青海高原牦牛mtDNA Cytb基因和D-loop区遗传多样性及系统进化分析

为探究青海高原牦牛的遗传多样性和起源进化关系,本研究通过测定和分析青海高原牦牛155个个体细胞色素b基因(Cytb)和D-loop区全序列,分析多态性及构建系统进化树.结果表明:青海高原牦牛Cytb基因全序列长度为1 140 bp,个体间序列长度无差异,4种核苷酸T、A、G、C的含量分别为26.26％、31.73％、13.09％、28.920％;发现13个SNP位点,全部为转换,符合Cytb基因保守的特征;核苷酸多样性(Pi)为0.002 88,单倍型数(H)为9,单倍型多样性(Hd)为0.645;D-loop区序列长度在892～895 bp之间,不同个体间存在序列长度差异;4种核苷酸T、A、G、C的平均比例分别为28.69％、32.22％、13.75％、25.34％,A+T含量为60.91％,G+C含量为39.09％.在155个个体中共统计出41个SNP位点,其中转换38个,颠换3个,缺失5个,插入3个.其核苷酸多样性(Pi)为0.012 44,分析得到的单倍型数(H)为38,单倍型多样性(Hd)为0.881.在基于Cytb基因的系统进化树中,青海高原牦牛首先与野牦牛和巴州牦牛聚在一起,紧接着与美洲野牛聚在一起;在D-loop区的系统进化分析中,青海高原牦牛首先与西藏牦牛和野牦牛聚在一起,展示出丰富的遗传多样性,同时进一步支持了牦牛和野牦牛划为牛亚科牦牛属(Bovidae)的观点.本研究结果为牦牛改良及育种工作提供了科学依据.     

日本条螽线粒体基因组序列的测定与分析

日本条螽完整的线粒体基因组序列长16 281 bp,包括13个蛋白质编码基因、22个tRNA基因、2个r RNA基因和1个D-loop区,其基因次序和方向与祖先序列相同。该线粒体基因组排列紧凑,但在ND2和tRNA~(Trp)之间有一段长为650 bp的基因间隔区。为研究螽斯科的系统发育关系,本研究选取日本条螽及其它17个螽斯科物种线粒体基因组的蛋白质编码基因和r RNA基因序列构建贝叶斯系统发生树。     

新西兰白兔线粒体基因组全序列的测定与分析

本研究旨在获得新西兰白兔(New Zealand white rabbit)线粒体DNA基因组全序列(mtDNA).根据GenBank已经公布的近缘物种穴兔mtDNA全基因组序列(GenBank登录号:AJ001588.1),设计12对可覆盖新西兰白兔mtDNA全序列的引物,通过PCR扩增、测序、拼接,获得新西兰白兔线粒体全序列,并分析其特点.新西兰白兔线粒体基因组全序列为17 418 bp,A+T含量高,为59.72％,蛋白编码基因数量为13个,rRNA基因数量为2个,tRNA基因数量为22个和1个非编码控制区(D-loop区),与其他兔属动物线粒体全基因组排列顺序一致.分析4种特殊的tRNA二级结构,发现tRNA-Ser(AGY)为二叶草型,缺失DHU臂,其余三种tRNA均为三叶草型.与其他哺乳动物线粒体基因组的D-loop区相比,新西兰白兔与格拉达野兔(Le-pus granatensis)核苷酸组成、编码偏好性和氨基酸组成较为相近.相比穴兔的线粒体基因组,新西兰白兔具有一定的保守性和异质性,该结果为其遗传种质资源保护和利用提供基础资料.     

