牛亚科物种TRs分布特点及着丝粒区卫星DNA进化研究

旨在研究牛亚科物种间串联重复序列（tandem repeats sequence,TRs）的分布特点及着丝粒区卫星DNA的进化关系。本研究基于普通牛、瘤牛、牦牛、水牛、野牛、独龙牛6个牛亚科物种的基因组序列,研究了不同物种间TRs的组成、分布及结构特点,并分析了6个牛亚科物种染色体着丝粒区卫星DNA的进化关系。结果表明：1）TRs在牛亚科物种中平均占比为2.03%,平均长度54.93 Mb,其中普通牛的占比最高3.42%（93.00 Mb）,瘤牛最低1.42%（37.88 Mb）。2）微卫星DNA在3类TRs中位点数最多,为483 405,占TRs总位点数85.64%,高于小卫星DNA（43 026,7.62%）和卫星DNA（38 180,6.75%）。3）通过对微卫星DNA丰度和平均长度分析发现,二碱基微卫星DNA在牛亚科物种中丰度最高,为70.93 loci/Mb,且以AC拷贝类别为主。4）通过构建着丝粒区1.715和1.723卫星DNA的系统发育树发现,1.715卫星DNA普遍存在于牛亚科物种的基因组中,而1.723卫星DNA在牦牛中不存在,两类卫星DNA在不同物种间或不同染色体上存在不同程度分化,具有较明显的种间特异性。TRs在牛亚科6个物种中平均占比为2.03%,微卫星DNA为TRs主要序列,且二碱基微卫星丰度最高,并以AC拷贝类别为主;1.715卫星DNA普遍存在于6个牛亚科物种的基因组中,但在物种间或染色体间存在不同程度分化。本研究结果为牛亚科物种间TRs分布特征及进化关系研究提供了重要理论依据。     

牦牛KAP1家族基因长度多态研究

试验旨在研究牦牛角蛋白关联蛋白1（keratin associated protein 1,KAP1）家族基因长度多态与重复序列特点。研究对牦牛和黄牛KAP1家族基因进行测序,并与绵羊已知序列进行比较分析。结果发现,牛KAP1家族位于19号染色体,根据绵羊KAP1家族基因在染色体上的位置与相似性,重新命名了牛KAP1家族基因B2D、B2A、KAP1-1和B2C为KAP1-4、KAP1-1、KAP1-2、KAP1-3（按照染色体上的基因顺序）。KAP1家族基因之间在3'和5'端区域高度保守,中间有重复序列长度差异,其中牦牛KAP1-KAP4基因发现有30 bp的长度多态。研究其蛋白序列发现5个氨基酸为基序的重复序列B（CCQTS）A₁（CCQPT）,以及一个新的重复序列C（SIQTS）。本研究结果说明重复序列是KAP1家族基因间和基因内的主要差异区域,这可能与其角蛋白结合螺旋数相关。     

利用TSPY基因鉴定奶牛早期胚胎性别的研究

TSPY（testis—specific protein）是Y染色体上特异的基因。公牛Y染色体上的TSPY基因的拷贝数达200个。本研究主要探讨利用TPSY基因鉴定胚胎性别的可能性。利用TSPY特异引物,通过PCR技术扩增牛血样DNA的结果为公牛血样均为阳性,母牛为阴性。结果表明,TSPY基因是一个很好的雄性特异标记。PCR扩增TSPY特异序列鉴定牛性别的灵敏度试验表明,DNA的最低需要量为20pg/μL,提示,TSPY特异基因具有鉴定牛早期胚胎性别的可能。     

鉴别牛早期胚胎性别PCR方法引物的设计与筛选

根据牛Y-染色体特异重复序列、睾丸特异蛋白基因以及性别决定基因序列设计合成5对公牛Y-染色体特异引物，依据牛骨胳肌α肌动蛋白前体基因和微卫星DNA序列设计合成4对牛DNA特异引物(内标引物)。单重PcR扩增牛基因组DNA，筛选出4对牛Y-染色体特异引物和1对牛DNA特异内标引物。将不同的Y-染色体特异引物与内标引物组合，多重PCR扩增牛基因组DNA、已知性别的牛成纤维细胞和克隆胚胎，筛选出2个可用于牛早期胚胎性别鉴别的PCR引物组合：B34／A12和B78／A12。     

