DNA条形码技术在中缅树鼩隆安种群和昆明种群鉴定中的研究

目的:本研究利用线粒体细胞色素C氧化酶亚基I基因(CO1)的一段保守区域作为DNA条形码技术的研究序列,探讨DNA条形码技术对中缅树鼩隆安种群和昆明种群进行分类鉴定的可行性。方法:对22只广西隆安树鼩和21只昆明树鼩样本的CO1基因进行PCR扩增、测序,应用MEGA V5软件对序列进行比对及分析其遗传距离,采用NJ法构建系统发育树。结果:中缅树鼩种群中,隆安种群、昆明种群和海南亚种的种内遗传距离为0.00%-0.79%,种群间遗传距离为9.71%-13.59%,中缅树鼩与普通树鼩的种间遗传距离为20.43%-24.11%,存在条形码间隔。系统发育树显示:隆安种群、昆明种群及海南亚种分别聚为一小支,分支置信度高达100%。结论:本研究结果表明DNA条形码技术有助于树鼩种群和亚种的分类鉴定,经CO1基因的测序分析证实广西隆安树鼩和昆明树鼩分属不同的种群。     

云南不同地区中缅树鼩头骨形态特征的比较

中缅树鼩为东洋界热带亚热带特有类群.本研究采用几何形态测量法对分布于滇中高原(禄劝县)和横断山地区(剑川县、丽江市和云龙县)的中缅树鼩头骨侧面、腹面、背面及下颌侧面的形态进行了初步研究.结果表明,主成分分析和判别函数分析显示头骨侧面和下颌侧面更适宜于区分滇中高原和横断山地区的中缅树鼩;经过薄片样条法分析显示形变多集中在鼻骨和臼齿,这可能与中缅树鼩生存的气候和地理环境相适应;经多维尺度分析显示滇中高原和横断山地区的中缅树鼩头骨有明显差异,这可能与中缅树鼩生活环境的经度和纬度有关.因此,滇中高原和横断山地区的中缅树鼩从头骨形态上可以区分开来,并且差异仅仅发生在种群水平,这可能反映了其对特定生态环境的形态适应.     

云南中缅树鼩线粒体细胞色素b和D-loop区遗传多样性研究

对3个地方种群的49只样本的线粒体Cyt b基因全序列（1140 bp）及33只样本的控制区D-loop基因区段（745 bp）进行了序列测定。结果表明：Cyt b基因多态性位点有47个,其中单变异位点位点23个,简约信息位点24个。共定义了24个单倍型,其中种群间的共享单倍型有2个（8.33%）,其余均为某个种群所特有,单倍型多样度范围为0.80952（剑川种群）~0.91532（禄劝种群）,核苷酸多样度指数介于0.00326（禄劝种群）~0.00635（剑川种群）之间;D-loop基因多态性位点有18个,其中单变异位点8个,简约信息位点10个。共定义了16个单倍型,无种群间的共享单倍型,单倍型多样度范围为0.76615（禄劝种群）~0.93333（丽江种群）,核苷酸多样度指数介于0.00269（禄劝种群）~0.00583（丽江种群）之间。从各单倍型的TCS网络进化图显示横断山种群位于分支的末端,表现出中缅树鼩由南向北的扩散模式,支持＂岛屿起源＂假说。     

云南昆明和禄劝中缅树鼩体温调节和产热特征

为探讨不同地区中缅树鼩Tupaia belangeri的生理生态适应特征,对其体温调节和产热特征进行了测定,代谢率采用开放式呼吸仪进行测定。结果显示:A组中缅树鼩(禄劝县屏山镇)的体温(T b)与环境温度(T a)的关系为T b=38.0+0.07T a;B组中缅树鼩(昆明团结乡)的体温与环境温度的关系为T b=38.3+0.05T a;热中性区分别为30³5℃和27.535℃和27.5³5℃;基础代谢率分别为(1.40±0.03)mL/(g·h)和(1.66±0.06)mL/(g·h);平均最小热传导为(0.14±0.0034)mL/(g·h·℃)和(0.15±0.0041)mL/(g·h·℃);热中性区内F值,即(RMR/Kleiber期望RMR)/(C/Bradley期望C),分别为0.91±0.01和1.14±0.03。结果表明,昆明中缅树鼩较禄劝中缅树鼩有较高的基础代谢率和较宽的热中性区,并且有较好的调节体温的能力;它们的这种产热特征和体温调节方式的不同可能与它们的生活史和栖息地环境有关。     

