团头鲂PGC1β基因克隆及高糖胁迫、葡萄糖和胰岛素负荷对其mRNA表达的影响

为探讨过氧化物酶体增殖活化受体 γ 辅激活因子 1β (peroxisome proliferator activated receptor γ coactivator 1β, PGC1β)基因在鱼类糖代谢中的作用, 本研究采用 RACE 方法获得了团头鲂 PGC1β 的 cDNA 片段序列, 利用生物信息学技术对该基因及其所编码蛋白的结构特征进行了分析; 采用实时定量 PCR 技术, 检测了高糖(45%)饲喂、葡萄糖及胰岛素负荷对团头鲂肝脏和肌肉中该基因表达的影响。结果显示, 团头鲂 PGC1β cDNA 片段长为 1759 bp, 其中开放阅读框为 1719 bp, 编码 573 个氨基酸; 同源性分析结果显示, 团头鲂 PGC1β 与其他鲤科鱼类的 PGC1β 高度同源(氨基酸相似度 78%以上), 并与草鱼 PGC1β 的进化关系最近; 高糖组鱼肝脏和肌肉中 PGC1β 的表达量高于对照组, 但无显著差异; 此外, 葡萄糖及胰岛素负荷后, 肝脏中 PGC1β 基因的表达量在 2 h 内显著降低至最小值, 随后逐渐升高至基础水平。以上结果提示, PGC1β 可能在调节鱼类糖代谢中发挥重要作用。     

草鱼NADPH氧化酶2个调节亚基cDNA的克隆及表达特征

为了解草鱼(Ctenopharyngodon idellus)NADPH多酶复合体的基因结构及其在机体的防御体系中的功能,利用RT-PCR结合RACE-PCR的方法,克隆草鱼NADPH氧化酶的2个调节亚基p40phox和p47phox的cDNA,对其编码的氨基酸序列进行了同源性比较和功能域分析,同时对这两个亚基在草鱼不同组织中的差异性表达进行分析。结果显示:p47phox亚基cDNA序列全长为1 589 bp,开放阅读框长度为1 233 bp,编码410个氨基酸;p40phox亚基cDNA序列全长为2 103 bp,开放阅读框为1 068 bp,编码355个氨基酸。这两个亚基编码的氨基酸序列与其他鱼类的同源性在68%~96%,具有其它鱼类类似的PX,SH3,PB1和PC功能域。组织差异性表达分析结果表明:2个调节亚基在草鱼胸腺、心脏、头肾、鳃、肠、肝、肾、脾和皮肤组织中均有表达,在不同组织中的表达强度略有差异,在心脏、胸腺中表达水平最高,在肝脏中表达水平较低,但是不同组织之间的表达水平差异不显著(P>0.05)。     

草鱼APN基因的克隆及表达特征

氨肽酶N(APN)是肽酶M1家族的成员之一,在蛋白质的消化中发挥重要作用。采用同源克隆和RACE技术首次克隆草鱼APN基因的全长cDNA序列。该cDNA全长为3258bp,包含27bp的5UTR序列,552bp的3UTR序列,2679bp开放阅读框,编码892个氨基酸;草鱼与斑马鱼基因同源性和编码氨基酸同源性分别为81.5%和75.4%,与其他动物同源性分别为58.8%～61.2%和54.3%～60.2%。经预测,其编码蛋白的分子量为100.61ku,等电点为5.14,该蛋白具有与哺乳动物十分相似的1个螺旋跨膜结构,但跨膜区氨基酸同源性较低;系统进化分析表明,草鱼APN基因与斑马鱼的亲缘关系最近;利用Real-timePCR技术检测了该基因的发育表达,结果显示草鱼出膜4d后APNmRNA表达量相对稳定;APN在草鱼前肠、中肠和后肠均有较高的表达量,以前肠组织表达量最高;昼夜节律研究发现,肠道APN基因06:00-18:00的表达量较18:00-06:00高。     