西藏金丝野牦牛的遗传分类地位初步分析

在西藏羌塘国家级自然保护区发现有种群数量约200头的全身被毛为金色的野牦牛。为探究该种金丝野牦牛与普通野牦牛(Bos grunniens mutus)和家牦牛(Bos grunniens)的遗传差异,本研究通过分析金丝野牦牛线粒体DNA中的细胞色素b(Cyt b)和D-loop区基因序列差异,初步探讨了金丝野牦牛遗传分类地位。结果表明:金丝野牦牛的这两个基因片段的序列结构特征、长度、核苷酸组成与其他牦牛相似;金丝野牦牛与普通野牦牛的遗传距离最小,在系统树中,它们聚集为一支,说明金丝野牦牛与普通野牦牛亲缘关系最近;但金丝野牦牛与普通野牦牛的遗传距离较普通野牦牛个体间平均遗传距离大,加上毛色等形态区别,我们认为金丝野牦牛为野牦牛的一个亚种或者重要保护单元。但由于样本数量有限,仍需进一步深入研究确定。     

北京鸭线粒体基因组全序列测定和分析

线粒体DNA作为遗传标记,已在家鸡(Gallus gallus)和家鹅(Anser anser)的研究中取得了重大进展,而对家鸭(Anas platyrhychos domesticus)的研究却很少.本研究参照近源物种线粒体基因组序列设计15对引物,通过PCR扩增、测序、拼接,获得北京鸭(A.platyrhychos)线粒体基因组全序列,初步分析其特点和各基因的定位.结果显示,北京鸭线粒体基因组全长16 604 bp,碱基组成为29.19%A、22.20%T、15.80%G、32.81%C,包含13个蛋白质编码基因、2个rRNA基因、22个tRNA基因和1个非编码控制区(D-loop),基因组成及排列顺序与其他鸟类相似.基于线粒体D-loop区全序列,用N-J法构建了7种雁形目鸟类系统进化树,结果表明,北京鸭与绿头鸭(A.platyrhychos)系统进化关系较近.     

藏鸡线粒体全基因组序列的测定和分析

通过PCR扩增,测序,拼接,获得藏鸡（Tibetan Chicken）线粒体全基因组序列并进行数据分析处理。藏鸡线粒体全基因组序列全长16783bp,共有13个蛋白质编码基因、2个rRNA基因、22个tRNA基因和1个D-loop区。模拟电子酶切结果显示,藏鸡DraI酶的酶切结果和先前报道的原鸡,茶花鸡,尼西鸡和大理漾濞黄鸡的酶切结果都不相同,为藏鸡特有。基于D-loop区全序列和13个蛋白质编码基因序列,采用N-J算法与原鸡属4个种,3个亚种和3个家鸡品系构建系统进化树：初步确定藏鸡起源于红原鸡,与家鸡中的来航鸡、白洛克鸡亲缘关系最近,但是藏鸡的进化与来航鸡、白洛克鸡这两个家鸡品系又显得相对独立。推测可能原因是藏鸡的祖先在进入高原以后处于相对封闭的环境,从而形成了独特群体遗传特性。     

齿缘摄龟线粒体基因组全序列与系统进化

采用常规PCR扩增并测序获得了齿缘摄龟(Cyclemys dentata)线粒体DNA(mtDNA)全序列,并研究了其基因组结构特点;根据20种龟的线粒体基因组重链蛋白编码基因序列,分别利用最大简约法(MP)、最大似然法(ML)和贝叶斯法(Bayesian)构建系统进化树,探讨这些龟鳖物种之间的系统进化关系。结果显示,齿缘摄龟线粒体基因组全序列长为16489 bp(GenBank登录号为JX455823),A+T含量为61.51%,编码37个基因,包括13个蛋白质编码基因,2个rRNA,22个tRNA和1个控制区(Dloop),基因组成与其他龟鳖类动物相似;非编码区D-loop长973 bp,包含1个中央保守区(CD),2个扩展终止结合序列区(ETASs),3个保守盒(CSBs);构建的MP树、ML树和Bayesian树的拓扑结构相似,闭壳龟属7种龟聚为一枝,拟水龟属6种龟聚为一枝,齿缘摄龟与黑桥摄龟聚在一枝,3种进化树均支持这些龟鳖物种现有的分类学地位。     

