【作者】 姜俊；

【导师】 周小秋；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2009， 博士

【摘要】 本研究率先研究建立的鲤鱼(Cyprinus carpio)悬浮原代肠上皮细胞(IECs)研究模型,进一步利用该研究模型结合同位素示踪技术,分子克隆、生物信息学和荧光定量方法,克隆了IECs谷氨酰胺酶(GLS)和蛋白合成信号调控分子雷帕霉素靶(TOR)基因cDNA序列,研究了GLS和TOR基因在鲤鱼肠道不同部位的表达及GLS活力分布,探索了Gln对鲤鱼肠道不同肠段IECs蛋白质合成能力的影响及机理。结果如下:1.研究建立了鲤鱼悬浮原代IECs研究模型。结果表明:分离鲤鱼IECs呈圆形,可见明显两极特征,纯度95%,活力93%,分离IECs提取的DNA纯度更高,提取的总RNA结构完整。结果表明:该模型可应用于PCR分子生物实验研究。2.克隆了鲤鱼IECs GLS基因cDNA序列。该序列长1788bp,包含从起始密码子1位到末端终止密码子1788位的开放阅读框,编码595个氨基酸残基的GLS蛋白前体,分子量65.96kDa。核苷酸序列与斑马鱼的相似性为89.9%,与原鸡的相似性为69.4%,与小鼠K-和L-GLS的相似性分别为70.2%和65.2%,与大鼠K-和L-GLS的相似性分别为70.4%和65.9%,与人K-和L-GLS的相似性分别为69.8%和64.3%。蛋白质结构分析表明:鲤鱼GLS氨基酸残基序列含有两个非常保守的区域,分别为171～457位氨基酸残基的GLS蛋白家族区域和484～570位氨基酸残基的锚定蛋白重复序列;氨基酸残基中118、355、409、438和502位的丝氨酸残基磷酸化位点,205位的苏氨酸残基磷酸化位点和393、511位的酪氨酸残基磷酸化位点高度保守,是GLS蛋白功能调控位点;含有10个高度保守的半胱氨酸残基,位于GLS蛋白家族区域和锚定蛋白重复序列,与GLS蛋白分子结构稳定性和亚细胞定位密切相关。3.克隆了鲤鱼IECs TOR基因cDNA序列。该序列长7548bp,包含从起始密码子1位到末端终止密码子7548位的开放阅读框,编码2515个氨基酸残基的TOR蛋白,分子量为286.03kDa。核苷酸序列与斑马鱼相似性为92.0%,与原鸡相似性为78.0%,与鸭嘴兽、小鼠和大鼠相似性分别为74.5、78.4和78.5%,与人相似性为78.6%。蛋白质结构分析表明:鲤鱼TOR蛋白的1～348氨基酸残基为HEAT重复序列;349～2515位氨基酸残基为PIKKs蛋白质家族结构域,包括1365～1948位的FAT结构域,1985～2078位的FRB结构域(FKBP12-RAP复合物结合区域),2119～2397位的激酶催化区域,2485～2515位的FATC结构域,这些结构域序列非常保守,对TOR蛋白空间构象和生物活性非常重要;N-端931～1039氨基酸残基序列含有内质网和高尔基体的定位序列,对TOR蛋白定位于内质网和高尔基体膜非常重要。4.研究了鲤鱼不同肠段谷丙转氨酶(AlaAT)、谷草转氨酶(AspAT)、谷氨酸脱氢酶(GDH)、苹果酸脱氢酶(MDH)和乳酸脱氢酶(LDH)活力大小。结果表明:AlaAT活力,5肠段显著高于其它各肠段(P＜0.05),3肠段显著高于1～2、4和6～7肠段(P＜0.05),2、4肠段显著高于1、6～7肠段(P＜0.05),1与6～7肠段差异不显著(P＞0.05):AspAT活力,2肠段显著高于其它各肠段(P＜0.05),3～5肠段显著高于1、6～7肠段,1与6～7肠段差异不显著(P＞0.05):GDH活力,3肠段显著高于其它各肠段(P＜0.05),2与4～6肠段差异不显著(P＞0.05),但显著高于1、7肠段(P＜0.05),1与7段差异不显著(P＞0.05);MDH活力,3肠段显著高于其它各肠段(P＜0.05),2肠段显著高于1、4～7肠段(P＜0.05),4肠段显著高于1、5～7肠段(P＜0.05),1与5肠段差异不显著(P＞0.05),但显著高于6～7肠段(P＜0.05),6与7肠段差异不显著(P＞0.05);LDH活力,2肠段显著高于其它各肠段(P＜0.05),4肠段显著高于1、3和5～7肠段(P＜0.05),3肠段显著高于1、5～7肠段(P＜0.05),1与5～7肠段差异不显著(P＞0.05)。结果说明:AlaAT、AspAT、GDH、MDH和LDH活力均以鲤鱼中肠(2～5肠段)较高。5.研究了鲤鱼不同肠段GLS活力大小和基因表达。结果表明:不同肠段GLS活力,3～4肠段显著高于1～2肠段和6～7肠段(P＜0.05),5肠段显著高于1、6～7肠段(P＜0.05),2肠段显著高于1、7肠段(P＜0.05),6肠段显著高于1、7肠段(P＜0.05),1肠段显著高于7肠段(P＜0.05),5、2～4肠段之间及2、6肠段之间差异不显著(P＞0.05)。不同肠段GLS mRNA表达量,3～4肠段显著高于7肠段(P＜0.05),其余各肠段差异不显著(P＞0.05),其分布规律与GLS活力基本相同。结果说明:鲤鱼中肠(2～5肠段)GLS活力和mRNA表达量均较高。6.研究了生长期不同体重鲤鱼不同肠段TOR基因表达量。结果表明:1肠段,18.1g组TOR mRNA相对表达量显著低于其它各体重组(P＜0.05),18.1、31.1g组之间以及40.6、58.0、73.9g组之间差异不显著(P＞0.05);2～5肠段和6～7肠段,18.1g和31.1g组TOR mRNA相对表达量显著低于其它各体重组(P＜0.05),18.1、31.1g组之间以及40.6、58.0、73.9g组之间差异不显著(P＞0.05)。40.6、58.0、73.9g组TOR mRNA相对表达量各肠段之间差异不显著(P＞0.05),但是18.1g组1肠段显著高于6～7肠段,31.1g组1肠段则显著高于2～5肠段和6～7肠段(P＜0.05)。结果说明:随体重增加,鲤鱼肠道TOR mRNA表达量增加,同时在幼龄阶段(30g前)前中肠表达量高。7.研究了鲤鱼肠道不同肠段IECs蛋白质合成能力及Gln对不同肠段IECs合成能力的影响。结果表明:蛋白质合成率2～3肠段显著高于其它各肠段(P＜0.05),1肠段显著高于4～7肠段(P＜0.05),4肠段显著高于5～7肠段(P＜0.05),5～7肠段差异不显著(P＞0.05);1mg/L Gln组1～3肠段IECs蛋白质合成率显著高于0mg/L Gln组(P＜0.05),但4～7肠段则差异不显著(P＞0.05)。结果说明:鲤鱼前中肠(1～3肠段)蛋白合成能力高于后肠(5～7肠段),Gln可提高前中肠(1～3肠段)IECs蛋白质合成能力。8.TOR抑制剂RAP、GLS抑制剂DON和Gln对鲤鱼IECs蛋白质合成的影响。