构树种质资源遗传多样性SRAP分子标记研究

构树（Broussonetia papyrifera L.）为桑科（Moraceae）构树属（Broussonetia）落叶乔木，广泛分布于亚洲东部及太平洋岛屿。它是我国重要的经济林木，具有重要的经济价值，其树皮纤维品质优良，自古就是造纸的优良原料；叶片可用作饲料；果实具有重要的药用价值；环境适应性强，是迅速绿化荒山、荒滩和盐碱地的理想树种。因此对构树这些特性的深入研究和开发利用具有非常重要的实际应用价值。本研究以日本和国内构树主要分布区域的的10种生态型及杂交构树共23份材料，摸索并改进了构树DNA的提取方法，建立了稳定的SRAP分子标记体系，以杂交构树组培苗为材料从120对引物组合中筛选出条带信息较高的17对SRAP引物，以这些引物对23份构树材料进行PCR扩增和标记分析。 本研究取得的主要结果如下： （1）本实验首次将SRAP技术应用于构树的研究中，建立起构树稳定的SRAP-PCR反应体系；实验中对影响扩增的5个主要因素进行了优化，确定20 μL PCR反应体系中各因素最适浓度：模板DNA浓度80 ng/(20 μL)，Mg2+浓度2.0 mM，dNTP浓度0.6 mM，引物浓度0.8 mM，Taq 酶浓度1.5 U/(20 μL)。 （2）从120对引物组合中筛选出来的17对SRAP引物对21份不同生态型构树样本（21份材料指的是除两份杂交构树材料外的其它生态型构树，以下同）进行PCR扩增，共扩增出439条带，平均每对引物25.5条，其大小介于100～1,000 bp之间，其中多态性条带319条，占总数的72.67%。 （3）用Popgene1.32软件进行分析，计算出Shannon信息指数（I）值为0.2275（0.2042），物种水平的Nei基因多样性（H）值为0.1336（0.1436），表明各生态型构树之间的平均遗传多态性不高，中国大陆各生态型构树Shannon信息指数（I）值仅为0.1675（0.2271），物种水平的Nei基因多样性（H）值为0.1039（0.1540），群体遗传多样性较低，构树的遗传分化主要存在于中国和日本之间。 （4）用NTSYS-2.10e软件进行聚类分析，发现不同生态型构树按距离关系远近及分布区域可划分为不同类群。在遗传相似系数0.57附近，对21份材料进行聚类分析发现其可分为两个群体，一类为日本生态型，另一类为中国生态型，表明日本构树与中国野生种构树种源遗传相似性较小。中国各生态型构树在遗传相似系数0.91处可分为5类，总体而言，中国各地区之间的构树遗传相似度较高。对杂交构树分析表明，其亲缘关系与日本构树（母本）更接近。 （5）日本及杂交构树的SCAR标记。本研究找到两条日本及杂交构树的特异性条带，回收、测序，再根据序列往里重新设计引物，转变成稳定性更好，更直观的SCAR标记，这为挑选性状优良的日本及杂交构树提供重要的参考，对其育种有一定的指导意义。其中一条片段经与NCBI数据库比对发现与拟南芥磺基转移酶家族基因具有较高的同源性。

Fibre optic distributed sensor system for condition monitoring of synthetic ropes

Reliability of the measuring devices is very important problem. Optical fibre sensors are very efficient. The use of optical fibre sensors for monitoring the physical and chemical parameters has been expanding over resent years. These sensors are applied for monitoring the structural integrity of long, parallel lay synthetic ropes. Such ropes are corrosion free, however, their operational lifetime under cyclic load is not well understood and premature failure can occur due to slippage and breakage of yarns within the rope. The monitoring system has been proposed which is based on acoustic detection of yarn breakage. Monitoring the strain and temperature is performed using the array of fibre gratings distributed along the rope length.