赤麂线粒体全基因组的序列和结构

提取赤麂细胞株总DNA,参照我们实验室已测定的同属动物小麂线粒体全基因组序列设计引物,PCR扩增、测序、拼接,获得赤麂线粒体全基因组序列并进行生物信息学分析。赤麂线粒体全基因组序列全长16354bp。定位了22个tRNA基因、2个rRNA基因、13个蛋白编码基因和1个D-loop区。赤麂与小麂及其它哺乳动物线粒体的基因组结构相同,它们的序列同源性都较高。     

麦穗鱼线粒体基因组序列测定及分析

利用麦穗鱼Pseudorasbora parva和相关鱼类的部分线粒体基因序列,设计出2对长批引物和30对短批引物,采用基于长PCR的2次PCR扩增法测定并注释麦穗鱼线粒体基因组全序列。结果表明,麦穗鱼线粒体基因组长16600bp,A+T含量为58.9%,37个基因位置及组成与其它硬骨鱼一致,均由13个蛋白编码基因、22个tRNA、2个rRNA基因和1个控制区(D-loop)组成。其中L链仅含8个tRNA(Pro、T yr、Ser、Ala、Asn、Cys、Glu、Gln)及ND6基因,其余基因皆由H链编码。基因排列紧密,间隔序列共计13处64bp,长度从1～32bp不等;基因重叠区7处23bp,重叠碱基数在1～7bp之间。13个蛋白编码基因中,除COI起始密码子为GTG外,其余均以ATG为起始密码子;有8个基因(ND1、ND2、COI、ATP6、ATP8、ND4L、ND5、ND6)3’端有完全的TAA或TAG终止密码子,其它5个基因终止密码子为不完整的TA(ND3和ND4)或T(COⅡ,COⅢ,Cyt b)。除tRNASer(AGY)外,其余21个tRNA基因的二级结构均为典型的三叶草结构。预测的lrRNA二级结构共有6个结构域,53个茎环结构,srRNA二级结构包含43个茎环结构。控制区(D-loop)存在3个结构区:终止序列区(TAS)、中央保守区(CSB-F、CSB-D)和保守序列区(CSB-1、CSB-2、CSB-3),其中TAS与DNA复制终止相关,出现茎环结构。     

SEQUENCE AND ANALYSIS OF COMPLETE MITOCHONDRIAL GENOME OF PSEUDORASBORA PARVA

利用麦穗鱼Pseudorasbora parva和相关鱼类的部分线粒体基因序列,设计出2对长批引物和30对短批引物,采用基于长PCR的2次PCR扩增法测定并注释麦穗鱼线粒体基因组全序列.结果表明,麦穗鱼线粒体基因组长16600 bp,A+T含量为58.9％,37个基因位置及组成与其它硬骨鱼一致,均由13个蛋白编码基因、22个tRNA、2个rRNA基因和1个控制区(D-loop)组成.其中L链仅含8个tRNA(Pro、Tyr、Ser、Ala、Asn、Cys、Glu、Gln)及ND6基因,其余基因皆由H链编码.基因排列紧密,间隔序列共计13处64 bp,长度从1～32 bp不等;基因重叠区7处23 bp,重叠碱基数在1～7bp之间.13个蛋白编码基因中,除COI起始密码子为GTG外,其余均以ATG为起始密码子;有8个基因(ND1、ND2、COI、ATP6、ATP8、ND4L、ND5、ND6)3端有完全的TAA或TAG终止密码子,其它5个基因终止密码子为不完整的TA (ND3和ND4)或T(COⅡ,COⅢ,Cyt b).除tRNAser(AGY)外,其余21个tRNA基因的二级结构均为典型的三叶草结构.预测的lrRNA二级结构共有6个结构域,53个茎环结构,srRNA二级结构包含43个茎环结构.控制区(D-loop)存在3个结构区:终止序列区(TAS)、中央保守区( CSB-F、CSB-D)和保守序列区(CSB-1、CSB-2、CSB-3),其中TAS与DNA复制终止相关,出现茎环结构.     

