大熊猫生长激素受体(GHR) cDNA 的克隆与序列分析

根据已报道的若干物种GHR 基因cDNA 序列设计引物, 利用RT- PCR 技术首次从大熊猫肝脏组织总RNA中扩增出GHR 基因编码区全长cDNA 序列, 克隆于pGEM®-T 载体后进行测序和序列分析。结果表明,大熊猫GHR 的ORF为1 917 bp , 编码638 个氨基酸的前体蛋白, 由18 个氨基酸的信号肽和620 个氨基酸的成熟肽组成,与人、狗、猪GHR 结构相似, 大熊猫GHR 成熟肽由246 个氨基酸的胞外区、24 个氨基酸的跨膜区和350 个氨基酸的胞内区组成, 并具GHR 的特征性结构。序列相似性比较显示, 大熊猫GHR 与哺乳类GHR 具有69 %～93 %的高序列相似性, 与爬行类和鸟类的序列相似性也达到60 % , 而与鱼类的序列相似性较低, 仅为30 %左右。与其它哺乳动物GHR 相比, 大熊猫GHR 在氨基酸序列上也存在明显的特异性。     

藏绵羊GHR基因5′侧翼区序列特征分析

对欧拉型藏绵羊生长激素受体(GHR)基因5′侧翼区(包括P1启动子和外显子1A)进行了T-A克隆和序列测定(GenBank accession No. EF116490), 在分析其序列结构特征的基础上与GenBank中摩弗伦羊、山羊、普通牛、欧洲野牛进行了比较基因组学和系统进化研究, 结果表明: (1)欧拉型藏绵羊GHR基因启动子P1区存在C/EBP、C/EBPb、SP1、Cap、USF、HFH-2、HNF-3b、Oct-1等多个潜在的转录因子结合位点, 可能与GHR基因的转录调控和起始以及特异表达有关。在该非编码序列中, 重复序列所占比率为2.55%, 不存在SINEs、LINEs、LTR类反转录元件和DNA转座子元件, 而发现存在一(TG)11微卫星位点; (2)在启动子P1区, 藏绵羊与摩弗伦羊、山羊、普通牛、欧洲野牛各物种间同源性大小分别为99.7%、94.2%、85.9%、86.5%; 而在外显子1A区段, 藏绵羊与摩弗伦羊、山羊、普通牛、欧洲野牛各物种间同源性大小分别为 99.0%、97.0%、92.7%、94.6%。物种间欧拉型藏绵羊与摩弗伦羊同源性最高, 而欧拉型藏绵羊与普通牛最低。(3)邻接法(即NJ法)构建的分子系统进化树聚类结果表明, 欧拉型藏绵羊与摩弗伦羊先聚为一类, 再与山羊聚类形成一个大分支, 而普通牛和欧洲野牛先聚类形成另一大分支, 两大分支最后再聚为一起, 其聚类结果与线粒体DNA和动物学分类的研究结果一致。     

山羊磷脂氢谷胱甘肽过氧化物酶生物信息学分析

旨在克隆山羊磷脂氢谷胱甘肽过氧化物酶(PHGPx)基因cDNA全序列并进行序列分析.提取山羊睾丸中总RNA,利用RT-PCR和RACE技术,扩增山羊PHGPx基因cDNA序列并对其生物信息学进行分析.结果表明,山羊PHGPx基因cDNA序列全长844 bp,共编码199个氨基酸;山羊与牛、猪、人和小鼠的氨基酸序列同源性均大于90％;山羊PHGPx蛋白二级结构功能区域属谷胱甘肽过氧化物酶家族,预测23-24和28-29氨基酸位点有潜在的信号肽位点;UPGMA算法构建该物种间分子系统进化树,山羊与牛先聚为一类,再分别与猪、鼠、人、鸡聚类,最后与蜜蜂聚类,与物种动物学分类基本吻合.首次克隆了山羊PHGPx基因,具有GSH-Px家族典型特征,研究结果将为PHGPx基因表达分子调控研究提供一定的理论依据.     

