斿�氶泦涓�鍦ㄥ浐鏈夊眰鍜岀矘鑶滀笅灞，

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