剑尾鱼卵黄蛋白原C(Vg C)全长cDNA的克隆及表达

利用逆转录聚合酶链式反应(RT-PCR)和cDNA末端快速扩增(RACE)等方法,克隆获得剑尾鱼卵黄蛋白原C(Vg C)基因的全长cDNA序列。剑尾鱼Vg C基因cDNA序列全长4 011 bp,其5′非编码区包含12 bp和3′非编码区包含246 bp;含有一个3 753 bp的开放阅读框(ORF),编码1 250个氨基酸,推测其编码氨基酸分子量大小为141.7 ku,编码氨基酸序列与其他鱼类卵黄白原C编码氨基酸序列相似性在44%～85%。荧光定量PCR结果显示,Vg C在剑尾鱼肝脏中表达量最高,脾、肾、卵巢中有微量表达,脑、肌肉、鳃中几乎没有检测到表达;对不同时间暴露在雌激素中剑尾鱼肝脏进行实时荧光定量PCR表达分析的结果表明,Vg C在剑尾鱼肝脏中第5天表达量最高,随后降低,第9天后维持相对较低的表达量。研究首次克隆了剑尾鱼Vg C基因全长cDNA序列,并对Vg C在剑尾鱼体内表达组织器官分布及雌激素诱导后不同时间表达谱进行了初步研究,为剑尾鱼生殖生理及环境污染物监测应用等不同领域研究打下重要基础。     

草鱼Mx蛋白基因的克隆与原核表达

根据已报道的Mx蛋白cDNA序列设计合成特异引物，应用逆转录-聚合酶链式反应(RT-PCR)方法，从草鱼呼肠孤病毒(GCRV)诱导的草鱼(Ctenopharyngodon idellus)肝脏总RNA中扩增获得Mx蛋白cDNA，回收纯化后克隆到pGEM-T Easy Vector系统的T载体上。重组子的序列分析表明：所克隆的Mx蛋白cDNA长722bp，编码240个氨基酸，包括1个三联GTP结合区域和1个发动蛋白(dynamin)族特征的序列。与已知鱼类Mx蛋白基因序列比较表明，草鱼Mx蛋白基因序列与其它鱼类Mx基因相应序列具有较高的同源性，其中与斑马鱼Mx蛋白E型基因碱基序列比较同源性最高，为87．1％。将此基因改造后定向克隆至原核表达质粒pBV220，构建成重组草鱼Mx蛋白基因表达质粒pBVgcMx，并在大肠杆菌中获得高效表达，重组草鱼Mx蛋白的表达量占菌体总蛋白的26．6％。Q-sephrose FF层析柱分离纯化的重组草鱼Mx蛋白纯度达95．6％。     

草鱼小肽转运载体PepT1基因的克隆与表达特征

采用同源克隆和RACE技术克隆草鱼(Ctenopharyngodon idellus)PepT1基因的全长cDNA序列。该cDNA全长为2 762 bp,包含141 bp的5′UTR序列,479 bp的3′UTR序列,2 142 bp开放阅读框,编码713个氨基酸;草鱼与鲫(Carassius auratus)、斑马鱼(Danio rerio)的核苷酸同源性分别为77.6%和74.0%,氨基酸同源性分别为78.0%和76.7%;与其他物种的核苷酸同源性为53.9%～59.1%,而氨基酸的同源性为57.2%～61.8%。经预测,其编码蛋白的分子量为79.29 kD,等电点为5.87,该蛋白具有与哺乳动物十分相似的11个螺旋跨膜结构,跨膜区氨基酸高度保守;系统进化分析表明,草鱼PepT1基因与鲫鱼和斑马鱼的亲缘关系最近;利用Real-time PCR技术检测了该基因的时空表达,结果显示,PepT1在草鱼前肠组织表达量最高,其次是肌肉组织;草鱼出膜7 d后PepT1 mRNA表达量相对稳定;昼夜节律研究发现,肠道PepT1基因夜间的表达量较白天高。本研究旨在为小肽转运载体PepT1介导肠道转运小肽调控草鱼对饲料蛋白消化吸收的分子机理提供理论基础。     

