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1.
采用逆转录聚合酶链式反应(RT-PCR)方法,从哲罗鲑(Hucho taimen)肝脏的总RNA中扩增出胰岛素样生长因子-Ⅰ(IGF-Ⅰ)的c DNA开放阅读框(Open reading frame,ORF)序列,运用软件对其进行生物信息学分析,并利用荧光实时定量PCR技术检测了哲罗鲑成鱼不同组织中IGF-Ⅰ mRNA的表达情况。结果显示,IGF-Ⅰ基因的c DNA开放阅读框为573 bp,编码190个氨基酸,蛋白质等电点为9.21,氨基酸结构由信号肽、B、C、A、D结构域及E肽组成;氨基酸序列与其他鲑科鱼类具有较高的同源性,其中与北极红点鲑的IGF-Ⅰ同源性最高(99.2%);组织表达分析显示,哲罗鲑IGF-Ⅰ mRNA在肝脏中表达量最高,在鳃、前肠中次之,在脑、头肾、脾、心、胃和肌肉等组织中的表达量较低。  相似文献   

2.
利用RACE-PCR技术,从牙鲆(Paralichthys olivaceus)的肝脏组织总RNA中克隆得到胰岛素样生长因子结合蛋白-1(insulin-like growth factor binding protein 1, IGFBP-1)基因的全长cDNA序列,该cDNA全长为1070 bp,开放阅读框为729 bp,编码242个氨基酸。通过系统进化树分析,牙鲆IGFBP-1与鱼类IGFBP-1基因聚为一支;通过同源性比对,牙鲆IGFBP-1基因的核苷酸序列与大菱鲆同源性最高,为95%,而其推导的氨基酸序列与其它鱼类如大菱鲆、五条鰤、黄金鲈、红点鲑、鲤鱼和斑马鱼的同源性分别为89%、89%、84%、79%、67%和67%。半定量RT-PCR分析表明,牙鲆IGFBP-1基因存在母源转录本,合子基因在孵化前的胚胎阶段及早期仔鱼中仅有较低水平的表达,在后期仔鱼中表达逐渐增高;牙鲆IGFBP-1基因在肝脏中表达量最高,在胃、脾、肠、性腺、肾、鳃、脑、心脏和肌肉中也有不同程度的表达。  相似文献   

3.
类胰岛素生长因子结合蛋白(IGFBP)是IGF系统的一部分,主要参与IGF的运输、定位和生物活性调节。本研究采用RACE技术和长PCR技术,克隆了缢蛏IGFBP基因的cDNA和DNA全长序列,应用荧光定量PCR技术分析了缢蛏不同发育时期和不同组织中IGFBP mRNA的表达特征,并进一步筛选了IGFBP基因与生长性状相关的SNP位点。序列分析表明,缢蛏IGFBP cDNA序列全长631 bp,包括5'端非编码区60 bp,3'端非编码区136 bp和开放阅读框435 bp,编码144个氨基酸。该基因含有保守的IGFBP-N端,包含12个半胱氨酸残基,其中1~18个氨基酸为信号肽,属于分泌型蛋白。IGFBP DNA全长3 122 bp,其中包含1个内含子(2 687 bp)和2个外显子(200和235 bp)。荧光定量PCR结果显示,IGFBP mRNA在消化腺组织中表达量最高;在缢蛏的稚贝期,IGFBP mRNA呈现高表达,而在其他发育时期表达量低。在IGFBP基因中筛选到4个SNP位点,其中1个SNP位点与缢蛏的壳长和体质量呈显著相关。  相似文献   

4.
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)。  相似文献   

5.
补体调节因子H(Complement factor H,CFH)为一类重要的补体调节蛋白,在调节补体激活反应和抵御病原感染过程中发挥重要作用。为研究黄河鲤(Cyprinus carpio YR)CFH基因在病原感染中的作用,本文克隆获得了体质量(20±2)g黄河鲤CFH基因开放阅读框序列(命名为CcCFH),进行生物信息学分析后,用荧光定量PCR(RT-PCR)技术分析其在肝脏、脾脏、头肾、鳃、心脏、皮肤、肠和体肾等组织中的表达水平以及腹腔注射100μL浓度为0.2 mg/mL的LPS溶液、100μL浓度为0.2 mg/mL的Poly I:C溶液和100μL生理盐水(对照组)后3 h、6 h、12 h、24 h、48 h和72 h脾脏、头肾和体肾等组织的表达变化。结果显示:CcCFH基因开放阅读框全长2 817 bp,编码939个氨基酸,包含一个信号肽和15个CCP重复结构域。同源性比对和系统进化分析显示:CcCFH基因与鲤科鱼类相似性最高。组织分布结果表明:CcCFH基因广泛分布在健康黄河鲤各组织器官中,在肝脏和皮肤中表达量最高。LPS和Poly I:C刺激后,CcCFH基因在脾脏中...  相似文献   