Complete sequence of the yak (Bos grunniens) mitochondrial genome and its evolutionary relationship with other ruminants

The yak (Bos grunniens) is the most important domesticated species in the Qinhai-Tibetan Plateau. In present study, the complete sequence of the yak mitochondrial genome was determined. Sequence analysis revealed that there are no differences with cattle in the yak mitochondrial genome organization. Interestingly, within the D-loop, the conserved sequence blocks are less conserved than surrounding regions. Neighbor-Joining (NJ) trees based on single genes, gene sets and concatenated genes of mitochondrial genome were constructed. The analysis identified the yak as a sister group of a cattle/zebu clade. Based on substitutions in 22 tRNA genes, 12S rRNA gene and 16S rRNA gene, the dating of divergence between yak and cattle/zebu, and yak and water buffalo, was proposed to have occurred 4.38-5.32 and 10.54-13.85 million years before present, respectively. This is consistent with the paleontologyical data. Yak and sheep/goat divergent dating predicts that their divergence occurred at 13.14-27.99 million years before the present day.     

青海省可可西里地区几种有蹄类动物的食物重叠初步分析

2005年7月和2006年1月,应用粪便显微组织分析法测定了可可西里地区的藏野驴、藏羚、藏原羚、野牦牛,以及家牦牛和藏羊在冷季(1月)和暖季(7月)的食物构成.用Schoener's Index计算了这些同域分布动物种间的食物重叠度.结果表明,藏野驴分别与藏羚羊、野牦牛、藏原羚在暖季的食物重叠度各为63.0%、48.4%和24.1%,在冷季的重叠度为别为71.6%、42.0%和11.4%;藏羚羊与野牦牛、藏原羚在暖季的食物重叠度分别为52.0%和33.4%,其在冷季的食物重叠度各为50.3%和29.3%;野牦牛与藏原羚在冷暖季的食物重叠度分别为13.1%和15.9%.在可可西里地区吲域分布野牛有蹄类动物种间,藏原羚与其他有蹄类动物食物重叠较少,而藏羚羊、藏野驴、野牦牛之间则存在不同程度的食物重叠,且随季节不同而变化,反映了这些动物之间复杂的竞争和共存关系.此外,家牦牛和藏羊均与这些野生有蹄类动物存在高度的食物重叠.     

牦牛的分类学地位及起源研究:mtDNA D-loop序列的分析

牦牛的起源与属级分类学地位至今仍然存在一定的争议。我们测定了家养牦牛和野生牦牛线粒体控制区(D-loop)序列,并以此构建牦牛和牛属、野牛属、水牛属以及非洲水牛属相关种的系统发育树。研究结果表明线粒体D-loop区与Cytb基因序列在构建牛族的系统发育具有同样重要的价值。系统发育关系显示野牛属的灭绝种草原野牛与现存种美洲野牛先聚合为一单系群,然后再和牦牛形成一单系分支,表明牦牛与野牛属的草原野牛、美洲野牛亲缘关系最近,具有最近的共同祖先,而与牛属的其它亚洲物种亲缘关系较远。因此,本研究不支持将牦牛独立为牦牛属———Poephagus,牛属与野牛属在分类上也应合并为一个属。基于上述研究结果和化石证据,我们进一步对牦牛起源的历史背景进行了讨论,认为牦牛与野牛属的分化是由于第四纪气候变化在欧亚大陆发生的,野牛通过白令陆桥进入北美;冰期结束后,由于欧亚大陆其它地区温度升高,牦牛只能局限分布在较为寒冷的青藏高原;而野牛属在北美先后分化为草原野牛和美洲野牛,前者可能是后者的直接祖先。     