牦牛Myf-5基因的克隆测序及其系统进化研究

本研究对牦牛(九龙牦牛)的生肌决定因子5(Myf-5)基因进行了T-A克隆测序和分析,并与多个物种的相应基因编码区核苷酸序列、氨基酸序列进行了比对分析,构建了物种间的系统进化树.结果 表明:①牦牛的Myf-5基因大小为3313 bp,由3个外显子和2个内含子组成,与普通牛等9个物种比较,在基因大小上有较大的差异,但外显子和内含子的组成一致.②牦牛、大额牛、瘤牛、水牛、普通牛、人、恒河猴、黑猩猩、狗、家鼠、软体贝壳、鸡、斑马鱼等物种间Myf-5基因编码区的核苷酸序列同源性较高,其中,牦牛、大额牛、瘤牛、水牛、普通牛间的同源性最高,达98.4%以上,说明Myf-5基因编码区核苷酸序列在动物物种间具有较高的保守性.③牦牛、大额牛、瘤牛、水牛、普通牛、黑猩猩、恒河猴、人、狗、家鼠、软体贝壳、鸡、斑马鱼等物种间Myf-5基因编码蛋白的氨基酸序列具有较高的同源性,保守性强,这一结果与编码区核苷酸序列的比对结果基本一致.④根据核苷酸序列,用NJ法构建的牦牛、大额牛、瘤牛、水牛、普通牛、人、恒河猴、黑猩猩、狗、家鼠、软体贝壳、鸡、斑马鱼等13个物种的分子系统进化树显示:牦牛、普通牛、瘤牛、水牛和大额牛在较近的亲缘关系下聚为一大类,人、黑猩猩和恒河猴聚为另一大类,然后这两类再和其他物种相聚.这一分类结果与各物种的动物学分类结果和血液蛋白、mtDNA水平上的聚类结果基本一致,支持牦牛、普通牛和瘤牛3个物种间不应该是属间或亚属间关系,而应是同一属下的不同种,将牦牛、普通牛和瘤牛划分在同一个属--牛属(Bos),而将水牛划分在另一个单独的属的观点.同时也显示该基因序列适合用于动物学分类.     

鳜鱼CRP cDNA和斜带石斑鱼AAT cDNA的克隆与序列分析

采用RT-PCR及RACE法分别克隆得到鳜鱼(Siniperca chuatsi)C-反应蛋白(C-reactive protein,CRP)cDNA全序列和斜带石斑鱼(Epinephelus coioides)α1-抗胰蛋白酶(α1-antitrypsin,AAT)cDNA全序列.鳜鱼CRP基因cDNA全长为914 bp,其中5'非翻译区(5'-UTR)为49 bp,3'非翻译区(3'-UTR)为199 bp,开放阅读框(ORF)为666 bp,编码222个氨基酸.序列同源性分析发现,推测的鳜鱼CRP氨基酸序列与小鼠(Mus musculus)、人类(Homo sapiens)、大鼠(Rattus,norvegicus)、非洲蟾蜍(Xenopus tropicalis)和中国鲎(Tachypleus tridentatus)的CRP氨基酸同源性分别为33.2%、32.4%、31.5%、24.9%和22.4%.斜带石斑鱼肝脏ATT基因cDNA全序列长1 785 bp,其中,5'-UTR为13 bp,3'-UTR为530 bp,ORF为1 242 bp,编码414个氨基酸.序列同源性分析发现,推测的斜带石斑鱼AAT氨基酸序列与斑马鱼(Danio rerio)、非洲爪蟾(X.laevis)、楔齿蜥(Sphenodon punctatus)、大鼠、人类、狒狒(Papio papio)和小鼠的AAT氨基酸序列同源性分别为59.2%、40%、38.6%、38.5%、37.7%、37%和36%.鳜鱼CRP基因和斜带石斑鱼AAT基因cDNA全序列的获得为其疾病相关分子机理研究奠定了基础,对今后进一步进行种苗育苗的研发,并以此为依据提高其人工育苗仔鱼成活率有重要意义.     

牦牛hnRNP K基因的克隆及序列分析

为研究hnRNP K基因的生物学功能及其在牦牛中的特异性,利用RT-PCR和粘性末端连接法,分两段克隆了牦牛hnRNP K基因cDNA序列。序列分析结果表明,牦牛hnRNP K基因cDNA序列长11706bp,开放阅读框(ORF)长1389bp,编码463个氨基酸。序列比对结果表明,牦牛与黄牛hnRNP K cDNA序列的同源性达99.1%,编码的氨基酸同源性达到97.0%;在牦牛氨基酸序列中有15个突变。通过同源建模的方法成功构建了牦牛hnRNP K蛋白质三级结构,结果表明牦牛hnRNP K属于A型结构,而黄牛hnRNP K蛋白属于B型结构,其差异是由第459-463位氨基酸序列由"ADVEG"突变为"SGKFF"所致。乙酰化分析结果显示,牦牛hnRNP K对基因转录的影响水平跟黄牛是一致,表明不同物种hnRNP K功能的差异可能跟其氨基酸序列的差异有关。成功克隆的牦牛hnRNP K基因的cDNA序列为进一步分析该基因的功能提供参考。     