6.
为了探究补体C9在尼罗罗非鱼(Oreochromis niloticus)免疫中发挥的作用,本研究克隆并分析了尼罗罗非鱼补体C9基因(OnC9)。结果显示:OnC9的cDNA序列全长2 502 bp,包含1 761 bp的开放阅读框(ORF),编码586个氨基酸。氨基酸序列同源性分析表明,OnC9与牙鲆(Paralichthys olivaceus)补体C9氨基酸相似性最高,达73.0%,与其他鱼类补体C9的相似性介于49.3%~71.4%之间。实时荧光定量PCR检测结果表明,OnC9基因在所检测的各个组织或器官中表达水平有明显差异,在肝脏中表达量最高,其次是肠道、后肾、皮肤、肌肉、鳃,在脑、脾脏、心脏、头肾、胸腺和血液中表达量最低。在无乳链球菌(Streptococcus agalactiae)感染鱼体后,肝脏、后肾等组织中OnC9表达量表现为在感染12 h、48 h(或36 h)、120 h先后出现三个峰值的波动表达的规律。说明OnC9在无乳链球菌感染尼罗罗非鱼后的免疫应答中发挥作用。  相似文献   

7.
主要组织相容性复合体(major histocompatibility complex,MHC)是一类编码细胞表面糖蛋白的基因,在所有硬骨鱼的适应性免疫系统中起着至关重要的作用,而关于MHC基因的研究一直是鱼类分子免疫学和鱼类抗病辅助育种的研究热点之一。本研究首次分析了大弹涂鱼(Boleophthalmus pectinirostris)MHCIα基因的cDNA序列特征,构建了系统发生树,评估了大弹涂鱼MHC Iα基因mRNA在健康个体不同组织中的表达差异,研究了注射病毒拟似物poly(I:C)后MHC Iα基因在机体主要免疫器官肝和脾的表达情况。结果显示,大弹涂鱼MHC Iα基因具有由1101个碱基组成的开放阅读框(ORF),共编码366个氨基酸残基,具有3个蛋白激酶C-磷酸化位点、1个酪蛋白激酶Ⅱ磷酸化位点和1个N-糖基化位点。系统发育分析显示与大弹涂鱼MHCIα基因亲缘关系最密切的是河川沙塘鳢(Odontobutis potamophila)。RT-PCR分析显示,MHCIα基因mRNA在不同组织中均有表达,其中肾和脾组织中表达量最高,鳃和肠组织中表达次之。大弹涂鱼在腹腔注射poly(I:C)后,肝和脾组织中mRNA表达量明显上升,在12 h时, MHC Iα基因mRNA表达量在肝和脾中均达到峰值。本研究结果表明, MHC Iα基因参与了大弹涂鱼在高盐胁迫下的免疫应答。  相似文献   

8.
利用中国大鲵(Andrias davidianus)性腺转录组测序获得的生长激素受体(GHR)基因部分序列,克隆获得基因全长2992 bp,开放阅读框1812 bp,编码604个氨基酸,该蛋白具有高度保守的FGEFS基序与BOX框。系统进化分析结果显示,中国大鲵GHR氨基酸序列与两栖类非洲爪蟾(Xenopus laevis)同源性最高,蜥形纲锦龟(Chrysemys picta bellii)次之,哺乳类水牛(Bubalus bubalis)和鱼类半滑舌鳎(Cynoglossus semilaevis))最低。实时定量PCR结果表明,GHR基因在肝中表达最高,肌肉、垂体、肾、性腺中的表达量次之,其他各组织表达量较低。在精巢发育早期GHR表达较高,随后表达量逐渐降低,在卵巢中表达量随时间增长而增加;17α-甲基睾丸酮(MT)与芳香化酶抑制剂来曲唑(LE)短暂处理后GHR基因在脑与卵巢中表达量发生变化,MT处理后,脑与卵巢中GHR表达量增加,LE处理后脑与卵巢中表达量降低。研究表明,GHR基因在大鲵性腺发育中可能发挥作用,且MT与LE可能通过不同的途径调节GHR基因的表达。  相似文献   