MULTIPLE GENOTYPES OF MITOCHONDRIAL-DNA WITHIN A HORSE POPULATION FROM A SMALL REGION IN YUNNAN PROVINCE OF CHINA

mtDNA genotypes of six domestic horses (three adult short horses whose heights are under 1 m and three common domestic horses) from a small region of 15 km(2) in Malipo county of Yunnan province of China were investigated by the technique of restriction fragment length polymorphism (RFLP) with restriction endonucleases which recognize 6-bp sequences. An average of fragments for an individual was obtained. Unlike other domestic animals, this population of horses exhibits high mtDNA genetic diversity. Each of the six horses has a specific mtDNA genotype showing a pattern of multiple maternal origins, as suggested by fossil and literature records. We think the population of horses is an amazing seed-resource pool of horses and hence deserves to be paid more attention from the view of conservation genetics. However it is also remarkable that we did not find any typical mtDNA genetic markers which would discriminate between short horses and common domestic horses.

Genetic diversity and population history of the red panda (Ailurus fulgens) as inferred from mitochondrial DNA sequence variations

The red panda (Ailurus fulgens) is one of the flagship species in worldwide conservation and is of special interest in evolutionary studies due to its taxonomic uniqueness. We sequenced a 236-bp fragment of the mitochondrial D-loop region in a sample of 5

The mitochondrial genome organization of the rice frog, Fejervarya limnocharis (Amphibia : Anura): a new gene order in the vertebrate mtDNA

The mitochondrial DNA of the rice frog, Fejervarya limnocharis (Amphibia, Anura), was obtained using long-and-accurate polymerase chain reaction (LA-PCR) combining with subcloning method. The complete nucleotide sequence (17,717 bp) of mitochondrial genome was determined subsequently. This mitochondrial genome is characterized by four distinctive features: the translocation of ND5 gene, a cluster of rearranged tRNA genes (tRNA(Thr), tRNA(Pro), tRNA(Leu) ((CUN))) a tandem duplication of tRNA(Mer) gene, and eight large 89-bp tandem repeats in the control region, as well as three short noncoding regions containing two repeated motifs existing in the gene cluster of ND5/tRNA(Thr)/tRNA(Pro)/tRNA(Leu)/tRNA(Phe). The tandem duplication of gene regions followed by deletions of supernumerary genes can be invoked to explain the shuffling of tRNAM(Met) and a cluster of tRNA and ND5 genes, as observed in this study. Both ND5 gene translocation and tandem duplication of tRNA(Met) were first observed in the vertebrate mitochondrial genomes. (c) 2004 Elsevier B.V. All rights reserved.

Phylogenetic study of complete cytochrome b genes in musk deer (genus Moschus) using museum samples

As an endangered animal group, musk deer (genus Moschus) are not only a great concern of wildlife conservation, but also of special interest to evolutionary studies due to long-standing arguments on the taxonomic and phylogenetic associations in this group. Using museum samples, we sequenced complete mitochondrial cytochrome b genes (1140 bp) of all suggested species of musk deer in order to reconstruct their phylogenetic history through molecular information. Our results showed that the cytochrome b gene tree is rather robust and concurred for all the algorithms employed (parsimony, maximum likelihood, and distance methods). Further, the relative rate test indicated a constant sequence substitution rate among all the species, permitting the dating of divergence events by molecular clock. According to the molecular topology, M. moschiferus branched off the earliest from a common ancestor of musk deer (about 700,000 years ago); then followed the bifurcation forming the M. berezouskii lineage and the lineage clustering M. fuscus, M. chrysogaster, and M. leucogaster (around 370,000 years before present), interestingly the most recent speciation event in musk deer happened rather recently (140,000 years ago), which might have resulted from the diversified habitats and geographic barriers in southwest China caused by gigantic movements of the Qinghai-Tibetan Plateau in history. Combining the data of current distributions, fossil records, and molecular data of this study, we suggest that the historical dispersion of musk deer might be from north to south in China. Additionally, in our further analyses involving other pecora species, musk deer was strongly supported as a monophyletic group and a valid family in Artiodactyla, closely related to Cervidae. (C) 1999 Academic Press.