中缅树鼩消化道长度和重量变化

为探讨栖息于昆明禄劝地区中缅树鼩(Tupaia belangeri)的消化道特征与环境之间的适应关系,在2008年6月和12月(夏季和冬季)分别对自然环境中缅树鼩的胃、小肠、大肠、盲肠的长度、含内容物重、去内容物重、干组织重等消化道指标进行了测定。结果表明,中缅树鼩消化道特征冬季和夏季存在变化,随着温度降低、食物质量下降,中缅树鼩的小肠长度和重量增加;各器官重量均在冬季最大;中缅树鼩在受到低温、食物质量下降等因子胁迫下,通过调节消化道长度和重量来满足能量需求的增加,维持正常的生理机能。中缅树鼩的消化道在冬季和夏季中表现出的变化模式及消化对策对其在自然环境中的生存是至关重要的。     

中缅树鼩微卫星分子标记的筛选

目的 筛选中缅树鼩微卫星分子标记,逐步填补中缅树鼩特异性遗传标记的空白.方法 建立中缅树鼩基因小片段插入文库,利用5’端地高辛标记的(CA)15探针从约1500个菌落中选出36个阳性克隆.对这些克隆进行测序,发现其中15个含有重复序列,其中1个为重复克隆,1个因两端序列太短而不能设计引物.结果 用Primer3软件设计...     

大麝鼩韩国种群与俄罗斯远东种群之间的遗传分化:基于线粒体Cyt b 基因的分析

为重新分析韩国大麝鼩种群与相邻的俄罗斯远东种群之间的遗传分化情况,我们获得9 条来自韩国4 个地点和俄罗斯3 个地点的大麝鼩线粒体细胞色素b 基因(Cyt b)全序列,并将其与来自GenBank 的4 条Cyt b 全序列和4 条Cyt b 部分序列进行比较。结果发现韩国的大麝鼩并非只有一种基因型,因此,在利用Cyt b 部分序列进行种群遗传学分析时需格外注意。基于Cyt b 全序列分析,发现韩国的大麝鼩与俄罗斯远东地区的大麝鼩之间存在1.08% 的平均JC 距离和7 个位点的差异,推测韩国的大麝鼩与俄罗斯远东地区的大麝鼩在包括末次冰盛期在内的很长时期都没有进行过遗传交流。目前的测序结果不支持当前认为大麝鼩为单系群的亚种分类理论,支持韩国大麝鼩是C. l. thomasi 亚种的分类理论,但还需进一步对中国东北地区的样品进行测序分析后才能最终确认。     

雀形目15种鸟类Co Ⅰ与Cyt b基因序列的比较

对雀形目Passeriformes 6科15种鸟类线粒体DNA的Cyt b基因全序列(1 143 bp)和CoⅠ基因部分序列(1 176 bp)进行了比较,结果显示Cyt b和CoⅠ基因序列的变异位点分别为454个和366个,简约信息位点为337个和303个,而且CoⅠ基因比Cyt b基因略微保守,进化速率也较低.采用邻接法、最大简约法、最大似然法和贝叶斯法分别构建了CoⅠ和Cyt b基因两组数据集的分子系统发生树及其合一树,并对建树结果进行了比较分析.基于以上两点,本文认为CoⅠ基因比Cyt b基因更适合于确定雀形目科级阶元之间的系统发生关系,而且它也能够作为雀形目物种鉴定的分子标记,但在物种鉴定方面不如Cyt b基因稳定和准确.     