9.
为解析黄鳝(Monopterus albus)性逆转机制,实验以雌、雄、间性发育黄鳝为研究对象,分析不同组织、不同发育时期性腺、甲基睾丸酮处理性腺及Zebularine处理性腺原代细胞后dynlt3基因表达模式的变化及其在性腺中的表达定位。性腺转录组测序结果显示,基因全长1 082 bp,开放阅读框354 bp,编码117个氨基酸。生物信息学分析显示,dynlt3基因编码蛋白质的二级结构包含37.96%的α-螺旋,29.20%的β-折叠,32.85%的无规则卷曲。系统进化分析结果显示,黄鳝DYNLT3氨基酸序列与硬骨鱼纲中底鳉(Fundulus heteroclitus)同源性最高。实时荧光定量PCR结果表明,dynlt3基因在黄鳝肌肉和脑具有较高表达,心脏次之,在其它各组织表达量较低。在性腺的不同发育时期,在间性后期和雄性中表达量显著性高于雌性与间性早期。甲基睾酮处理后黄鳝卵巢组织结构发生明显退化,卵母细胞退化且数量减少,结缔组织间出现空泡结构;dynlt3基因在卵巢中表达量显著性下调。原位杂交分析dynlt3基因在性腺组织中的表达定位结果显示,在不同性腺发育时期均检测到dynlt3...  相似文献   

10.
为探索文蛤GRB2基因的结构和功能,本实验通过已构建的文蛤转录组文库,利用SMART RACE技术扩增得到了文蛤GRB2基因的cDNA全长序列,对其生物信息学、组织及发育阶段表达特征进行了分析,并利用直接测序方法在外显子中筛选SNP位点。结果显示,GRB2基因cDNA全长1791bp,开放阅读框669bp,编码223个氨基酸,蛋白由SH-SH2-SH3三个结构域组成;氨基酸序列比对发现,文蛤GRB2与泥蚶的同源性最高,达到65.6%,与脊椎动物的同源性都在60%以上,说明GRB2基因在进化过程中比较保守。qRT-PCR结果表明,GRB2在文蛤6个组织和10个发育时期中均有表达,但不同组织间的表达量并没有显著差异(P>0.05);发育时期中的表达量呈现逐渐升高的趋势,在壳顶幼虫时期达到最高,之后表达量又有所降低。SNP位点的筛选结果表明,在GRB2基因的外显子区域共发现16个SNP位点。  相似文献   

11.
The role of growth hormone (GH) in regulating hepatic mRNA expression of insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) in yellowtail Seriola quinqueradiata was examined using in vivo and in vitro assays. Yellowtail hepatic IGF-I, IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-5 mRNAs were measured by real-time quantitative RT-PCR. Intraperitoneal injection of recombinant GH of chum salmon Oncorhynchus keta (rsGH) at a dose of 1 μg/g body weight resulted in a significant increases in hepatic IGF-I, IGFBP-3, and IGFBP-5 mRNA levels, whereas significant reductions in hepatic IGFBP-1 and IGFBP-2 mRNA levels were observed. For in vitro assays, liver slices were incubated with rsGH at different concentrations (doses: 0, 1, 10, 100, 500, and 1,000 ng/ml). Liver slices incubated with 100 ng/ml rsGH elicited a significant increase in IGF-I mRNA level. Similarly, a slight increase in IGFBP-3 and IGFBP-5 mRNA levels were also observed in liver explants incubated with rsGH. In contrast, a significant decline in IGFBP-1 mRNA levels was observed in liver slices incubated with 1,000 ng/ml rsGH. A slight decline in the level of IGFBP-2 mRNA was noted in liver explants with rsGH treatment. This study demonstrates the modulating effect of GH on the IGF system.  相似文献   

12.
Insulin-like growth factors I and II (IGF-I and IGF-II) are two highly homologous mitogenic peptides that are expressed ubiquitously and show diverse effects on development, growth, and metabolism. The cDNA encoding IGF-I of a teleost, the orange spotted grouper (Epinephelus coioides) was produced from liver by RT-PCR, and rapid amplification of cDNA ends, RACE. Typically, the deduced 186 amino acid protein contains a signal peptide, B, C, A, D and E domains. On the amino acid level, grouper IGF-I shares 97.3% similarity with black seabream (Sparus macrocephalus) with the differences focusing on the B and C domains. The analysis of the E domain showed that grouper IGF-I belonged to Ea-4 type. When mature amino acid sequence was compared with other vertebrates, it revealed higher similarity with black seabream and halibut, while lower similarity with human and mouse. The expression of IGF-I mRNA in adult tissues was studied using RT-PCR. IGF-I mRNA expression level in the liver was significantly higher than those in the brain and muscles. In other tissues, low amount of IGF-I mRNA expression was also detected. The coding region of IGF-I cDNA for mature IGF-I protein was subcloned into an expression plasmid pTRX and fused with E. coli thioredoxin (Trx). Moreover, we have successfully developed an expression system in E. coli to overproduce recombinant grouper IGF-I. Using western blotting, we found that the fusion protein could blot with antiserum to barramundi IGF-I further confirming the immunoactivity of the recombinant IGF-I.  相似文献   