眼镜王蛇泛素融合蛋白基因和核糖体蛋白L30 基因的克隆及分析

从广西产眼镜王蛇( Ophiophagus hannah) 毒腺中抽提总RNA , 经mRNA 纯化后构建眼镜王蛇毒腺 cDNA 文库。从所构建的cDNA 文库中, 随机筛选200 个克隆测序, 得到两个在进化上高度保守的基因: 泛素融 合蛋白基因(GenBank 登录号为AF297036) 和核糖体蛋白L30 基因(GenBank 登录号是AF297033) 。前者cDNA 的开放阅读框为387 bp , 后者为348 bp 。前者编码128 个氨基酸残基组成的泛素融合蛋白前体; 后者编码115 个氨基酸残基组成的核糖体蛋白L30 前体。由cDNA 序列推导出的氨基酸序列分析表明, 泛素融合蛋白前体包 括N - 末端的泛素结构域(76 个氨基酸残基) 和C - 末端的核糖体蛋白L40 结构域(52 个氨基酸残基) 。该蛋 白为一高碱性蛋白, C 末端含有一个“锌指”模式结构。与16 个物种比较的结果表明, 眼镜王蛇与脊椎动物的 泛素融合蛋白氨基酸序列相似度较高, 具有高度的保守性。

Cloning and characterization of a blood coagulation factor IX-binding protein from the venom of Trimeresurus stejnegeri

A blood coagulation factor IX-binding protein (TSV-FIX-BP) was isolated from the snake venom of Trimeresurus stejnegeri. On SDS-polyacrylamide gel electrophoresis, TSV-FIX-BP showed a single band with an apparent molecular weight of 23,000 under non-reducing conditions. and two distinct bands with apparent molecular weights of 14,800 and 14,000 under reducing conditions. cDNA clones containing the coding sequences of TSV-FIX-BP were isolated and sequenced to determine the structure of the precusors of TSV-FIX-BP subunits. The deduced amino acid sequences of two subunits of TSV-FIX-BP were confirmed by N-terminal protein sequencing and trypsin-digested peptide mass fingerprinting. TSV-FIX-BP was a nonenzymatic C-type lectin-like anti-coagulant. The anti-coagulant activity of TSV-FIX-BP was mainly caused by its dose dependent interaction with blood coagulation factor IX but not with blood coagulation factor X. (C) 2003 Elsevier Science Ltd. All rights reserved.

Molecular cloning and characterization of a platelet glycoprotein Ib-binding protein from the venom of Trimeresurus stejnegeri

A platelet glycoprotein Ib-binding protein, termed TSV-GPIb-BP, was isolated from the venom of Trimeresurus stejnegeri. On SDS-polyacrylamide gel electrophoresis, TSV-GPIb-BP showed a single band with an apparent molecular weight of 28,000 and two distinct bands with apparent molecular weights of 16,000 and 15,000 under non-reducing and reducing conditions, respectively. cDNA clones containing the coding sequences for both TSV-GPIb-BP subunits were isolated and sequenced. The deduced amino acid sequences of TSV-GPIb-BP subunits were confirmed by N-terminal protein sequencing and trypsin-digested peptide mass fingerprinting. Interestingly, the a subunit of TSV-GPIb-BP is identical to that of alboaggregin-B, and the sequence identity of their beta subunits is 94.3%. TSV-GPIb-BP inhibited ristocetin-induced human platelet agglutination in platelet-rich plasma under lower dosages (<5 mug/ml). On the other hand, it directly aggregated washed human platelets in the absence of additional Ca2+ or any other cofactors under higher dosages (>5 mug/ml). This platelet aggregation activity was dose-dependently inhibited by specific GPIbalpha antibodies, but not by those antibodies against platelet GPIa, GPIIa, GPIIb and GPIIIa. (C) 2003 Elsevier Science Ltd. All rights reserved.