草鱼AdipoR1-B基因克隆、组织分布及其表达的营养调控

为探讨Adipo R1-B在鱼类糖类和脂质代谢中的作用,本实验采用RACE方法获得了草鱼Adipo R1-B的全长c DNA序列(登录号:KP733846),利用生物信息学技术对该基因及其所编码蛋白的结构特征进行了分析;采用实时定量PCR技术,检测了Adipo R1-B在19个不同组织中的表达特性以及高糖(45%)、高脂(8%)和高糖高脂饲喂对草鱼肝脏中该基因表达的影响。结果显示,草鱼Adipo R1-B c DNA全长2186 bp,其中开放读码框为1122 bp,编码373个氨基酸;跨膜结构分析表明草鱼Adipo R1-B为典型的7次跨膜蛋白;同源性分析结果显示,草鱼Adipo R1-B与其他物种的Adipo R1-B高度同源(氨基酸相似度78%以上),并与斑马鱼Adipo R1-B的进化关系最近;组织分布结果显示,Adipo R1-B在草鱼肝脏中表达量最高,中枢神经系统和红肌次之;此外,高脂和高糖高脂饲喂均能够显著提高草鱼肝脏中Adipo R1-B的表达水平。因此,草鱼肝脏中Adipo R1-B的表达水平受到日粮中脂类水平的调控,推测该受体可能在调节鱼类脂质代谢中发挥重要作用。     

虎皮鱼热激蛋白PtHsp70基因序列与低温表达分析

通过同源克隆和cDNA末端快速克隆技术扩增获得狭温热带鱼类虎皮鱼热激蛋白70(PtHsp70)基因的cDNA序列,BLAST比对分析基因序列同源性,预测其编码氨基酸序列和结构,实时荧光定量PCR技术分析虎皮鱼PtHsp70基因组织特异性表达谱和低温胁迫下PtHsp70基因的表达模式。研究结果表明,PtHsp70基因cDNA序列全长2317 bp,其中5′非编码区120 bp,3′非编码区266 bp,编码区1932 bp,对应的开放阅读框为121～2052 bp,预测编码643个氨基酸。PtHsp70基因mRNA在成年虎皮鱼不同组织中表达水平依次为肝脏肌肉鳃脑,其中肝脏PtHsp70基因本底表达水平显著高于其他组织(P0.01),表明其在肝脏中具有重要的生物学功能。而随温度降低,肝脏PtHsp70基因表达水平呈先降后升的趋势,表明肝脏PtHsp70基因参与虎皮鱼应对低温胁迫的过程;随温度降低,虎皮鱼脑、鳃和肌肉组织PtHsp70基因mRNA水平显著上调(P0.01),说明低温胁迫诱导PtHsp70基因mRNA表达。本研究克隆并鉴定了PtHsp70基因序列和组织表达谱,分析了低温胁迫下PtHsp70基因表达模式,研究结果显示,狭温热带鱼类虎皮鱼的PtHsp70基因具有组织特异性和温度诱导型表达特征。研究发现,PtHsp70基因在响应低温胁迫过程中具有重要作用,具有作为虎皮鱼低温胁迫分子标志物的潜能。     

草鱼C1qC基因的克隆及表达分析

为了研究C1qC基因在草鱼(Ctenopharyngodon idella)免疫过程中所起的作用,利用RT-PCR和RACE方法克隆获得了C1qC基因cDNA全长序列,经序列分析表明,所克隆的C1qC cDNA全长为916 bp,包括开放阅读框(open reading frame,ORF)735 bp,5′端非编码区(untranslated region,UTR)89 bp和3′端非编码区(UTR)92 bp。735 bp的ORF共编码244个氨基酸,相对分子量为26 162.5 U。同源性分析表明,草鱼与斑马鱼(Danio rerio)的相似度最高,达到71%。经草鱼呼肠孤病毒(grass carp reovirus,GCRV)诱导后,草鱼C1qC基因在鳃、皮肤、肌肉、肝、中肾、心脏、头肾等组织中的mRNA表达水平均显著上调。在草鱼胚胎发育的各个阶段都能检测到C1qC mRNA的表达,说明该基因可能在草鱼胚胎和鱼苗的免疫反应和早期发育中起重要作用。本研究将为今后在草鱼免疫功能方面深入研究C1qC基因提供基础资料。     