13.
为进一步了解哲罗鲑IGF-I,实验采用逆转录聚合酶链式反应(RT-PCR)方法,从哲罗鲑肝脏的总RNA中扩增出胰岛素样生长因子-I(IGF-I)的c DNA开放阅读框(open reading frame,ORF)序列。利用原核表达载体p S构建重组表达质粒(IGF-I/p S),并将其转化到宿主大肠杆菌Rosetta后经IPTG诱导获得重组哲罗鲑IGF-I蛋白。经SDS-PAGE电泳检测,在35~50 ku有条带与预期相符,且重组蛋白以包涵体的形式存在。包涵体经变性/复性实验后,获得纯化的IGF-I融合蛋白。ELISA鉴定结果显示,目的蛋白能特异性识别抗鱼类IGF-I抗体,表明获得了具有免疫活性的哲罗鲑IGF-I蛋白。细胞增殖实验(MTT法)结果显示,重组IGF-I蛋白对大麻哈鱼胚胎细胞(CHSE-214)、鲤上皮细胞(EPC)及虹鳟性腺细胞(RTG-2)均有显著促增殖作用,表明获得的重组IGF-I蛋白具有细胞水平的生物活性。本研究为深入了解IGF-I在哲罗鲑生长发育中的调控机制及绿色高效促生长制剂的研发奠定基础。  相似文献   

14.
草鱼胰岛素样生长因子_基因克隆及序列分析   总被引:4,自引:1,他引:3  
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。  相似文献   

15.
IGF-I is a mitogenic polypeptide that is an important regulator of growth in fish. The potential of IGF-I mRNA abundance as a rapid growth indicator in the Nile tilapia, Oreochromis niloticus, was evaluated. Hepatic IGF-I cDNA was isolated and partially cloned. The partial sequence having 539 bases encodes for the signal peptide, mature protein and a portion of the E domain. The deduced 68 amino acid sequence for mature IGF-I showed 84–90% and 77–79% sequence identity with fish and mammalian counterparts, respectively. The deduced amino acid sequence for domains B and A was most conserved (93–97%) relative to other fishes. A sensitive TaqMan real time qRT-PCR assay for O. niloticus was developed based on the mature IGF-I peptide for measures of hepatic IGF-I mRNA levels. Hepatic IGF-I mRNA levels were found to be significantly correlated with growth rate of fish reared under different feeding regimes and temperature conditions. Higher feed consumption and water temperature produced faster-growing fish and increased hepatic IGF-I mRNA expression. These findings suggest that hepatic-derived IGF-I plays a key role in controlling growth in O. niloticus and indicates that IGF-I mRNA quantification could prove useful for the rapid assessment of growth rate in this species.  相似文献   

16.
魏巍  刘红柏  王荻  卢彤岩  尹家胜 《水产学报》2009,33(6):996-1002
采用RT-PCR技术从哲罗鱼主要免疫器官脾脏总RNA中获得免疫球蛋白(immunoglobulin, Ig)轻链可变区cDNA克隆,随机挑取51个阳性克隆菌落进行测序,得到46条完整的不重复序列,用以确定哲罗鱼IgL的家族种类,为哲罗鱼病毒性疾病的疫苗研制提供理论依据。经过BLUST网络对比,结果显示:其与GenBank报导的虹鳟(序列号为X68517.1和X68519.1)序列的相似性均达90%以上,46条序列的核苷酸相同率分别为最低86.1%(P4与P43)至最高的99.8%(P21与P38)之间不等;经DNAstar 5.0 软件包MegAling中Jotun Hein方法对所得序列进行氨基酸同源性比较,得到两个不同的家族;通过可变性参数计算方法可知哲罗鱼IgL的可变性主要集中在CDRs区,可变性最高的区域为CDR3区;对可变区中CDR3序列进行分析后,得出哲罗鱼Ig轻链可变区的重排方式是Jκ基因片段位于Vκ基因片段的3′端的结论,这种重排方式增加了核苷酸不同的连接方式,使哲罗鱼抗体具有更多的可变性空间;对照其亲水性分布图可知:可变区的亲水性氨基酸主要分布于LP前端,FR1末端,完整的FR2,FR3两端,以及CDR1,CDR2和CDR3前端。  相似文献   