Methods of patterning magnetic fields using bulk superconductors

High Temperature superconductors are able to carry very high current densities, and thereby sustain very high magnetic fields. There are many projects which use the first property and these have concentrated on power generation, transmission and utilization, however there are relatively few which are currently exploiting the ability to sustain high magnetic fields. There are two main reasons for this: high field wound magnets can and have been made from both BSCCO and YBCO but currently their cost is much higher than the alternative provided by low Tc materials such as Nb3Sn and NbTi. An alternative form of the material is the bulk form which can be magnetized to high fields and using flux pumping this can be done in situ. This paper explores some of the applications of bulk superconductors and describes methods of producing field patterns using the highly uniform magnetic fields required for MRI and accelerator magnets as the frame of reference. The patterns are not limited to uniform fields and it is entirely possible to produce a field varying sinusoidally in space such as would be required for a motor or a generator. The scheme described in this paper describes a dipole magnet such as is found in an accelerator magnet. The tunnel is 30 × 50 × 1000 mm and we achieve a uniformity of better than 200 ppm over the 1000 mm length and better than 1 ppm over the central 500 mm region. The paper presents results for both the overall uniformity and the integrated uniformity which is 302 ppm over the 1000 mm length. © 2010 IEEE.

Identification and characterization of novel reptile cathelicidins from elapid snakes

Three cDNA sequences coding for elapid cathelicidins were cloned from constructed venom gland cDNA libraries of Naja atra, Bungarus fasciatus and Ophiophagus hannah. The open reading frames of the cloned elapid cathelicidins were all composed of 576 bp an

Molecular Cloning and Sequence Analysis of Two cDNAs Encoding Metalloproteinase2disintegrin from Trimeresurus jerdonii

　从菜花烙铁头蛇的毒腺中,利用RT2PCR 进行体外扩增,克隆到2 个金属蛋白酶2去整合素基 因,命名为TJM21、TJM22. TJM21 cDNA 全长为1 528 bp ,编码481 个氨基酸;TJM22 cDNA 全长为1 578 bp ,编码484 个氨基酸. TJM21 和TJM22 都属于Ⅱ型蛇毒金属蛋白酶,由信号肽、前肽、金属蛋白酶、 间隔肽和去整合素5 部分组成. Ⅱ型蛇毒金属蛋白酶氨基酸序列的比较及进化分析显示,它可进一 步分为两类,一类包括大多数Ⅱ型蛇毒金属蛋白酶(其中含有TJM21) ,而TJM22 和agkistin 则组成了 另一类. 并且TJM22 和agkistin 的第407 位和第426 位残基都是半胱氨酸,而在其它Ⅱ型金属蛋白酶 的相应位置,407 位是丝氨酸,426 位则缺失. TJM22 和agkistin 均有可与整合素α2 Ⅰ区域特异性结合 的片段QPNRKRHDNAQ(残基276～284) ,这个片段在其它Ⅱ型金属蛋白酶中并没有发现. 因此推 断, TJM22 和agkistin 可能属于一类新型的Ⅱ型蛇毒金属蛋白酶.

四个烙铁头蛇毒凝集素样蛋白基因的克隆与序列分析

从烙铁头蛇（Trimeresurus mucrosquamatus）的毒腺中提取mRNA，利用RT-PCR进行体外扩增，获得凝集素样蛋白基因，克隆至PMD18-T载体中，筛选出4种凝集素样蛋白基因（命名为TML-1、TML-2、TML-3和TML-4）。由基因序列推导出的氨基酸序列表明：TML-1，2，3，4序列中均有CRD结构。序列同源性比较和Cys位点分析推测：TML-1和TML-2可能分别是类似于flavocetin-A的蛇毒凝集素样蛋白的#alpha#亚基和#beta#亚基；TML-3可能类似于GPIb-bp的蛇毒凝集素样蛋白的#alpha#亚基，TML-4则可能是类似于IX/X-bp的蛇毒凝集素样蛋白的β亚基。