草鱼碱性氨基酸转运载体y+LAT2 cDNA基因克隆与表达研究

利用逆转录聚合酶链式反应(RT-PCR)和cDNA末端快速扩增(RACE)方法,克隆y+LAT2基因cDNA序列,并利用实时荧光定量PCR探讨y+LAT2在草鱼(Ctenopharyngodon idellus)各组织中的表达情况以及饥饿对其mRNA的影响。结果表明,获得的y+LAT2基因的cDNA序列片段为1849bp,含1371bp的核心序列,编码456个氨基酸。预测草鱼氨基酸序列与斑马鱼(Danio rerio)的同源性高达96.5%,与哺乳动物和两栖动物的同源性在78%～83%,构建的系统进化树与传统形态学分类一致。在草鱼的肌肉、脑、鳃、心、肾脏、肝、后肠、中肠、前肠、脾脏组织均检测到y+LAT2基因的表达。草鱼脾脏、肾脏以及前肠、中肠的y+LAT2 mRNA表达水平对禁食的反应不同,在短期饥饿(14d)期间,其y+LAT2 mRNA表达水平均呈下降趋势。草鱼碱性氨基酸转运载体y+LAT2基因全长cDNA序列的获得,有利于进一步探讨鱼类氨基酸的吸收转运机制。     

草鱼胰岛素样生长因子_基因克隆及序列分析

采用逆转录－聚合酶链式反应（ＲＴ－ＰＣＲ）方法,从草鱼肝脏的总ＲＮＡ中扩增出胰岛素样生长因子－Ｉ（ＩＧＦ－Ｉ）基因序列,定向克隆至质粒pUC18,测宇了该基因序列,推导期编码的蛋白序列,克隆的cDNA序列编码包括Ｂ,Ｃ,Ａ,Ｄ和Ｅ五个区域的１７个氨基酸,与鲤ＩＧＦ－Ｉ成熟肽比较,核酸序列和氨基酸序列的同源性分别为９３．８％和９７．１％,E区域分析结果表明,所克隆的草鱼ＩＧＦ－Ｉ序列属于ＩＧＦ－ＩＥa-2亚型。     

草鱼ghrelin基因的分子克隆与组织分布及其摄食调控作用分析

ghrelin是一种在脊椎动物摄食调节过程中起重要作用的脑肠肽,具有明显的摄食促进作用。实验利用同源克隆技术获得了草鱼ghrelin基因的cDNA序列和DNA序列,其中cDNA序列全长506 bp,包括90 bp的5′端非编码区(5′-untranslated region,5′UTR),312 bp的开放阅读框(open reading frame,ORF),以及104 bp的3′端非编码区(3′-untranslated region,3′UTR)。开放阅读框编码的103个氨基酸的ghrelin前体肽,经剪切加工后形成含有19个氨基酸的成熟肽。氨基酸序列分析结果显示,草鱼ghrelin与硬骨鱼类ghrelin相似度最高,而与其他脊椎动物相似度较低,同时草鱼ghrelin成熟肽N端的"活性中心"(active core)为鲤科鱼类中常见的GTSF形式。与大多数硬骨鱼类的ghrelin基因结构相同,草鱼ghrelin基因也包括4个外显子和3个内含子。荧光定量PCR检测到ghrelin mRNA大量分布于草鱼的前肠和脾,脑、肾、肝、肌肉、皮和鳔等组织也有ghrelin mRNA分布。草鱼脑和肠中的ghrelin表达水平在摄食后下降,随着饥饿时间的延长表达水平逐步升高,最后维持在较高水平,表明ghrelin作为摄食启动信号对草鱼的摄食活动起到了促进作用。     

草鱼LPL基因的表达及饥饿和再投喂对其影响

获得了草鱼脂蛋白脂酶(lipoprotein lipase, LPL)部分cDNA序列（GenBank注册号为FJ612596），并进行了序列同源性分析；采用半定量反转录聚合酶链式反应（SQ-RT-PCR）方法，检测了LPL基因在草鱼不同组织和不同发育阶段脂肪细胞中的表达状况；研究了饥饿和再投喂对草鱼肝胰脏LPL基因表达的影响。结果显示，所获得的草鱼LPL部分cDNA序列长度为360 bp，与其它物种的同源性为70%~89%； LPL基因在肝胰脏、肌肉、心脏、鳃、腹腔脂肪组织中均表达，其中在腹腔脂肪组织中表达丰度最高，在鳃中表达丰度最低；在成熟脂肪细胞中的表达丰度显著高于基质脉管组分（stromal vascular fraction,SVF）细胞；草鱼LPL基因表达水平在饥饿48 h后显著升高，再次投喂后12 h，回复到0 h的表达水平。研究表明，草鱼LPL在不同组织及脂肪细胞分化的起始和终末阶段均有表达，显示其在草鱼不同组织和脂肪细胞分化中具有重要作用，同时，其表达水平受营养状况的调控。论文首次克隆得到草鱼LPL基因部分cDNA序列，并对其进行了表达分析，研究结果可为LPL在鱼类脂质代谢中的作用研究提供参考和依据。     