斜带石斑鱼生长激素/催乳素家族基因cDNAs的分子克隆及其进化意义

从斜带石斑鱼垂体提取总。RNA,再取其50ng合成SMART cDNA。从所构建的垂体SMART cDNA质粒文库中筛选到生长激素／催乳素基因家族的2个成员的全长cDNA片段：生长激素(GH)基因全长为938bp,编码204个氨基酸;催乳素基因(PRI．)全长为1429bp,编码212个氨基酸。采用计算机软件Mega 2和CLUSTAL W1．64b对9种鱼的生长激素／催乳素基因家族的3个成员(GH、PRL和生长催乳素SL)的氨基酸序列进行系统分析,构建NJ分支系统树,对于序列中的插入／缺失位点则采用Pairaise Deletion,1000次自展(Bootstrap)分析计算各节点支持率。根据3个基因的氨基酸序列构建的系统树表明,石斑鱼与金头鲷、金鲈和牙鲆聚成一类,虹鳟与大马哈鱼聚成一类,鲫鱼与鲶鱼聚成一类,鳗鲡成另外一类。根据石斑鱼全长cDNA推断的氨基酸序列比较表明,SL相对GH和PRL有较高的保守性。石斑鱼的GH、PRL和SL的氨基酸同源性在24％～31％,但其C-端的氨基酸同源性较高,尤其是C-端的3个Cys是严格保守的。其中SL与GH的同源性(30．8％)高于与PRL的同源性(25．6％),GH和PRL的同源性最低(24．1％)。     

菜心苯丙氨酸解氨酶基因克隆与序列分析

通过同源克隆和RACE相结合的方法,首次从菜心中克隆了苯丙氨酸解氨酶(PAL)基因的全长cDNA,命名为BcPAL1(GenBank登录号为GU245694).BcPAL1全长2 476 bp,开放阅读框2 166 bp,编码721个氨基酸.经氨基酸序列多重比较发现,BcPAL1编码的氨基酸序列与拟南芥、烟草、甘蓝型油菜等植物的PAL氨基酸序列同源性大于90%,BcPAL1编码的氨基酸序列包含与拟南芥、烟草PAL蛋白质相同的脱氨基位点和催化活性位点.基于氨基酸序列的PAL系统进化树分析结果显示,BcPAL1编码的氨基酸序列与十字花科类植物(如拟南芥)的PAL聚为一支,说明两者的亲缘关系较近.     

斜带石斑鱼2种胰脂肪酶基因全长cDNA序列的克隆与分析

利用RT-PCR和RACE方法克隆得到斜带石斑鱼（Epinephelus coioides）肝胰脏中胆盐活化的胰脂肪酶(bile salt-activated lipase,BSAL)和依赖于辅酶的胰脂肪酶(colipase-dependent pancreatic lipase,PL)基因的全长cDNA序列.BSAL基因全长cDNA序列1 796 bp,编码558个氨基酸,该蛋白序列含有BSAL的全部特征结构区,与其他脊椎动物BSAL的氨基酸序列同源性为49.9%～57.3%.PL基因的全长cDNA序列1 503bp,编码465个氨基酸,该蛋白序列含有PL全部的特征结构区,与其它脊椎动物PL的氨基酸同源性为49.1%～73.9%.系统树分析表明,斜带石斑鱼BSAL和PL与其它物种BSAL、PL和胰脂肪酶相关蛋白(PL-RP)聚于进化树的两个不同分支,属于2种不同的胰脂肪酶.结果证实,在同一鱼类体内也存在BSAL和PL两种胰脂肪酶基因.     

能源植物续随子延伸因子EF1A基因cDNA序列的克隆及分析

利用同源克隆和cDNA末端快速扩增技术(RACE)克隆得到能源植物续随子延伸因子EFIA基因的cDNA序列(命名为ElEF1A,GenBank登录号为KT892703)。序列分析表明,ElEF1A编码序列(CDS)长1344bp,编码447个氨基酸,氨基酸序列具有典型的EF1-alpha、EF1-alpha-Ⅱ和EF1-alpha-Ⅲ结构域。ElEF1A与其他植物EF1A基因的核苷酸序列同源性达到88%以上,推导的氨基酸序列的同源性达95%以上。     

Polymorphism of prion protein gene in Arctic fox (<Emphasis Type="Italic">Vulpes lagopus</Emphasis>)