结果表明:1mg/L Gln组蛋白质合成率显著高于0mg/L Gln组(P＜0.05);1mg/L Gln+RAP、1mg/L Gln+DON和1mg/L Gln+RAP+DON组均显著低于1mg/L Gln组(P＜0.05),高于0mg/L Gln组(P＜0.05);1mg/L Gln+DON组显著高于1mg/L Gln+RAP和1mg/L GlnRAP+DON组(P＜0.05);1mg/L Gln+RAP组显著高于1mg/L Gln+RAP+DON组(P＜0.05)。结果说明:GLS和TOR信号分子参与了Gln提高鲤鱼IECs蛋白质合成的调控。9.研究了Gln对鲤鱼IECs GLS基因表达的影响。结果表明:30～120min,1mg/L Gln组GLS mRNA相对表达量显著高于0mg/LGln组(P＜0.05);1mg/LGln组中,120min GLSmRNA相对表达量显著低于70min(P＜0.05),与30min差异不显著(P＞0.05):0mg/LGln组中,30～120min,GLS mRNA相对表达量差异不显著(P＞0.05)。结果说明:Gln促进了鲤鱼IECs GLS基因表达。10.研究了Gln、TOR抑制剂RAP和GLS抑制剂DON对鲤鱼IECs TOR基因表达的影响。结果表明:1mg/L Gln组IECs TOR mRNA相对表达量显著高于0mg/L Gln组(P＜0.05);1mg/L Gln+RAP组显著低1mg/L Gln组(P＜0.05),与0mg/L Gln差异不显著(P＞0.05);1mg/L Gln+DON组与1mg/L Gln差异不显著(P＞0.05),但显著高于1mg/LGln+RAP组和0mg/L Gln组(P＜0.05)。Gln作用时间,15min,30min组IECs TOR mRNA相对表达量显著高于5min组(P＜0.05);30min与15min组之间差异不显著(P＞0.05)。结果说明:Gln可通过TOR信号分子调控TOR基因表达,提高了TOR mRNA表达量;GLS没有参与Gln对鲤鱼IECs TOR基因表达的调控过程。综上所述:在鲤鱼肠道存在IECs蛋白合成信号调控分子TOR,随着体重增加,TORmRNA在肠道中表达量逐步增加,30g前幼龄鲤鱼前中肠表达量高;鲤鱼前中肠IECs蛋白质合成能力强;Gln提高前中肠IECs蛋白质的合成能力,Gln提高其蛋白质合成能力受到TOR和GLS的调控。更多还原

【Abstract】 In this study,using research model of suspension primary carp intestinal epithelial cells (IECs) by established in this study,combining with isotope tracer technique,molecular cloning, bioinformatics and fluorescence quantitative PCR method,the gene eDNA sequence of target of rapamyein(TOR),which is a signal regulation molecular of protein synthesis in intestinal epithelial cells(IECs),and glutaminase(GLS) were cloned,GLS and TOR gene expression and the GLS activity in the carp intestinal tract different section,further the influence of L-glutarnine(Gin) on carp intestines different section IECs ability of protein synthesis and mechanism were studied.The results are as follows:1.Established the research model of carp suspension primary IECs.The results showed that IECs isolated from carp intestine were round under light microscopy,the polarization characteristics can be seen clearly.A high proportion of IECs,representing the total number of cells,95%,cell viability,93%.And the extraction DNA from isolation IECs is purity,the extraction total RNA structure is complete.2.The carp GLS eDNA was cloned.This sequence contains from start eodon 1 to the terminal termination codon 1788bp opening reading frame,coded includes 595 amino acid residue GLS protein precursor,molecular weight 65.96kDa.The nucleotide sequence with the zebrafish homology is 89.9%,with the original chicken homology is 69.4%,with mouse K-and the L-GLS homology respectively is 70.2%and 65.2%,with rat K-and the L-GLS homology respectively is 70.4%and 65.9%,with human K-and the L-GLS homology respectively is 69.8%and 64.3%.The protein structure analysis indicated that carp GLS amino acid sequence includes two very conservative regions,respectively GLS protein family region from 171 to 457 amino acid residue and anchor protein repetitive sequence from 484 to 570 amino acid residues.118,355,409,438 and 502 serine residue phosphorylation position spot,205 threonine residue phosphorylation position spot and 393,511 tyrosine residue phosphorylation position spot of GLS are highly conservative and important to the GLS function regulation.The GLS protein includes 18 cysteine residues,and has highly conservative 10 cysteine residues,which locates at GLS protein family region and anchor protein repetitive sequence,relates with GLS protein molecular structure,stable and subcellular localization. 