草鱼SGLT1/2基因克隆及葡萄糖处理对其mRNA表达的影响

作为重要的转运载体,SGLTs(钠依赖性葡萄糖协同转运蛋白,sodium-glucose cotransporters)在物质的吸收过程中发挥着关键的作用。SGLT1和SGLT2作为SGLTs家族的重要成员,参与调节体内的葡萄糖吸收,从而维持血糖的稳态。为研究SGLT1和SGLT2在草鱼葡萄糖吸收过程中的作用,本实验利用RT-PCR技术从草鱼前肠和肾脏中分别克隆了基因sglt1和sglt2,对其进行生物信息学分析,并利用荧光定量PCR技术检测其mRNA的表达。结果显示,草鱼sglt1和sglt2的开放阅读框(open reading frame,ORF)分别为1 977和1 989 bp,并分别编码658和662个氨基酸。经过预测,草鱼SGLT1和SGLT2蛋白均为14次跨膜结构。氨基酸序列比对及系统进化树分析结果显示,草鱼sglt1和sglt2与金鱼、金线鲃中sglt1和sglt2的亲缘关系最近。荧光定量PCR结果显示,sglt1在草鱼肠道和肾脏组织中表达量较高,在其他组织中表达量较低;sglt2在草鱼肾脏组织中表达量最高,在其他组织中表达量较低。葡萄糖灌喂实验结果显示,草鱼前肠中sglt1和肾脏中sglt2的表达量在灌喂1 h后显著增加。在离体实验中,葡萄糖处理CIK细胞能够显著增加sglt1和sglt2的表达量。本研究为完善SGLTs在调控鱼类血糖稳态方面具有的功能提供了理论依据。     

Cloning, tissue distribution and hormonal regulation of stearoyl-CoA desaturase in tilapia, Oreochromis mossambicus

The stearoyl-CoA desaturase cDNA in tilapia (Oreochromis mossambicus) was cloned by RT-PCR and RACE, and it was compared with those in grass carp, common carp and milkfish. Nucleotide sequence analysis revealed that the full length of cDNA (1172 bp) clone encompasses 1008 bp open reading frame (ORF) encoding 336 amino acid residues. The deduced amino acid sequence shares 78–82% identity with the teleosts and 64–66% with mammals compared, and like these fish, the cloned tilapia stearoyl-CoA desaturase amino acid sequence conserves three histidine cluster motifs (one HXXXXH and two HXXHH), which functioned as non-heme iron binding sites, essential for stearoyl-CoA desaturase activity. RT-PCR and Northern blot analysis reveal that tilapia stearoyl-CoA desaturase is expressed only in liver, but the stearoyl-CoA desaturase expression in multiple tissues was observed in milkfish, grass carp and carp. Further, the hormonal regulation of stearoyl-CoA desaturase gene expression was investigated by a single injection of 17β-estradiol and testosterone. The results showed that the administration of 17β-estradiol to tilapia led to a greater increase in desaturase activity than testosterone, and higher doses of steroids produced greater increases in enzyme activity. The comparative RT-PCR analysis showed that the stearoyl-CoA desaturase mRNA level increased significantly in 17β-estradiol treated animals, especially in the groups receiving a single injection of 50 mg 17β-estradiol. This was reflected in the decrease in the saturated fatty acids and the increase in the monounsaturated fatty acids. The proportion of the polyunsaturated fatty acids was not affected.     