Mitochondrial DNA sequence diversity and origin of Chinese domestic yak

In order to clarify the origin and genetic diversity of yak in China, we analysed mitochondrial DNA (mtDNA) control region sequences (approximately 891 bp) in 52 individuals from four domestic yak (Poephagus grunniens) breeds, as well as from a hybrid between yak and cattle (Pianniu). Twenty-five samples were further selected for partial (420 bp) cytochrome b sequencing based on control region sequence information. Two yak samples shared sequences with Chinese cattle (Bos taurus); the remaining yak mtDNAs converged into two major clades in the phylogenetic analysis. Genetic diversity varied substantially among the breeds, with the hybrid Pianniu yak demonstrating the highest diversity. Our results suggest that the Chinese yak was domesticated from two distinct matrilineal sources or from a heterogeneous pool containing both divergent lineages, with occasional gene introgression from cattle.     

Phylogeny of bovine species based on AFLP fingerprinting

The Bovini species comprise both domestic and wild cattle species. Published phylogenies of this tribe based on mitochondrial DNA contain anomalies, while nuclear sequences show only low variation. We have used amplified fragment length polymorphism (AFLP) fingerprinting in order to detect variation in loci distributed over the nuclear genome. Computer-assisted scoring of electrophoretic fingerprinting patterns yielded 361 markers, which provided sufficient redundancy to suppress stochastic effects of intraspecies polymorphisms and length homoplasies (comigration of non-homologous fragments). Tree reconstructions reveal three clusters: African buffalo with water buffalo, ox with zebu, and bison with wisent. Similarity values suggest a clustering of gaur and banteng, but bifurcating clustering algorithms did not assign consistent positions to these species and yak. We propose that because of shared polymorphisms and reticulations, tree topologies are only partially adequate to represent the phylogeny of the Bovini. Principal-coordinate analysis positions zebu between a gaur/banteng cluster and taurine cattle. This correlates with the region of origin of these species and suggests that genomic distances between the cattle species have been influenced by genetic exchange between neighbouring ancestral populations.     

Species authentication of commercial beef jerky based on PCR-RFLP analysis of the mitochondrial 12S rRNA gene

In this study,we determined species-specific variations by analyzing the mitochondrial 12S rRNA gene sequence variation(～440 bp) in 17 newly obtained sequences and 90 published cattle,yak,buffalo,goat,and pig sequences,which represent 62 breeds and 17 geographic regions.Based on the defined species-specific variations,two endonucleases,Alu I and Bfa I,were selected for species authentication using raw meat/tissue samples and the PCR-RFLP method.Goat and pig were identified using the Alu I enzyme,while cattle,yak,and buffalo were identified by digestion with Bfa I.Our approach had relatively high detection sensitivity of cattle DNA in mixed cattle and yak products,with the lowest detectable threshold equaling 20% of cattle DNA in a mixed cattle/yak sample.This method was successfully used to type commercial beef jerky products,which were produced by different companies utilizing various processing technologies.Our results show that several yak jerky products might be implicated in commercial fraud by using cattle meat instead of yak meat.     

Interferon-alpha genes from Bos and Bubalus bubalus

Interferon-a genes were cloned from six breeds of three species of two genera (three Chinese native cattle breeds of yellow cattle, wild yak and HuanHu domestic yak, one European breed of Holstein cow, and two water buffalo breeds of FuAn water buffalo and FuZhong water buffalo) by direct PCR. The PCR products were directly inserted into the expression vector to be sequenced and expressed. Sequence analysis showed that IFN-a genes of six clones were composed of 498 nucleotides, encoding a mature polypeptide with 166 amino acids. Compared with the published BoIFN-a subtypes, the IFN-a gene of Holstein cow had only one point mutation with the BoIFN-aA subtype. The IFN-a gene of yellow cattle was similar to the BoIFN-aD subtype with amino acid identity of 97.0% and may be considered as a new subtype, namely, BoIFN-aD1. The other four IFN-a genes, cloned from wild yak and HuanHu domestic yak, FuAn water buffalo, and FuZhong water buffalo, represented four new subtypes, namely, BoIFN-aI, BoIFN-aJ, BuIFN-a1, and BuIFN-a2, respectively. Each of the six clones was expressed in E. coli with molecular weight of approximately 20 kDa by SDS-PAGE and Western blot analyses. Antiviral activity assays showed that the six recombinant IFN-a (rIFN-a) all exhibited 1,000 times higher antiviral activity in the MDBK/VSV cell line than in the CEF/VSV one. Moreover, the rIFN-as could inhibit infectious bovine rhinotracheitis virus replication in the MDBK cell line using CPE inhibition method. The results suggested that rIFN-as a potential agent for clinical application against virus diseases in cattle industry.     

