梅花鹿Ghrelin cDNA的克隆及序列分析

为了扩增梅花鹿Ghrelin基因的cDNA序列,根据已发表的驯鹿生长素Ghrelin基因的cDNA序列设计并合成1对特异性引物,以梅花鹿皱胃组织中提取的总RNA为模板,通过反转录-聚合酶链式反应(RT-PCR)进行cDNA扩增,获得了300 bp片段,重组到pBlueselect T载体,经限制性内切酶谱分析和DNA序列分析,确认PCR产物为Ghrelin cDNA。     

反向嵌套PCR技术扩增驯鹿Ghrelin cDNA 5′末端序列

为了研究Ghrelin在驯鹿生长发育过程中的作用和功能,以驯鹿为研究对象,采用反向嵌套PCR RACE技术原理,根据已知的序列设计1条5′末端磷酸化的特异性反转录引物和2对特异性反向嵌套PCR引物,首先进行反转录(RT),然后将反转录成的cDNA进行环化,最后进行反向嵌套巢式PCR,成功地扩增了驯鹿Ghrelin cDNA 5′末端序列。与锚定PCR相比,反向嵌套PCR法具有特异性强、扩增效率高等优点,是一种非常有效的扩增cDNA 5′末端序列的方法。     

反向嵌套PCR技术扩增驯鹿Ghrelin cDNA 5'末端序列

为了研究Ghrelin在驯鹿生长发育过程中的作用和功能,以驯鹿为研究对象,采用反向嵌套PCR RACE技术原理,根据已知的序列设计1条5'末端磷酸化的特异性反转录引物和2对特异性反向嵌套PCR引物,首先进行反转录(RT),然后将反转录成的cDNA进行环化,最后进行反向嵌套巢式PCR,成功地扩增了驯鹿Ghrelin cDNA 5'末端序列.与锚定PCR相比,反向嵌套PCR法具有特异性强、扩增效率高等优点,是一种非常有效的扩增cDNA 5'末端序列的方法.     

驯鹿Ghrelin基因组DNA克隆及序列分析

为了得到驯鹿Ghrelin基因组DNA的全序列,试验根据驯鹿Ghrelin DNA外显子1~4序列设计特异性引物,利用PCR技术对驯鹿的Ghrelin基因进行扩增,经基因组染色体步移和克隆技术获得Ghrelin基因组DNA全序列,并分析其序列结构特点。结果表明:成功克隆出全长为4 076 bp的驯鹿Ghrelin基因组DNA全序列(GenBank accession No.KX857495),其由4个外显子和3个内含子构成,4个外显子片段大小分别为154,114,109,148 bp,3个内含子片段大小分别为198,2 868,485 bp。     

中国驯鹿生长素(ghrelin)全长cDNA的克隆及序列分析

采用cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE),在本课题组已获得的驯鹿ghrelin cDNA的部分片段的基础上进行3′和5′RACE。结果成功克隆出驯鹿ghrelin cDNA的3′和5′末端序列,从而得到604bp的驯鹿ghrelin cDNA全序列,其中包含57bp 5′非翻译区(UTR)、405bp的开放读码框(ORF)、128bp的3′UTR和poly(A)14。405bp的ORF编码134个氨基酸残基的前原ghrelin(preproghrelin),其中包含41个氨基酸残基的N末端信号肽,27个氨基酸残基的成熟肽及66个氨基酸残基的C末端肽。序列同源性比较显示驯鹿ghrelin cDNA与山羊、绵羊和牛的同源性分别是92.0%、90.8%和89.5%;与猪和人的同源性分别是69.5%和65.2%;与鸡的同源性仅为33.8%。表明ghrelin的结构具有明显的种属特异性。驯鹿Ghrelin cDNA全序列的获得为进一步研究其生理作用奠定了基础。     

Ghrelin在驯鹿胃肠道的表达及定位检测

通过实时荧光定量PCR(RT-qPCR)和免疫组织化学技术对Ghrelin在驯鹿胃肠道的表达及定位进行初步研究。RT-qPCR结果显示,Ghrelin在驯鹿胃肠道内均有表达,且在皱胃内表达量极显著高于其他组织(P0.01),其次是十二指肠、回肠和结肠;免疫组织化学结果显示,Ghrelin免疫阳性细胞在皱胃内主要分布于黏膜层、黏膜下层和肌层;在瘤胃、网胃、瓣胃的黏膜层、黏膜下层中也可见Ghrelin的免疫阳性细胞,但肌层中未见表达;在十二指肠、空肠、回肠、结肠的黏膜层、黏膜下层和肌层均有Ghrelin免疫阳性细胞分布,且在肠绒毛和黏膜下层分布较多。RT-qPCR和免疫组织化学结果显示,Ghrelin在胃肠道内表达量及分布范围基本一致,这表明Ghrelin对驯鹿的消化可能具有潜在的调控作用。     