牛分枝杆菌广西分离株MPB70基因的克隆及序列分析

利用牛分枝杆菌MPB70基因特异性引物,以2株牛分枝杆菌广西分离株的染色体DNA为模板,扩增MPB70基因,产物经纯化后与载体PMD18-T连接,然后转化至大肠杆菌DH5α。提取转染后大肠杆菌的质粒进行双酶切和PCR鉴定,鉴定为阳性的质粒进行序列测定。测序后通过序列分析软件DNAstar MegAlign对MPB70基因核苷酸序列及推导的氨基酸序列进行分析,并与GenBank上已发表的8株牛分枝杆菌的MPB70基因参考序列进行比较。结果显示广西分离株mt359、mt370与已发表的7株参考株序列比较,其核苷酸序列和氨基酸同源性在77.4%~95.9%之间。这一结果表明广西奶水牛分枝杆菌分离株与参考的其他牛分枝杆菌毒株的MPB70基因没有太大的差异,说明牛分枝杆菌分泌蛋白MPB70基因十分保守,从而为检测跟踪毒株的变异,研制牛分枝杆菌亚单位疫苗提供基础。     

应用AML基因进行牛性别鉴定的研究

本文以染色体牙釉蛋白基因(AML)对牛肌肉基因组进行性别鉴定，为应用此基因进行胚胎鉴定提供理论依据。本研究应用奶牛染色体牙釉蛋白基因的DNA序列，设计了牛染色体特异性引物P1和P2，并通过聚合酶链反应(PCR)扩增牛肌肉组织中的X和Y染色体上的牙釉蛋白基因内第五内含子。最后依据其分子量的差异对电泳后的性染色体所呈现的不同区带进行区分，从而达到性别鉴定的目的，具有很高的应用价值。     

牛成纤维细胞生长因子受体3(FGFR3)基因对日本褐牛软骨发育异常性侏儒症的影响

成纤维细胞生长因子受体 3(FGFR3)是成纤维细胞4个不同跨膜酪蛋白激酶受体之一。它是软骨骨化的负调控因子 ,FGFR3的突变体已在软骨发育异常的病人身上检测到。最近 ,将与日本褐牛侏儒症相关的位点定位到牛 6号染色体端粒区 ,该区域与 FGFR3基因紧密相邻。因此 ,FGFR3成为这种侏儒症的定位候选基因。本研究中 ,作者分离克隆了包括 FGFR3基因全部编码区的 c DNA。通过对软骨发育正常和异常牛核苷酸序列的比较分析 ,发现它们的氨基酸序列无特异性差别。进一步利用放射杂交定位的方法对 FGFR3进行精细定位 ,结果FGFR3基因位点与这种疾病位点截然不同。因此 ,认为日本褐牛软骨发育异常性侏儒症不是 FGFR3基因突变引起的     

一株多重耐药大肠杆菌耐喹诺酮gyrA基因突变的研究

临床获得1株鸡致病性大肠杆菌CE01，进行药每、质粒转化、耐药质粒消除试验，结果该菌对11种受试抗菌药物全部呈高度耐药，其中氧氟沙星的MIC≥50μg/ml，且含有喹诺酮抗生质粒，溴化乙淀和黄连素作为消除剂作用48小时可使其喹诺酮抗性质粒消除，耐药水平显著下降。聚合酶链反应（PCR）扩增该菌gyrA基因喹诺酮耐药决定区（QRDR），质粒DNA和染色体DNA均可扩增出长度为668bp的PCR产物片段。经测序分析，两者基因序列相同率为98.17%，相同位点突变相同的碱基有5个，相同位点突变不同的碱基有2个；与基因数据库Swanberg等报道的大肠杆菌gyrA基因相应序列比较，该菌质粒DNA PCR产物同源率97.8%，有13个位点发生突变，3个氨基酸被替换；染色体DNA PCR产物同源率98%，有12个位点发生突变，2个氨基酸被替换。用放射性同位素α-^32P分别标记CE01菌株染色体DNA和质粒DNA的PCR扩增片段制备探针，对CE01菌株进行检测。质粒DNA和染色体DNA分别与质粒控针和染色体探针杂交出现典型的阳性杂交斑。证实该菌株质粒和染色体上同时存在耐喹诺酮gyrA突变基因，对喹诺酮的耐药性是质粒介导和染色体突变共同作用的结果。     