牦牛分类地位研究概述

牦牛的分类地位一直存在着争议,牦牛究竟是属于牛亚科牛属还是属于牛亚科牦牛属,到目前为止还没有形成一个明确的定论.本文通过对牦牛与牛亚科其他属在古生物学证据、形态学特征、血液蛋白多态性、微卫星多态性、mtDNA序列变异、rDNA的RFLP数据和功能基因序列信息等各方面研究资料的比较分析,发现牦牛无论在古生物学证据、形态学特征,还是在分子生物学特征上均表现出与牛属中的普通牛Bos taurus、瘤牛Bos indicus不同,而与美洲野牛Bison bison的亲缘关系更近一些,因此将牦牛划分为牛亚科中1个独立属(即牦牛属),似乎比将牦牛作为牛属中的1个亚属或1个种更合适.     

Origin of mitochondrial DNA diversity of domestic yaks

Background  

The domestication of plants and animals was extremely important anthropologically. Previous studies have revealed a general tendency for populations of livestock species to include deeply divergent maternal lineages, indicating that they were domesticated in multiple, independent events from genetically discrete wild populations. However, in water buffalo, there are suggestions that a similar deep maternal bifurcation may have originated from a single population. These hypotheses have rarely been rigorously tested because of a lack of sufficient wild samples. To investigate the origin of the domestic yak (Poephagus grunnies), we analyzed 637 bp of maternal inherited mtDNA from 13 wild yaks (including eight wild yaks from a small population in west Qinghai) and 250 domesticated yaks from major herding regions.     

Interferon-α Genes from Bos and Bubalus bubalus

Interferon-α genes were cloned from six breeds of three species of two genera (three Chinese native cattle breeds of yellow cattle, wild yak and HuanHu domestic yak, one European breed of Holstein cow, and two water buffalo breeds of FuAn water buffalo and FuZhong water buffalo) by direct PCR. The PCR products were directly inserted into the expression vector to be sequenced and expressed. Sequence analysis showed that IFN-α genes of six clones were composed of 498 nucleotides, encoding a mature polypeptide with 166 amino acids. Compared with the published BoIFN-α subtypes, the IFN-α gene of Holstein cow had only one point mutation with the BoIFN-αA subtype. The IFN-α gene of yellow cattle was similar to the BoIFN-αD subtype with amino acid identity of 97.0% and may be considered as a new subtype, namely, BoIFN-αD1. The other four IFN-α genes, cloned from wild yak and HuanHu domestic yak, FuAn water buffalo, and FuZhong water buffalo, represented four new subtypes, namely, BoIFN-αI, BoIFN-αJ, BuIFN-α1, and BuIFN-α2, respectively. Each of the six clones was expressed in E. coli with molecular weight of ～ 20kDa by SDS-PAGE and Western blot analyses. Antiviral activity assays showed that the six recombinant IFN-α (rIFN-α) all exhibited 1000 times higher antiviral activity in the MDBK/VSV cell line than in the CEF/VSV one. Moreover, the rIFN-αs could inhibit infectious bovine rhinotracheitis virus replication in the MDBK cell line using CPE inhibition method. The results suggested that rIFN-αs a potential agent for clinical application against virus diseases in cattle industry.