Prion diseases are fatal neurodegenerative disorders of humans and certain other mammals. Prion protein gene (Prnp) is associated with susceptibility and species barrier to prion diseases. No natural and experimental prion diseases have been documented to date in Arctic fox. In the present study, coding region of Prnp from 135 Arctic foxes were cloned and screened for polymorphisms. Our results indicated that the Arctic fox Prnp open reading frame (ORF) contains 771 nucleotides encoding 257 amino acids. Four single nucleotide polymorphisms (SNPs) (G312C, A337G, C541T, and A723G) were identified. SNPs G312C and A723G produced silent mutations, but SNPs A337G and C541T resulted in a M–V change at codon 113 and R–C at codon 181, respectively. The Arctic fox Prnp amino acid sequence was similar to that of the dog (XM 542906). In short, this study provides preliminary information about genotypes of Prnp in Arctic fox.     

Phylogenetic implications of the 38 putative ancestral chromosome segments for four canid species.

Chromosome homologies between the Japanese raccoon dog (Nectereutes procyonoides viverrinus, 2n = 39 + 2-4 B chromosomes) and domestic dog (Canis familiaris, 2n = 78) have been established by hybridizing a complete set of canine paint probes onto high-resolution G-banded chromosomes of the raccoon dog. Dog chromosomes 1, 13, and 19 each correspond to two raccoon dog chromosome segments, while the remaining 35 dog autosomes each correspond to a single segment. In total, 38 dog autosome paints revealed 41 conserved segments in the raccoon dog. The use of dog painting probes has enabled integration of the raccoon dog chromosomes into the previously established comparative map for the domestic dog, Arctic fox (Alopex lagopus), and red fox (Vulpes vulpes). Extensive chromosome arm homologies were found among chromosomes of the red fox, Arctic fox, and raccoon dog. Contradicting previous findings, our results show that the raccoon dog does not share a single biarmed autosome in common with the Arctic fox, red fox, or domestic cat. Comparative analysis of the distribution patterns of conserved chromosome segments revealed by dog paints in the genomes of the canids, cats, and human reveals 38 ancestral autosome segments. These segments could represent the ancestral chromosome arms in the karyotype of the most recent ancestor of the Canidae family, which we suggest could have had a low diploid number, based on comparisons with outgroup species.     

北极狐GHR基因单核苷酸多态性及其与生长性状的相关性分析

文章采用单链构象多态性(PCR-SSCP)和DNA测序的方法检测了北极狐生长激素受体(Growth hormne receptor, GHR)基因的单核苷酸多态性(SNPs), 并针对该群体的特点建立合适的统计分析模型, 对GHR基因多态性与生长性状的相关性进行了分析。结果表明, 在北极狐GHR基因的外显子1和外显子5上发现了4个多态位点, 分别为5′UTR上的G3A和外显子1上的C99T突变, 外显子5上的T59C和G65A突变; GHR基因G3A和C99T多态性与母狐的体重性状显著相关(P<0.05), T59C和G65A多态性与公狐的体重性状显著相关(P<0.05), 与母狐的皮张长度性状极显著相关(P<0.01)。因此, 可以利用以上点突变对北极狐的体重及皮张长度性状进行标记辅助选择研究, 以达到快速选育出快大、优质的北极狐的目的。     

The isozyme spectra of lactate dehydrogenase in the tissues of the raccoon dog <Emphasis Type="Italic">Nyctereutes procyonoides</Emphasis> in the autumn

An electrophoretic assay of lactate dehydrogenase (EC 1.1.1.27) isozymes in the tissue homogenates of cardiac and skeletal muscles, kidney, lungs, spleen, and liver of the raccoon dog Nyctereutes procyonoides from two different geographic zones, viz., northwestern Russia and Poland, as well as the Arctic blue fox Alopex lagopus L. and the red fox Vulpes vulpes L. was performed during the preparatory period to the winter season. Raccoon dogs, which hibernate under natural conditions, differ from other canids (the red fox and Arctic blue fox) to which they are close taxonomically by their body weight and by the higher proportion of aerobic H subunits of lactate dehydrogenase in all organs except for the heart. A higher content of “fast” anode fractions, lactate dehydrogenase-1 and lactate dehydrogenase-2, in the heart, kidney, lungs, liver, and spleen was detected in the raccoon dogs from the northern region compared to those from the southern geographic zone. The shift in the reaction catalyzed by lactate dehydrogenase towards the production of pyruvate indicates that this metabolite is necessary for the synthesis of fatty acids during lipogenesis in the autumn.     