耐氧氟沙星鸡大肠杆菌gyrA基因QRDR的扩增与测序

取临床分离的13株氧氟沙星（OFL）耐药菌，提取其质粒DNA，经纯化后人为模板，用PCR扩增gyrA基因喹诺酮耐药决定区（QRDR）；PCR阳性质粒的菌株，再扩增其染色体gyrA基因QRDR，直接测定质粒和染色体DNA扩增产物序列并进行分析。只有CE01菌株的质粒DNA和染色体DNA可扩增出长度为668bp的PCR产物片段，两者基因序列相同率为98．17％，相同位点突变相同的碱基有5个，相同位点突变不同的碱基有2个。与基因数据库中的大肠在杆菌gyrA基因相应序列比较，该菌质粒DNA PCR产物同源率为97．80％，有13个位点发生突变，3个氨基酸被替换；染色体DNA PCR产物同源率为97．80％，有13个位点发生突变，3个氨基酸被替换；染色体DNA PCR产物同源率98．00％，有12个位点发生突变，2个氨基酸被替换；都包括了国外资料报道的突变率较高的第83位氨基酸的突变。结果表明，该菌株质粒和染色体上都存在喹诺耐经基因，对OFL的耐药可能是质粒诱导和染色体突变共同作用的结果。     

牛白细胞黏附缺陷病液相基因芯片检测方法的建立

从福建某奶牛场200头进口荷斯坦牛采血提取牛血液DNA，根据已知牛染色体上CD18编码基因序列383位基由A变为G而引起牛白细胞黏附缺陷症（BLAD）设计特异性野生型和突变型引物，建立液相基因芯片检测方法用于牛白细胞黏附缺陷症（BLAD）的检测，结果检出2头母牛为BLAD携带者，检出率为0．67％，没有发现患病牛。结果证明建立的液相基因芯片检测方法是一种敏感性和特异性很高的筛选奶牛BLAD有害基因的新方法     

DNA extraction from bovine mummified fetuses and detection of factor XI gene deficiency in the mummies

Genomic DNA extracted from bovine mummified tissue is valuable material for detection of some genes that may contribute to fetal abnormalities. In this study bovine genomic DNA was extracted from the hardened tissue samples of ten bovine mummified fetuses. The amount of genomic DNA extracted from 2 g of the mummified tissues by the phenol/chloroform-ethanol method was low (less than 4 microg/ml) for all samples. The extracted DNA was then amplified by the GenomiPhi DNA amplification system. After amplification, the amount of DNA was increased to more than 100 microg/ml for all samples. This amplification system was shown to be a good tool for amplifying the genomic DNA of the mummified fetuses. The amplified genomic DNA was used for testing the mummies for Factor XI gene deficiency, an autosomal recessive deficiency involved in the early stages of the intrinsic blood coagulation pathway. Exon 12 of the Factor XI gene of the mummies was amplified by PCR. Two of the ten mummified fetuses were heterozygous for the Factor XI gene as indicated by the presence of two amplified DNA fragments of 320 bp and 244 bp. Factor XI deficiency has already been described in Holstein cattle. However, no report is available for bovine fetus. In this study, DNA was extracted and amplified from the bovine mummified fetuses, and the samples were successfully tested for Factor XI gene deficiency in the mummies.     

牛肌肉生长抑制素基因打靶载体的构建与鉴定

本试验旨在构建用于牛肌肉生长抑制素(MSTN)基因敲除的置换型打靶载体。基于已发布的MSTN基因序列,选取第3外显子约600 bp作为靶位点,在其上、下游设计2条同源臂,分别为4.4和1.4 kb。以pPNTⅢ为骨架载体,在其2个多克隆位点处插入同源臂,构建出置换型打靶载体MSTN-KO-pPNTⅢ。结果显示,经DNA测序及酶切鉴定证实1.4 kb同源短臂和4.4 kb同源长臂均正确插入基础载体中。结果表明,成功构建出牛MSTN-KO-pPNTⅢ打靶载体。     

Somatic cell mapping and restriction fragment length polymorphism analysis of bovine insulin-like growth factor I.

The DNA isolated from cow-hamster hybrid somatic cells segregating bovine chromosomes was analyzed by Southern blotting and hybridization with a heterologous [32P]-labeled porcine cDNA probe encoding insulin-like growth factor I (IGF-I). Thirteen of 25 cow-hamster hybrid cell lines exhibited the bovine-specific IGF-I fragment. Analysis for the retention or loss of bovine IGF-I with markers previously screened against the same panel of hybrid cells revealed a 100% concordance with lactate dehydrogenase B of bovine syntenic group U3 located on bovine chromosome 5. Restriction fragment length analyses of genomic DNA from animals representing five breeds (Angus, Polled Hereford, Simmental, Gelbvieh, and Belgian Blue) and from seven half-sib Angus calves indicated that polymorphisms for the genomic composition of the bovine IGF-I gene may exist in cattle populations.     