17.
The effects of fish protein hydrolysate (FPH) on growth, peptide and amino acid (AA) transporters, postprandial free AA and related gene expression of IGF‐1/AKT pathway were evaluated in turbot (Scophthalmus maximus). Three diets were formulated to contain the same low level of fishmeal; meanwhile 0, 45 and 180 g/kg FPH were, respectively, supplemented to the FF (FPH‐free), FL (FPH‐Low) and FH (FPH‐High) diets. Fish fed the FH diet improved the growth compared with the other groups. For peptide and AA transporters, PepT1, B0AT1, CAT1 and PAT1 mRNA levels in proximal or distal intestine decreased in fish fed the FH diet. The concentration of free total essential AAs in serum was higher in the FH treatment than that in the FF treatment at 2 and 6 hr after feeding. For IGF‐1/AKT pathway in muscle, IGF‐1, 4E‐BP1 and FoxO1 mRNA levels were the highest in the FH group, whereas IGF‐1R mRNA levels were the highest expression level in the FF group. In conclusion, down‐regulated AAs transport, alleviated the delayed postprandial peak of serum‐free AAs and increased muscle protein synthesis were observed to improve the growth when turbot was fed high FPH level diets containing a high plant protein.  相似文献   

18.
A full-length cDNA encoding the insulin-like growth factor binding protein-3 (IGFBP-3) was cloned from the liver of common carp (Cyprinus carpio) by RT-PCR. The IGFBP-3 cDNA sequence is 1,680 bp long and has an open reading frame of 882 bp encoding a predicted polypeptide of 293 amino acid residues. The deduced amino acid sequence contains a putative signal peptide of 25 amino acid residues resulting in a mature protein of 268 amino acids. A single band of approximate 1.9 kb was found in liver by Northern blot analysis. IGFBP-3 mRNA was observed in all regions of brain with high levels. In peripheral tissues, high levels of IGFBP-3 mRNA were found in retina, red muscle, liver, heart, posterior intestine, spleen, and testis. Relatively lower levels were found in white muscle, kidney, thymus gland, and ovary, while in head kidney, blood, skin, gill, middle intestine, and anterior intestine, the IGFBP-3 mRNA levels were much lower. IGFBP-3 mRNA was first detected in the blastula stage with significantly high level. The level sharply decreased in gastrula stage, and it became to increase in the following stages. During the reproductive cycle, the abundance of IGFBP-3 mRNA significantly decreased between the recrudescing stage and the matured stage in ovary, although in testis, IGFBP-3 mRNA expression level did not exhibit a significant change. The mRNA expression profiles in the present study imply that the IGFBP-3 may play important physiological functions in common carp development and reproduction.  相似文献   

19.
鲶生长激素基因cDNA的克隆和原核表达   总被引:5,自引:0,他引:5  
从脑垂体中提取总RNA,用RT-PCR扩增并克隆到鲶鱼的生长激素(GH)基因cDNA。分析其核苷酸序列和推测的氨基酸序列,结果显示:鲶鱼生长激素基因的开放阅读框(ORF)包括603个核苷酸;编码200个氨基酸;其中包括22个氨基酸的信号肽和178个氨基酸的成熟肽。把GH成熟肽的cDNA克隆入表达载体pET-28 a,用IPTG诱导重组蛋白的表达,其表达量超过细胞蛋白总量的50%,主要为不溶性的包含体。细菌裂解液沉淀溶于8 mol/L尿素后,用固定化金属配体亲和层析纯化,获得分子量为22.5 kD的单一蛋白带。  相似文献   

20.
Triangular bream (Megalobrama terminalis) IGF-I DNA and gene were cloned from triangular bream liver for the first time by RT-PCR. Sequence analysis indicated that the IGF-I cDNA consisted of 486 nucleotides encoding 117 amino acids which spanned the complete signal peptide, B, C, A, D and E domains. Analysis of the E domain indicated that triangular bream IGF-I belongs to the IGF-I Ea-2 subtype. Compared to bluntnose bream (Megalobrama amblycephala), another member of the same Megalobrama genus, triangular bream IGF-I shared 99.8% identity in cDNA sequence and 99.4% in predicted amino acid sequence. However, considerable differences were found in comparison to with common carp (Cyprinus carpio) and grass carp (Ctenopharyngodon idellus), which are members of the same family but of a different genus. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

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