Features of the architectonics of the bronchial tree of the polar fox as a manifestation of adaptation of the lungs to northern conditions

Peculiarities of the bronchial tree architectonics as a whole and in the pulmonary lobes have been studied in the animal of the Arctic zone--the Arctic fox in comparison with the dog. Certain species-specific differences in the form and branching of the bronchial tree have been stated. In the Arctic fox the subsegmentary bronchi are more developed, they are longer, with greater number of branchings and, thus, more interlobular bronchioles run off from them. Besides, up to 10% of interlobular additional bronchioles take their origin from the segmentary bronchi and as a result the bronchial tree of the Arctic fox is 1.5 times as thick as that of the dog. Complexes of the branches in the subsegmentary bronchi, or in large interlobular bronchioles of the Arctic fox are well isolated from each other and make the base of the subsegments of two types. The architectonic peculiarities are connected with genetically secured adaptation of the animal to low temperature of the environment, since they improve warming and mixing the inhaled cold air with the warm alveolar air.     

Association of MC3R gene polymorphisms with body weight in the red fox and comparative gene organization in four canids

There are five genes encoding melanocortin receptors. Among canids, the genes have mainly been studied in the dog (MC1R, MC2R and MC4R). The MC4R gene has also been analysed in the red fox. In this report, we present a study of chromosome localization, comparative sequence analysis and polymorphism of the MC3R gene in the dog, red fox, arctic fox and Chinese raccoon dog. The gene was localized by FISH to the following chromosome: 24q24‐25 in the dog, 14p16 in the red fox, 18q13 in the arctic fox and NPP4p15 in the Chinese raccoon dog. A high identity level of the MC3R gene sequences was observed among the species, ranging from 96.0% (red fox – Chinese raccoon dog) to 99.5% (red fox – arctic fox). Altogether, eight polymorphic sites were found in the red fox, six in the Chinese raccoon dog and two in the dog, while the arctic fox appeared to be monomorphic. In addition, association of several polymorphisms with body weight was analysed in red foxes (the number of genotyped animals ranged from 319 to 379). Two polymorphisms in the red fox, i.e. a silent substitution c.957A>C and c.*185C>T in the 3′‐flanking sequence, showed a significant association (P < 0.01) with body weight.     

Response to cold in the blue fox and raccoon dog as evaluated by metabolism, heart rate and muscular shivering: a re-evaluation

Oxygen consumption (ml kg-0.75/min) in relation to ambient air temperature at or below the lower critical temperature (Tlc) of the winter-furred raccoon dog (+10 degrees C) and the blue fox (-6 degrees C) is described by the equations y = 14.8-0.28x and y = 7.5-0.20x, respectively. Muscular shivering activity (integrated EMG) of both species increased below thermoneutrality parallel with increasing oxygen uptake and heart rate. Seasonal changes in measured metabolic parameters were evident for both species. The results suggest that the overall body insulation or the metabolic response to cold are not essentially worse in the raccoon dog as compared with the blue fox. It is concluded that earlier speculations of surprisingly wide thermoneutral zone and very low Tlc of the Arctic fox are not evident for the blue fox.     

狐、貉和水貂犬瘟热病毒受体SLAM 的基因克隆及其真核表达

信号淋巴激活分子(SLAM)为犬瘟热病毒(CDV) 感染其宿主动物识别的细胞受体。本试验应用RT -PCR 从狐狸、貉和水貂的外周血淋巴细胞中克隆到其相应SLAM 基因。基因测序比较发现,狐狸、貉与同科的犬SLAM 基因编码区长度均为1 029 bp,核苷酸同源性高于98.6% ;而水貂SLAM 基因编码区长度为1 020 bp,与以上三种动物遗传关系较远(核苷酸同源性< 83.7%),但与海豹SLAM 基因遗传关系较近(核苷酸同源性为91.4% )。基于不同动物SLAM 基因序列的系统进化树分析显示,犬、狐狸、貉、水貂和海豹在进化树上构成了以CDV 为感染宿主的遗传分支。氨基酸序列比较显示,该5 种动物SLAM 分子上均存在一个长度为26 个氨基酸的信号肽序列,且在空间结构上影响宿主--病毒特异性的8 个关键氨基酸均完全保守。通过构建表达该狐狸、貉、水貂SLAM 基因的三种真核表达质粒,分别转染CRFK 细胞后,应用CDV 强毒感染试验证实,CDV 均能在三种转染细胞上产生明显的细胞病变效应(CPE),而未转染CRFK 细胞对照无CPE 产生,由此证实作为CDV细胞受体的狐、貉和水貂的SLAM,体外表达后能明显增强犬瘟热强毒株对非敏感细胞的感染能力。