Detection and molecular characterisation of bovine polyomavirus in bovine sera in New Zealand

AIM: To investigate the prevalence of bovine polyomavirus (BPyV) DNA in commercial batches of bovine serum products, cell lines and cattle in New Zealand and to characterise the viral DNA detected. METHODS: Two nested polymerase chain reaction (PCR) assays were applied to detect BPyV in bovine sera. One was used to screen for the VP1 gene of BPyV DNA in: 140 batches of commercial bovine serum products, including 66 batches of fetal bovine serum (FBS), 34 batches of calf serum, and 40 batches of adult bovine serum (ABS)/plasma; 112 individual adult bovine sera; and 16 cell lines of various species origin. Fifty batches of serum samples were also tested, using the second nested PCR assay that screened for the Large T gene. Restriction fragment length polymorphism (RFLP) was conducted with 36 PCR products amplified from the VP1 gene of BPyV using EcoRI. Five selected VP1 PCR products were subjected to DNA sequencing and phylogenetic analysis. RESULTS: BPyV DNA was detected in 46 (70%) batches of FBS, 11 (32%) batches of calf sera and two (5%) batches of ABS/plasma, an overall prevalence of 42%. None of 112 adult bovine sera was BPyV-positive. RFLP analysis demonstrated a uniform digestion pattern in the majority (31/36) of amplicons tested, while the remaining PCR amplicons did not show enzyme cleavage. Sequence analysis of the PCR products (a 263 base pair (bp) fragment of the VP1 gene) obtained from five batches of FBS showed 96.2-98.9% homology to that of published sequences of BPyV. CONCLUSION: BPyV is a frequent contaminant of commercial bovine serum in New Zealand. The incidence of BPyV in adult bovine serum products is much lower than in FBS and calf serum. Genomic variations exist among different viruses. The clinical significance of the high prevalence of BPyV DNA in bovine serum products is yet to be determined.     

牛源犬新孢子虫NcSRS2-NcGRA7融合基因的克隆与生物信息学分析

为了解牛源犬新孢子虫NcSRS2-NcGRA7融合基因的生物学特性,本试验提取牛源犬新孢子虫基因组DNA,应用PCR技术扩增犬新孢子虫表面蛋白基因NcSRS2和致密颗粒蛋白基因NcGRA7,SOE-PCR技术拼接NcSRS2和NcGRA7基因,构建NcSRS2-NcGRA7融合基因重组克隆质粒,并进行PCR鉴定、双酶切鉴定及生物信息学分析。结果,NcSRS2基因扩增片段大小为1 061 bp,NcGRA7基因扩增片段大小为364 bp,NcSRS2-NcGRA7融合基因扩增片段大小为1 482 bp;获得重组克隆质粒pMD18-NcSRS2-NcGRA7经PCR鉴定、双酶切鉴定正确;测序分析表明,与Gen-Bank中已发表的美国株犬新孢子虫(AF061249、AF176649)核苷酸序列同源性为99%;经DNAman等软件分析,预测NcSRS2-NcGRA7融合蛋白抗原指数较高,融合蛋白二级结构以α-螺旋和β-折叠为主,三级结构中2种蛋白独立折叠,并借助Linker互相连接,功能互不影响。本试验为牛源犬新孢子虫NcSRS2-NcGRA7融合蛋白的免疫学研究奠定了基础。     

KIT基因多态性与荷斯坦牛着色性状的关联分析

牛的被毛颜色是重要的外貌性状。本研究采集北京地区401头荷斯坦母牛血样及21头公牛冻精样品．提取基因组DNA,选取牛6号染色体上KIT基因的4个SNPs位点,利用飞行时间质谱技术检测基因型。通过数码相机照相并进行图像分析计算牛的被毛黑白比例,记录乳头颜色。将基因型和表型数据进行关联分析,结果显示KIT基因的G72792875T住点对被毛比例有显著影响（P〈0．05）,推测KIT基因可能与荷斯坦牛的着色性状的形成相关。     

Quantitative real time polymerase chain reaction assays for the sensitive detection of Besnoitia besnoiti infection in cattle