PRDM16和BMP7基因表达量在中缅树鼩体重调节中的作用

为探讨季节性环境变化下中缅树鼩PRDM16(PR domain-containing 16)和BMP7(bone morphogenetic proteins 7)基因表达量对其生理适应性调节的作用,本研究测定了野外不同季节和实验室冷驯化条件下中缅树鼩的体重、静止代谢率(resting metabolic rate,RMR)、非颤抖性产热(nonshivering thermogenesis,NST)、摄食量、PRDM16和BMP7基因表达量的变化。结果表明:季节性变化过程中中缅树鼩的体重、RMR、NST、褐色脂肪组织(brown adipose tissue,BAT)重、白色脂肪组织(white adipose tissue,WAT)重和摄食量均是冬季显著高于夏季;中缅树鼩不同季节的PRDM16和BMP7基因表达量差异极显著,PRDM16表达量的季节变化趋势为:冬季秋季春季夏季;BMP7表达量的季节变化趋势为:冬季秋季夏季春季。冷驯化条件下,中缅树鼩的体重、RMR、NST、摄食量、BAT含量、大网膜WAT含量显著增加,PRDM16和BMP7基因表达量也显著增加。以上结果表明,中缅树鼩褐色脂肪细胞存在PRDM16和BMP7肌源性起源,即冬季或者低温条件下中缅树鼩PRDM16和BMP7表达量上调,促进褐色脂肪细胞形成,增加NST来弥补产热的不足,以适应冬季寒冷的环境。PRDM16和BMP7在中缅树鼩季节性产热调节和能量代谢中起着重要的作用。     

中缅树鼩头骨及下臼齿几何形态与环境的关系

共选取分布于云南、贵州和广西地区的190个中缅树鼩头骨和32颗臼齿(第二、第三下臼齿),运用几何形态测量法对其头骨侧面、腹面、背面及下颌侧面的形态特征进行主成分分析、薄片样条分析,同时运用统计软件SPSS15.0对头骨和臼齿进行多维尺度分析,以探讨头骨及臼齿形态与环境之间的关系.结果表明,头骨侧面在研究多种群头骨形态时更适宜,更有参考价值;经过薄片样条法分析显示形变多集中在鼻骨和臼齿,这可能与中缅树鼩生存的气候和地理环境相适应;经多维尺度分析显示云南、贵州和广西地区的中缅树鼩头骨具有明显的地区差异,同时,对臼齿的分析显示,臼齿形态具有地区差异,这种变化可能与中缅树鼩生活环境的经度和纬度有关.综上,云南、贵州和广西地区的中缅树鼩的头骨具有形态变异,并且差异仅仅发生在种群水平,这可能反映了其对特定生态环境的形态适应.     

昆明城郊中国树鼩群体线粒体DNA遗传多样性

由于树鼩是灵长类动物的近亲,且具有体型小、繁殖周期短、饲养管理成本低等优点,长期以来被认为有望替代灵长类动物用于人类疾病的动物模型研究.然而,目前对树鼩的群体遗传结构还知之甚少,这极大地限制了其在疾病动物模型研究的应用,也是其品系资源创制的瓶颈.本研究通过分析80只采自于云南省昆明周边地区的野生树鼩(Tupaia belangeri chinensis)线粒体DNA(mtDNA)多态性,结合国外报道的2个树鼩(Tupaia belangeri)序列比较后发现,在604 bp的mtDNA控制区片段中兵检测到29个核苷酸替代变异,这些变异共界定了13种单倍型,表现较高的群体遗传多样度.另外,昆明地区的树鼩与国外报道的2个树鼩间存在较大的遗传分化,mtDNA控制区单倍型之间的核苷酸替换数大于18个,远高于昆明地区树鼢群体内部不同单倍型之间的差异.选择含有代表性的mtDNA控制区单倍型的17个昆明地区树鼩个体进一步测定了细胞色素b基因片段(1134 bp),结合前人报道的数据分析,结果进一步支持mtDNA控制区数据反映的遗传格局及揭示的昆明地区树询与国外报道树鼩之间的明显差异.本研究结果提示,昆明地区树鼩与国外树鼩之间存在较大遗传差异,在将树鼩用于人类疾病动物模型研究中要注意这些遗传差别.昆明城郊的树鼩群体具有较高的遗传多样度,在开展近交系建立等工作时须考虑选取群体内部具有代表性的mtDNA世系.