绵羊卵巢上Ghrelin部分序列鉴定

为了研究GhrelinmRNA是否在绵羊卵巢表达,试验根据GenBank中报道的绵羊Ghrelin序列设计1对特异性引物,以绵羊卵巢所提取的RNA为模板,采用RT-PCR技术扩增出绵羊Ghrelin的cDNA,经DNA测序后进行分析。结果表明:扩增到的绵羊Ghrelin cDNA为Ghrelin的部分序列,将测序结果同已发表的绵羊Ghrelin序列进行比对,233个碱基中有1个碱基的差异,该差异不影响翻译后多肽的序列;该片段cDNA包含由231bp组成的开放读码框(ORF),编码77个氨基酸的前原绵羊Ghrelin多肽。     

Ghrelin在驯鹿器官内的表达及定位检测

为了探索Ghrelin在驯鹿体内的表达及定位,采用实时荧光定量PCR(Real-time PCR)和免疫组织化学技术对其进行检测。Real-time PCR结果显示Ghrelin在所检测的17种器官中均有转录,在皱胃内的转录量最高,其次是胰、十二指肠、睾丸和食管,且转录量显著高于其他器官(P0.05);免疫组化结果揭示Ghrelin的免疫阳性细胞在食管和皱胃内主要分布于黏膜层、黏膜下层和肌层;在瘤胃、网胃、瓣胃的黏膜层、黏膜下层中也可见Ghrelin的免疫阳性细胞,但肌层中未见表达;在肠道主要位于十二指肠、空肠、回肠、结肠的黏膜层、黏膜下层和肌层,尤其在肠绒毛和黏膜下层分布较多;在肝、胰、甲状腺、垂体前叶、睾丸、肺、肾、脾器官内均有Ghrelin的免疫阳性细胞。Real-time PCR和免疫组化结果显示Ghrelin在所检测的器官内均有分布,且在食管和胃肠道内表达量及分布范围最为广泛,这表明Ghrelin对驯鹿的消化系统可能存在一定的调节作用。     

驯鹿β-防御素reBD-1 cDNA的克隆及序列分析

从驯鹿舌黏膜上皮组织中提取总RNA，采用RT—PCR技术扩增出reBD-1的cDNA，并重组到pBlueselect T载体，经限制性内切酶谱分析和DNA序列测定，证实所克隆的reBD-1的cDNA为β-防御素，因为该cDNA包含由192个碱基组成的开放读码框（ORF），该ORF编码64个氨基酸残基的前原防御素，该前原防御素含有伊防御素特征性结构即6个在特定位置上的保守半胱氨酸残基。驯鹿β-防御素属首次发现，为更好地了解驯鹿黏膜防御机制有很大的帮助。     

水牛Ghrelin基因的克隆与序列分析

根据GenBank中公布的牛、人等物种的Ghrelin核苷酸序列设计合成一对引物,以水牛皱胃组织总RNA为模板,采用RT-PCR法,扩增出Ghrelin成熟蛋白编码cDNA序列,将此扩增产物克隆入pMD18-T载体,进行PCR、双酶切鉴定及序列测定与分析。结果表明,扩增的水牛Ghre-lin基因编码序列长为351 bp,编码116个氨基酸。同源性分析表明,水牛Ghrelin基因与牛、人、猪、小鼠及绵羊基因相应序列的同源性分别为97%、79%、81%、75%和95%,氨基酸同源性与牛、羊、人、猪、小鼠分别为96%,94%,73%,76%和75%,说明Ghrelin是一组在进化上高度保守的蛋白质。水牛Ghrelin成熟蛋白cDNA的成功克隆,为进一步研究水牛Ghrelin基因结构、基因表达与调控奠定了基础。     

驯鹿干扰素β1基因的克隆及遗传进化分析

试验旨在从驯鹿鹿茸顶端组织中克隆干扰素β1（interferon beta 1,IFNβ1）基因全长序列,分析其分子特性,为研究其生物学活性及实际应用奠定基础。根据GenBank数据库中牛、羊等近缘物种IFNβ1基因的保守序列设计1对引物,以驯鹿鹿茸顶端间充质层组织基因组DNA为模板,采用PCR技术扩增得到驯鹿IFNβ1基因并克隆、测序,利用相关生物信息学软件对该序列进行分析。结果发现,驯鹿IFNβ1基因全长561 bp,共编码186个氨基酸,含有3个糖基化位点;其编码蛋白含有4个α螺旋、4个β折叠区、7个β转角。将驯鹿IFNβ1基因推导的氨基酸序列与其他14种哺乳动物进行遗传进化分析发现,其同源性为45.1%~92.0%;驯鹿与其他动物IFNβ1基因序列分析和系统进化树分析表明,IFNβ1基因存在种属差异性,亲缘关系越近,同源性越高。本研究结果为驯鹿IFNβ1基因的进一步研究和应用提供了基础试验数据。     

