發(fā)布時(shí)間：2018-02-24 02:24

  本文關(guān)鍵詞： 企鵝貝 GLUT1 基因克隆 葡萄糖　出處：《上海海洋大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：葡萄糖轉(zhuǎn)運(yùn)蛋白(GLUTs)是能量代謝的關(guān)鍵因子,用于血液和組織間的葡萄糖轉(zhuǎn)運(yùn),維持機(jī)體能量代謝,為了探索GLUT1(glucose transporter type1)在企鵝珍珠貝中的功能,本文開(kāi)展了企鵝珍珠貝GLUT1的克隆與功能研究。主要結(jié)果如下:1.利用企鵝珍珠貝轉(zhuǎn)錄組序列數(shù)據(jù),篩選得到兩條GLUT1基因EST序列,利用RACE技術(shù)分別克隆出兩條企鵝貝GLUT1基因cDNA全長(zhǎng),一條蛋白分子量為57kDa,并命名為PpGLUT1,另一條蛋白分子量為58kDa。GLUT1(57kDa)基因cDNA全長(zhǎng)為2 068bp,ORF為1 569bp,5’UTR為216bp,3’UTR為276 bp。該基因編碼522個(gè)氨基酸,包含一個(gè)SP功能結(jié)構(gòu)域,12個(gè)跨膜螺旋區(qū),N端和C端都在胞質(zhì)側(cè),有多個(gè)磷酸化位點(diǎn),分子量為57.2264kDa,理論等電點(diǎn)為5.17。該基因序列與其他物種GLUT1序列一致性為57%-72%。不同組織熒光定量結(jié)果表明,該基因在閉殼肌、肝胰腺、腸、性腺、足、外套膜和鰓中都有表達(dá),在腸和外套膜中表達(dá)量最高,閉殼肌中的表達(dá)量顯著低于其他組織。50%的高濃度葡萄糖注射閉殼肌結(jié)果表明,在注射后30min內(nèi),腸PpGLUT1表達(dá)量升高,在30min之后,表達(dá)量逐漸降低,注射2h時(shí)表達(dá)量最低,2h之后表達(dá)量略有上升。不同濃度葡萄糖注射實(shí)驗(yàn)表明,腸道中的PpGLUT1表達(dá)量高于對(duì)照組的表達(dá)量,當(dāng)葡萄糖濃度為10%-40%時(shí),PpGLUT1表達(dá)量逐漸上升,濃度為40%時(shí),PpGLUT1表達(dá)量最高,40%之后,表達(dá)量逐漸降低。腸道組織的原位雜交顯色結(jié)果表明PpGLUT1在腸道組織中表達(dá)量很豐富且多集中在固有層和粘膜下層(基底膜附近)等處,肌層中表達(dá)量極少。2.GLUT1(58kDa)基因cDNA cDNA全長(zhǎng)為2 237 bp,開(kāi)放閱讀框?yàn)? 584bp,5’非編碼區(qū)(5’UTR)為494 bp,3’UTR為159 bp,包含有29個(gè)poly A尾巴,該基因編碼527個(gè)氨基酸,分子量預(yù)測(cè)為58.9388 kDa,理論等電點(diǎn)pI為8.16,親水性(GRAVY)總評(píng)均值為0.542,因此該蛋白為疏水性蛋白。該蛋白共有14個(gè)Ser、1個(gè)Thr和4個(gè)Tyr磷酸化位點(diǎn),5個(gè)糖基化位點(diǎn)(NYTF,NKTI,NKTS,NSTM和NISL),不存在信號(hào)肽序列�？缒そY(jié)構(gòu)域預(yù)測(cè)表明,該基因存在12個(gè)跨膜螺旋結(jié)構(gòu)域,N端和C端都在胞質(zhì)側(cè)。功能結(jié)構(gòu)域分析表明,該基因氨基酸序列的84aa-500aa處具有SP(sugar porter family)特征結(jié)構(gòu)域,并且還具有xylE同源結(jié)構(gòu)域和AraJ(arabinose efflux permease)透膜酶結(jié)構(gòu)域。多重序列比對(duì)分析表明,PpGLUT1有3個(gè)特征性保守序列(XXQQXSG,EXFXQXPR and PETKXXTFXEI),分別位于309-316aa,430-437aa and 490-500aa的氨基酸序列處,其中XXQQXSG位于跨膜螺旋區(qū)上,EXFXQXPR and PETKXXTFXEI位于胞質(zhì)側(cè)。同源性比對(duì)發(fā)現(xiàn),與長(zhǎng)牡蠣(Crassostrea gigas)的序列一致性為75%,與海蝸牛(Aplysia californica)的相似性達(dá)到83%,一致性為56%,與其他物種的比對(duì)結(jié)果表明,相似性都在36-50%,序列一致性多為58-70%,50%的高濃度葡萄糖注射實(shí)驗(yàn)結(jié)果顯示,在注射后30 min內(nèi),GLUT1(58kDa)表達(dá)量升高;在30 min之后,表達(dá)量逐漸降低;當(dāng)注射時(shí)間為2 h時(shí),GLUT1(58kDa)表達(dá)量最低;2 h之后,表達(dá)量逐漸上升,在4h時(shí),表達(dá)水平穩(wěn)定。葡萄糖注射實(shí)驗(yàn)結(jié)果表明,企鵝貝在注射不同濃度葡萄糖后,腸組織中的GLUT1(58kDa)表達(dá)量都比注射前增加,當(dāng)葡萄糖濃度小于0.2g/mL時(shí),表達(dá)量依次升高;當(dāng)濃度為0.2g/mL時(shí),GLUT1(58kDa)表達(dá)量達(dá)到最高,當(dāng)濃度大于0.2g/mL時(shí),表達(dá)量逐漸降低上述結(jié)果表明,企鵝珍珠貝GLUT1(57kDa)和GLUT1(58kDa)主要在腸和外套膜表達(dá),對(duì)葡萄糖注射有表達(dá)響應(yīng),表達(dá)量變化幅度相似,兩個(gè)葡萄糖轉(zhuǎn)運(yùn)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 緇撴灉琛ㄦ槑PpGLUT1鍦ㄨ偁閬撶粍緇囦腑琛ㄨ揪閲忓緢涓板瘜涓斿闆嗕腑鍦ㄥ浐鏈夊眰鍜岀矘鑶滀笅灞，

本文編號(hào)：1528518