3.The carp TOR cDNA was cloned.This sequence contains from start codon 1 to the terminal termination codon 7548bp opening reading frame,coded includes 2515 amino acid residue GLS protein,molecular weight 286.03kDa.The nucleotide sequence with the ,-’ebrafish homology is 92.0%,with the original chicken homology is 78.0%,with the platypus, mouse and rat homology respectively is 74.5,78.4 and 78.5%,and with human homology is 78.6%.The protein structure analysis indicated that Carp TOR protein includes 5 structure region.Between amino acid residues 1-348 of the HEAT repeat,between 349-2515 amino acid residue of PIKKs protein family’s domain.The PIKKs protein family’s domain includes between 1365-1948 amino acid residue is FAT.domain,between 1985-2078 amino acid residues is the FRB domain(FKBP 12-RAP complex binding domains),between 2119-2397 amino acid residues is kinase catalytic region,between 2485-2515 amino acid residue is FATC domain,which domains are very conservative and play vital role to the TOR protein’s spatial conformation and the biological activity.The N-terminal 93I-1039 amino acid residue sequence includes the endoplasmic reticulum and the golgiosome localization sequence, which is very important to TOR protein located in the endoplasmic reticulum and Golgi apparatus membrane. 4.Alanine aminotransferase(AIaAT),aspartate aminotransferase(AspAT),glutamate dehydrogenase(GDH),malate dehydrogenase(MDH) and lactate dehydrogenase(LDH) activities in carp different intestinal section have been determined.The results showed that the AIaAT activity in 5 intestinal section is significant higher than other various intestinal section (P＜0.05),2 intestinal section enzyme activity is significant higher than 1-2,4 and 6-7 intestinal sections(P＜0.05),2,4 sections activity is significant higher than 1,6-7 sections(P＜0.05),1,6-7 section activity is not significant difference(P＞0.05).The AspAT activity in 2 section is significant higher than other various sections(P＜0.05),3-5 sections is significant higher than 1,6-7 sections(P＜0.05),1,6-7sections is not significant difference(P＞0.05).The GDH activity in 3 section is signifcantly higher than other various intestines sections(P＜0.05),2, 4-6 sections is not significant difference(P＞0.05),but significant higher than 1 and 7 section (P＜0.05),1 and 7 section is not significant difference(P＞0.05).The MDH activity in 3 scection is significant higher than other various section(P＞0.05),2 section is significantyl higher than 1,4-7 sections(P＞0.05),4 section is significant higher than 1,5-7 sections (P＞0.05),1 and 5 section is not significant difference(P＞0.05),but is significant higher than 6-7 sections(P＞0.05),6-7 section is not significant difference(P＞0.05).The LDH activity in 2 scection is significantly higher than other various sections(P＞0.05),4 section is significantyl higher than 1,3,5-7 sections(P＞0.05),3 section is significantly higher than 1, 5-7 sections(P＞0.05),1 and 5-7 section is not significant difference(P＞0.05).The result indicated that AlaAT,AspAT,GDH,MDH and LDH activities are higher in carp midintestine (2-5 intestines sections).5.The GLS activity and gene expression in carp different intestines section was studied. The results showed that GLS activity in carp intestine 3-4 sections is significantly higher than 1-2 sections and 6～7 sections(P＜0.05),5 section is significantly higher than 1,6-7 sections (P＜0.05);2 section is significantly higher than 1,7 sections(P＜0.05);6 section is significantly higher than 1,7 sections(P＜0.05);1 section is significantly higher than 7 sections(P＜0.05).The GLS the mRNA expression quantity in 3-4 intestinal sections is significantly higher than 7 sections,other various sections is not signifcant difference.The result indicated that the GLS activity and the mRNA expression quantity in the carp midintestine(2-5 sections) are higher.6.The TOR gene expression in different intestinal section of different body weight carp was studied.The results showed that 1 section,18.1g group TOR the mRNA relative expression quantity is significantly lower than other weight groups(P＜0.05),between 18.1g and 31.1g group as well as between 40.6,58.0,and 73.9g groups were no significant differences(P＞0.05);2-5 sections and 6-7 sections,18.1g and 31.1g group TOR mRNA relative expression level was significantly lower than the other weight group(P＜0.05),18.1g and 31.1g group as well as between 40.6,58.0,and 73.9g groups were no significant differences(P＞0.05).40.6,58.0,73.9g group relative TOR mRNA expression in different section was no significant difference(P＞0.05),but 18.1g group,1 section was significantly higher than 6-7 sections,31.1g group 1 section was significantly higher than 2-5 sections and 6-7 sections(P＜0.05).The result indicated that with weight increasing,TOR the mRNA expression quantity in carp intestinal tract increased,simultaneously the fore and midgut expression quantity is higher in the young age stage(before 30g).7.The protein synthesis ability in different section IECs of the carp intestinal tract and the influence of Gin on IECs synthesis ability of the different intestines section were studied. The results showed that 2-3 sections is significantly higher than other section(P＜0.05),but not significant difference each other(P＞0.05);1 section is significantly higher than 4-7 sections(P＜0.05);4 section is significantly higher than 5-7 sections(P＜0.05),5-7 sections is not significant difference(P＞0.05).IECs protein synthesis rate of 1-3 sections,1mg/L Gin group