Morphological and molecular identification of three species of Sarcocystis in reindeer (Rangifer tarandus tarandus) in Iceland

Six Sarcocystis species have previously been described from reindeer in Norway based on sarcocyst morphology and DNA sequencing. The aim of this study was to determine whether reindeer in Iceland, which descend from reindeer imported from Norway in 1787, also were infected with Sarcocystis, and to identify and genetically characterise any species present. Muscle tissue from the heart, diaphragm and/or oesophagus was collected from 36 reindeer in Iceland. Pieces of all tissue samples were examined histologically. Frozen/thawed samples of cardiac muscle, oesophagus and/or diaphragm from 11 of the 36 reindeer were also examined under a stereoscopic microscope and sarcocysts present were identified to species either in situ or under a light microscope. Two cysts of each species, originating from two different reindeer were randomly selected for DNA analyses. The complete ssu rRNA gene was amplified by the polymerase chain reaction (PCR) and sequenced. In addition, two sarcocysts that could not be classified by microscopic examination were selected for partial ssu rRNA gene sequence analysis. By histology, sarcocysts were found in the diaphragm and/or oesophagus of 8 of 36 (22.2%) animals. By examination of fresh tissue, sarcocysts of Sarcocystis rangi, S. tarandivulpes and S. hardangeri were found in the oesophagus of seven of nine (77.8%) animals, suggesting a high prevalence of Sarcocystis in the Icelandic reindeer population. Cyst morphology and the ssu rRNA gene sequence of each of the three species were identical to isolates of the same species from Norwegian reindeer. DNA sequencing was useful in order to identify cysts with an ambiguous morphology. This is the first record of these Sarcocystis species in reindeer outside Norway.     

Toxin types of Clostridium perfringens isolated from free-ranging,semi-domesticated reindeer in Norway

Samples of faeces were taken from 166 healthy domesticated reindeer (Rangifer tarandus tarandus) from three flocks in different reindeer husbandry districts in northern Norway and examined bacteriologically for the presence of Clostridium perfringens. The organism was isolated from 98 (59 per cent) of the reindeer. The isolates were classified into C perfringens toxin types by PCR analysis specific for the genes encoding the four major toxins (alpha, beta, epsilon and tau) and were subclassified by the detection of the genes encoding C perfringens beta2-toxin and enterotoxin. All the isolates belonged to C perfringens toxin type A. In addition, 15 of the 98 isolates were PCR-positive for the beta2-toxin gene, and two of the isolates had the the gene encoding for enterotoxin.     

基于高通量测序技术的敖鲁古雅驯鹿鹿茸转录组分析

试验旨在通过高通量测序技术获得驯鹿的遗传信息和鹿茸组织的转录组特征,并进一步挖掘其中与鹿茸经济性状相关的关键基因信息。对驯鹿鹿茸组织提取RNA,检测RNA纯度、完整性及是否有污染,合格后构建文库,文库构建成功需要进行库检,库检合格后使用Illuminanovaseq平台进行转录组测序分析。利用Diamond、HMMER、KAAS、Blast2GO等软件对驯鹿鹿茸转录组序列进行了功能注释、基因丰度分析和其他分析。结果显示,测序获得47 818 202条raw reads,经过滤和质量检查得到了46 964 478条clean reads,组装后得到49 171个Unigenes,表明本次测序获得了高质量的鹿茸转录组。基于序列一致性分析,Unigenes与NR、Swiss-Prot、KOG、KEGG、Pfam、GO数据库比对注释成功总共占89.54%,共44 029条序列。Corset聚类后得到49 171个Unigenes,在这些Unigenes中共找到13 795个SNPs位点和18 446个SSRs位点。GO注释结果显示22 955个Unigenes得到注释,占总注释结果的46.68%。与KEGG数据库进行比对分析,发现49 171个Unigenes可能参与或涉及代谢途径,其中20 258个Unigenes共注释到32个KEGG代谢通路。此外,在转录组结果中发现了一些高丰度的基因,如OPN、TMSB4、TMSB10等,它们的功能可能与驯鹿生茸有关。本试验结果不仅对驯鹿的基因组数据进行了补充,而且还初步揭示了生长期驯鹿鹿茸的基因特征,为驯鹿分子遗传学研究、资源保护与利用及种群资源恢复提供了参考。     

驯鹿的皮蝇蛆线粒体CO1基因序列分析

利用分子生物学技术对内蒙古驯鹿未定种皮蝇蛆线粒体CO1基因种属特异性序列进行了研究.DNA核苷酸测序结果证实:该种皮蝇线粒体CO1种属特异性基因UEA7到UEA10特殊目标编码区域片段长度约为689 bp;种系发生进化树和同源性分析显示其与鹿皮蝇法国株同源性非常接近,因此确定内蒙古地区感染驯鹿的皮蝇蛆为鹿皮蝇(Hypo...     