Bovine besnoitiosis, an economically important disease in cattle in some countries of Africa and Asia, is emerging in Europe. The definitive host of Besnoitia besnoiti, the causative agent of bovine besnoitiosis, is unknown and the transmission of the parasite is not completely understood. Sensitive and quantitative DNA detection methods are needed to determine whether serologically positive animals are infectious and to examine the role of vectors (e.g. haematophagous insects) in the transmission of the parasite. To this end, we established two different 5'-nuclease quantitative assays to detect B. besnoiti infection in cattle and to estimate the parasite load in samples (BbRT1 and BbRT2). These PCRs are based on the sequence of the internal transcribed spacer region 1 (ITS-1) of the ribosomal RNA gene. Tests with serial dilutions of B. besnoiti genomic DNA in a buffer containing 100 ng/μl bovine DNA revealed a detection limit of 0.01 pg genomic B. besnoiti DNA. Reliable quantification was possible in samples containing ≥1 pg B. besnoiti genomic DNA with a coefficient of variation of ≤ 2%. To estimate the diagnostic sensitivity of the tests, skin biopsies and scrapings from the mucous membrane of the vestibulum vaginae (vaginal scrapings) were taken from cattle with clinical signs of chronic besnoitiosis. Regardless of the real time PCR assay used, 90.7% (39/43) of these animals were positive in at least one of two samples (skin or vaginal scrapings). Antibody titers, as determined by an immunofluorescent antibody test, and the threshold cycle values of the real time PCR obtained for skin samples and vaginal scrapings, were significantly correlated. The specificity of the PCRs was confirmed using genomic DNA from related parasites, including genomic DNA of Besnoitia spp., Neospora caninum, Toxoplasma gondii, Hammondia hammondi, Hammondia heydorni, Isospora spp., Sarcocystis spp., Eimeria bovis, Cryptosporidium parvum, and Trypanosoma brucei brucei. Since the sequence of the ITS-1 region of B. besnoiti is identical with that of Besnoitia species isolated from donkeys (Besnoitia bennetti), and reindeer (Besnoitia tarandi), both real time PCRs detected also DNA of these parasites. One of the B. besnoiti real time PCRs, BbRT1, but not BbRT2, cross-reacted with Besnoitia darlingi, Besnoitia oryctofelisi, and Besnoitia neotomofelis when large amounts of genomic DNA (10 ng) were used. The other B. besnoiti real time PCR assay (BbRT2) was specific for B. besnoiti, B. bennetti and B. tarandi, but did not react when 10 ng DNA of other related parasite species from the genus Besnoitia or other genera were subjected to analysis.     

Characterization and expression of the bovine growth hormone-releasing hormone (GHRH) receptor

The hypothalamic hormone, growth hormone-releasing hormone (GHRH) and its pituitary receptor are principal regulators of pituitary growth hormone (GH) synthesis and release. In the present study, we cloned and sequenced a complete bovine pituitary GHRH receptor cDNA in order to study its expression in cattle. The lengths of the exons in the bovine GHRH receptor gene were determined by comparison of the cloned cDNA with genomic sequences obtained from a bovine genomic library clone. As in other species, the bovine cDNA sequence encodes a 423-amino acid protein containing seven hydrophobic domains characteristic of a G protein-coupled receptor. The predicted bovine amino acid sequence shares 93, 90, 89, 87, and 85% identity with the ovine, porcine, human, rat and mouse sequences, respectively. Expression of the receptor in bovine ileum, ovary, anterior pituitary, testis, hypothalamus, pancreas and liver was examined by RT-PCR. Of those tissues examined, GHRH receptor expression was detected in the anterior pituitary gland and hypothalamus. To gain a better understanding of GHRH receptor gene regulation in ruminants, we examined the effect of bovine somatotropin (bST) treatment on pituitary GHRH receptor expression in dairy heifers using relative and real-time RT-PCR. In the present study, bST treatment of dairy heifers resulted in no significant decline in pituitary GHRH receptor expression.     

Characterization of the bovine immunoglobulin lambda light chain constant IGLC genes

To characterize the bovine immunoglobulin lambda light chain constant region (IGLC) genes, we have isolated a bacterial artificial chromosome (BAC) clone by a PCR based approach from a bovine genomic DNA library, constructed using a genital ridge cell line derived from a male Holstein fetus. The positive BAC clone, containing the bovine IGLC genes, was fully sequenced and had a 138 kb insert. Sequence analysis revealed that the bovine immunoglobulin lambda light chain locus consisted of four joining-constant gene recombination units spanning approximately 20 kb DNA in length. A detailed examination of the recombination signal sequences, RNA splicing sites and coding sequences of the four joining-constant gene recombination units suggested that only two IGLC genes (IGLC2 and IGLC3) were functional while the IGLC1 and IGLC4 appeared to be pseudogenes. This conclusion was further confirmed by a series of RT-PCR amplifications, which also showed that among these four genes the IGLC3 was preferentially expressed in cattle. Phylogenetic analysis indicated that the bovine IGLC genes were more closely related to their equivalents in sheep and goats than that to other mammals.