Abstract：

Due to their special phylogenetic position in the Euarchontoglires and close affinity to primates, tree shrews have been proposed as an alternative experimental animal to primates in biomedical research. However, the population genetic structure of tree shrews has largely remained unknown and this has hindered the development of tree shrew breeding and selection. Here we sampled 80 Chinese tree shrews (Tupaia belangeri chinensis) in Kunming, China, and analyzed partial mtDNA control region sequence variation. Based on our samples and two published sequences from northern tree shrews (T. belangeri), we identified 29 substitutions in the mtDNA control region fragment (～ 604 bp)across 82 individuals and defined 13 hapiotypes. Seventeen samples were selected for sequencing of the cytochrome b (Cyt b; 1134 bp) gene based on control region sequence variation and were analyzed in combination with 34 published sequences to solidify the phylogenetic pattern obtained from control region data. Overall, tree shrews from Kunming have high genetic diversity and present a remarkable long genetic distance to the two reported northern tree shrews outside China. Our results provide some caution when using tree shrews to establish animal models because of this apparent genetic difference. In addition, the high genetic diversity of Chinese tree shrews inhabiting Kunming suggests that systematic genetic investigations should be conducted before establishing an inbred strain for medical and biological research.     

Molecular cloning and nucleotide sequence of Thermus thermophilus HB8 trpE and trpG

The trpE gene of Thermus thermophilus HB8 was cloned by complementation of an Escherichia coli tryptophan auxotroph. The E. coli harboring the cloned gene produced the anthranilate synthase I, which was heat-stable and enzymatically active at higher temperature. The nucleotide sequence of the trpE gene and its flanking regions was determined. The trpE gene was preceded by an attenuator-like structure and followed by the trpG gene, with a short gap between them. No other gene essential for tryptophan biosynthesis was observed after the trpG gene. The amino-acid sequences of the T. themophilus anthranilate synthase I and II deduced from the nucleotide sequence were compared with those of other organisms.     

Comparative cytogenetic studies in tree shrews (Tupaia).

Through use of BrdU replication, RBA-banded karyotypes of Tupaia belangeri, T. chinensis, and T. glis were obtained. A chromosome number of 2n = 62 for T. belangeri is described here for the first time and is confirmed for T. chinensis. All chromosomes between these two phenotypically different species appear to have identical RBA banding patterns; in addition, there is no difference between T. belangeri and T. chinensis in the number and position of nucleolus organizer regions (NORs). The reduced chromosome number of 2n = 60 in T. glis can be explained by a Robertsonian translocation between two acrocentric chromosome pairs, Nos. 10 and 13, of T. belangeri and/or T. chinensis, resulting in the metacentric chromosome pair 1 of T. glis. Furthermore, two chromosome pairs each of T. glis and T. belangeri and/or T. chinensis are not homoeologous, as judged by their RBA patterns. Differences were also found in the number and position of NORs; whereas T. glis displays eight positively stained NORs after AgNO3 staining, there are only four silver-stained NORs in both T. belangeri and T. chinensis. The possibility of geographical isolation as an explanation for the lack of chromosomal differentiation between T. belangeri and T. chinensis is discussed.     

Characterisation of the beta-tubulin gene of Cylicocyclus nassatus

mRNA and genomic DNA were isolated from adult Cylicocyclus nassatus, and the mRNA was reverse transcribed. The cDNA was PCR amplified using degenerate primers designed according to the alignment of the β-tubulin amino acid sequences of other species. To complete the coding sequence, the 3′ end was amplified with the 3′-RACE, and for amplification of the 5′ end the SL1-primer was used. The cDNA of the β-tubulin gene of C. nassatus spans 1429 bp and encodes a protein of 448 amino acids. Specific primers were developed from the cDNA sequence to amplify the genomic DNA sequence and to analyse the genomic organisation of the β-tubulin gene. The complete sequence of the genomic DNA of the β-tubulin gene of C. nassatus has a size of 2652 bp and is organised into nine exons and eight introns. The identities with the exons of the gru-1 β-tubulin gene of Haemonchus contortus range between 79% and 97%.     

Saccharomyces cerevisiae ARS on a plasmid from Staphylococcus aureus

Staphylococcus aureus plasmid pC194 carries three sequences closely related to a consensus sequence defined previously by analysis of different genetic elements which replicate autonomously in yeast Saccharomyces cerevisiae. Two of these enable the plasmid to replicate in yeast, the third does not. A new consensus sequence A/T T T T A T R T T T, 1 bp shorter than the previous one, can be deduced from our results. Replacement of the T with G at the position 9 of the sequence abolishes its activity. The presence of the two active sequences on pC194 genome can be explained by the A + T-rich base composition of the plasmid.