is significantly higher than 0mg/L Gin group(P＜0.05),4-7 sections,lmg/L and 0mg/L Gln group is not significantly difference(P＞0.05).The result indicated IECs protein synthesis ability of the carp fore and midgut(1-3 section) is higher than hindgut(5-7 sections);Gin promoted fore and midgut(1-3 section) IECs protein synthesis ability,but has on influence on hindgut(5-7 sections).8.Studied the influence of TOR inhibitor RAP,GLS inhibitor DON and Gin on IECs protein synthesis ability.The results showed that protein synthesis rate of 1mg/L Gin group was significantly higher than 0mg/L Gin group(P＜0.05),lmg/L Gln+RAP,DON or RAP+DON group were significantly lower than 1mg/L Gln group(P＜0.05),higher than 0mg/L Gin group(P＜0.05).1mg/L Gln+DON group was significantly higher than 1mg/L GIn+RAP and 1mg/L Gin RAP+DON Group.1mg/L Gln+RAP group was significantly higher than 1mg/L Gln+RAP+DON Group.The results indicated that GLS and TOR signaling molecules are involved in regulation of Gin improving the ability of fish IECs protein synthesis.9.The influence of Gin on the GLS gene expression of carp IECs was studied.The results showed that 30-120min,1mg/LGln group GLS mRNA relative expression was significantly higher than 0mg/LGln group(P＜0.05).1mg/LGln group,120min GLS mRNA relative expression was significantly lower than 70min(P＜0.05),but no significant differences with 30min(P＞0.05);0mg/LGln group,30-120min,GLS mRNA relative expression difference was not significant(P＞0.05).The results indicated that Gln can influence GLS gene expression of carp IECs.10.The influence of Gln on the TOR gene expression of carp IECs was studied.The results showed that 1mg/L Gin group IECs TOR mRNA expression was significantly higher than 0mg/L Gin group(P＜0.05),Gln 1mg/L+RAP group was significantly lower than 1mg/L Gin group(P＜0.05),but no significant difference with 0mg/L Gln(P＞0.05),Gln 1mg/L+DON group was no significant difference with 1mg/L Gin group(P＞0.05),but significantly higher than Gin 1mg/L+RAP group and 0mg/L Gln group(P＜0.05).15min group IECs TOR mRNA relative expression was significantly higher than 5min group(P＜0.05);30min group was significantly higher than 5min group(P＜0.05),but was no significant difference with 15min group(P＞0.05).The results indicated that Gln through TOR signaling molecules regulating TOR gene expression,increase TOR mRNA expression levels. GLS did not take part in Gln on the regulation of TOR gene expression process of carp IECs.In summary,TOR,this is a signal regulation molecular of protein synthesis in IECs, esists in carp intestine.With the weight gain,TOR mRNA expression in the intestine gradually increase,the fore and midgut expression quantity is higher in the young age stage (before 30g).The carp fore and midgut IECs protein synthesis ability is higher,Gln promoted fore and midgut IECs protein synthesis ability.Gln improves IECs protein synthesis ability to receive TOR and the GLS regulation.更多还原