Molecular characterization of cholecystokinin in grass carp (Ctenopharyngodon idellus): cloning, localization, developmental profile, and effect of fasting and refeeding on expression in the brain and intestine

Cholecystokinin (CCK) is a multi-functional brain–gut peptide in fish and mammals. To investigate the role of CCK in appetite regulation in fish, a 770-bp full-length cDNA sequence of CCK gene was obtained by RT-PCR and rapid amplification of cDNA ends methods in grass carp Ctenopharyngodon idellus. Homology analysis showed that the CCK cDNA sequence of grass carp had the highest similarity (90 %) to that of goldfish Carassius auratus and a higher similarity (>70 %) to those of other teleosts than to mammals. The PCR amplification using genomic DNA identified that the CCK gene of grass carp was comprised of three exons and two introns. Real-time quantitative PCR was used to detect CCK mRNA expression in adult tissues. High levels of gene expression were found in the hypothalamus and pituitary; moderate levels in the intestine, muscle and white adipose tissue; and low levels in other tissues. During early development (i.e., fertilized eggs to 35-day post-hatching larvae) the levels of CCK mRNA expression were higher during embryonic developmental stages than during post-hatch larval stages. Fasting decreased CCK mRNA expression levels in the brain and intestine, whereas refeeding resulted in an increase of expression. The results suggest that CCK mRNA expression has obvious tissue specificity and may have a role in feed intake regulation in grass carp.     

Effect of waterborne zinc exposure on lipid deposition and metabolism in hepatopancreas and muscle of grass carp <Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis>

The aim of the present study was to explore the effect of waterborne zinc (control, 0.85, 2.20, 3.10 mg/l, respectively) exposure on lipid deposition and metabolism in the hepatopancreas and muscle of grass carp Ctenopharyngodon idella. The lipid content, Zn accumulation, and the activities and expression levels of several enzymes involved in lipid metabolism were determined in hepatopancreas and muscle. Waterborne Zn exposure reduced growth performance and increased Zn accumulation in both tested tissues. In hepatopancreas, Zn exposure increased lipid content, the activities of lipogenic enzymes, such as 6PGD, G6PD, ME, ICDH and FAS, as well as the mRNA expression level of G6PD, 6PGD, ICDH, FAS and SREBP-1. But the activity of CPT I and the mRNA expression of HSL, CPT Iα1a, CPT Iα2a and PPARα were down-regulated by Zn exposure. In contrast, in muscle, waterborne Zn exposure decreased lipid deposition, activities of 6GPD, ICDH and ME, as well as the mRNA expression level of G6PD, ICDH, ME, FAS and SREBP-1. However, the activity of CPT I as well as the mRNA expression level of PPARα, HSL, CPT Iα2a, CPT Iα1b and CPT Iβ were up-regulated by Zn exposure. Our results indicate that waterborne Zn increases lipid content by up-regulating lipogenesis and down-regulating lipolysis in hepatopancreas. But, in muscle, waterborne Zn reduces lipid accumulation by up-regulating lipolysis and down-regulating lipogenesis. Differential patterns of lipid deposition, enzymatic activities and genes’ expression indicate the tissue-specific regulatory mechanism in fish.     

Cloning, characterization and expression of the LECT2 gene in grass carp

An expressed sequence tag of grass carp leukocyte cell–derived chemotaxin 2 (LECT2) gene was screened from an established intestinal cDNA library. Rapid amplification of cDNA ends gave rise to a full-length LECT2 cDNA (gcLECT2) with a complete open-reading frame of 474 bp, encoding 158 amino acids about 17.9 kDa. Homology search and sequence alignment showed that this deduced protein sequence shared a high identity with LECT2 from other vertebrates. Western blotting indicated immunological cross-reactivity occurs between grass carp and human LECT2 protein. This gcLECT2 genomic sequence is 1,868 bp in size, which consists of five exons and four introns. Real-time quantitative PCR analysis revealed that gcLECT2 gene is ubiquitously expressed in different tissues of healthy grass carp including brain, gut, liver, spleen, kidney, muscle and heart, while the expression levels were significantly increased in liver and spleen followed by Aeromonas salmonicida infection. 992 bp 5′-flanking region sequence was cloned and analyzed, where one CAAT box and one GC island were found. Our results showed that the LECT2 is suggested to be most possibly involved in the grass carp’s immune response.