Bacteria of Pathogenic Importance in Faeces from Cadavers of Free-ranging or Corralled Semi-domesticated Reindeer in Northern Norway

There is little information on bacteria that have the potential to cause disease in reindeer husbandry. In this project, faecal samples from 35 free-ranging or corralled reindeer, adults and calves, that died in the winter of 2000 in northern Norway, were examined for the occurrence of Campylobacter spp., Clostridium perfringens, Listeria spp., Salmonella spp. and Yersinia spp. to evaluate the role of these microrganisms in loss and mortality in reindeer husbandry. In addition, 31 of these samples were examined for the occurrence of bacteria producing shigatoxin-1 and 2. C. perfringens was isolated in 20 (57.1%) of the faecal samples. In the free-ranging reindeer, 44% were positive carriers of C. perfringens and 90% of the corralled ones were positive for C. perfringens. In addition, the gene encoding for shigatoxin-1 was detected in one of the samples derived from a corralled reindeer. The other bacteria investigated were not found. Shigatoxin-1-producing bacteria were isolated for the first time from reindeer in Norway. However, no correlation between C. perfringens or shigatoxin-1-producing bacteria and mortality in the reindeer could be established.     

Theileriosis in a reindeer (Rangifer tarandus tarandus) associated with a potentially novel Theileria sp

A 5‐year‐old male neutered reindeer (Rangifer tarandus tarandus) from Missouri was presented with a 3‐week history of anorexia, respiratory distress, lethargy, and weight loss. Blood smear review revealed that a small percentage of RBCs contained small (1–2 μm in length) pleomorphic piroplasms (signet ring, rod‐ or pear‐shaped, and elongate forms) with an eccentric magenta nucleus and basophilic cytoplasm. Nested PCR to specifically amplify a portion of the piroplasm small subunit ribosomal RNA (SSU rRNA) gene was performed on DNA extracted from an EDTA specimen of whole blood. Subsequent sequence analyses showed similarity between the reindeer hemoparasite and Theileria spp SSU rRNA gene sequences in the GenBank database, with highest similarity to those of a Theileria sp in a White‐tailed deer from North Texas (AY735132, AY735133). The reindeer and North Texas Theileria sp are genetically distinct from, albeit closely related to, the White‐tailed deer Theileria sp (subsequently referred to as T cervi). To the authors' knowledge, this is the first identification of Theileria of this genotype in a reindeer. Historically, T tarandirangiferis infection was found with associated mortality in reindeer in Russia, but reports predate molecular characterization. Hence, the relationship of T tarandirangiferis with either T cervi or this agent remains unknown. T cervi is not typically pathogenic in White‐tailed deer in the US unless the animal is immune‐compromised by stress or disease; however, mortality from T cervi infection in reindeer has been reported.     

Changes in ghrelin levels of plasma and proventriculus and ghrelin mRNA of proventriculus in fasted and refed layer chicks

This is a test-report of ghrelin levels in plasma and proventriculus, the glandular portion of the avian stomach, by using a specific radioimmunoassay for acylated ghrelin, as well as the expression of the ghrelin gene in the proventriculus after a 12-h fasting period followed by a 6-h feeding period with 6-day-old layer chicks. After fasting, the plasma ghrelin levels increased from 21.3 ± 4.5 to 32.9 ± 5.0 fmol/ml, but once refed it returned to the control value. After fasting, the ghrelin mRNA and the peptide levels in the proventriculus increased, and ghrelin mRNA levels remained high but once refed the ghrelin content returned to the control level. Furthermore, in order to examine the effect of increased circulating ghrelin on food intake, a bolus intravenous injection of 500 pmol of chicken ghrelin was given to 8-day-old chicks. The ghrelin injection did not cause any significant changes in food intake. These results indicate that the levels of ghrelin and its mRNA with layer chicks are altered according to the feeding state and this in a similar manner as has been observed in mammals. Unlike in mammals, an increase in circulating ghrelin does not cause the promotion of food intake in chicks.