香港地区湍蛙遗传多样性研究

依据线粒体上ND2和CO1两个变异较大的基因序列分析了香港地区香港湍蛙7种群、华南湍蛙1种群，以及大陆其他地区华南湍蛙7种群，戴云湍蛙1种群，武夷湍蛙1种群的系统发育关系，进而探讨香港湍蛙的遗传多样性、香港湍蛙特有性、如何确定香港湍蛙最佳保护单元以及这四种湍蛙的物种分类地位。 1． 香港湍蛙保护遗传学研究 香港湍蛙核苷酸传多样性较低，从其遗传多样性信息、单倍型网络分析、中性检验值以及岐点分布结果一致显示香港湍蛙很可能经历了瓶颈后的扩张，种群正在由一个较小的有效种群大小迅速增长, 有足够的时间通过变异用于积累单倍型的多态性, 而对于提高核苷酸多样化而言, 时间尚短（Nei M et al，1975，Avise J C，2000；李明等，2003）。 分子变异分析结果显示香港湍蛙种群间存在较多的基因交流，且系统发育树上各种群间交叉在一起，没有形成与地理单元相关的分支，而从其单倍型网络看，他们源于共同的祖先，是一个单系群，与地理单元间没有形成显著的遗传分化。因此应作为一个进化显著单元（ESU）。结合其与其他湍蛙发育关系及遗传距离以及野外采集信息认为香港湍蛙只在香港地区有分布，属于香港特有种。该物种内遗传多样性较低，又属于世界自然保护联盟红皮书中的近危种，同时也是《野生动物保护条例》中的受保护野生动物，且由于香港城市建设等使得其栖息环境受到威胁，因此在香港特别行政区应该受到重点保护。 从单倍型分布和核苷酸多样性可以看出大榄涌种群和城门种群具有较高的单倍型多样性和核苷酸多样性，应该作为保护的重点区域。 2. 华南湍蛙东、南沿海种群间系统关系 华南湍蛙分布广，各种群存在着丰富的遗传多样性信息且中部种群广西龙胜和湖南张家界种群核苷酸多样性明显高于其他边缘种群华南湍蛙。种群间几乎没有基因交流，且各种群间无共享单倍型，可见已形成了显著的遗传分化。各种群间遗传距离都较远，其中广东南昆山种群以及福建三港种群与其他种群距离最远，因此可以推测其他种群（广东深圳、香港大屿山、广西龙胜和防城以及湖南张家界种群）可能为独立进化的种群。但是否是一新种或一隐存种，还需要结合形态学进行更深入的研究。 本研究中无论从系统关系看还是从遗传距离看，大屿山种群与深圳种群最近，支持陈坚峰等将其定为华南湍蛙，即华南湍蛙新增一个分布点：香港大屿山。 系统树上广西防城种群（支B）与龙胜和湖南种群（支A）形成姐妹群。香港大屿山种群与深圳种群先形成姐妹群（支C），但却没有与其距离很近的广东南岭及南昆山种群（支D）形成姐妹群，可能粤北和粤中的环境及气候较复杂因此与粤南其他种群形成了明显的隔离。同时可以看出华南湍蛙种群遗传分化与地理距离没有显著的相关性。 3. 四种湍蛙间的系统关系 根据线粒体CO1基因建立四种湍蛙间的系统关系及其遗传距离，很清楚地看到，香港湍蛙与戴云湍蛙关系很近，而华南湍蛙则与武夷湍蛙较近。然而，戴云湍蛙同一个种群内部共有两个单倍型DY1和DY2，且两个单倍型间遗传距离大于DY1与香港湍蛙间遗传距离，更远远大于香港湍蛙种群内部的距离，即戴云湍蛙内部两个单倍型间遗传距离达到了种级水平，同样在系统发育树上这两个单倍型与香港湍蛙形成并系。但是，戴云湍蛙种内在形态上差异不显著。因此，其是否属于萌芽物种分化形成（budding speciation）或已经完全分化为两个不同的种值得进一步研究？ 与戴云湍蛙香港湍蛙关系类似，从系统树上看华南湍蛙不形成单系，而是分成两个大支，与武夷湍蛙形成并系，且福建和南昆山的华南湍蛙与武夷湍蛙遗传距离远大于武夷湍蛙种内福建种群与浙江种群的遗传距离，达到了种级分化水平。由此，可以推断武夷湍蛙是有效种。系统树上广东深圳、香港大屿山、广西防城和龙胜以及湖南张家界种群与华南湍蛙福建及南昆山各种群间遗传距离已超出了种内各种群间的遗传距离，但是至于这一支是否应为另外一个种，有必要扩大采样，并结合核基因及形态信息进行进一步研究。 MtDNA of ND2 and CO1 gene were used to investigate genetic diversity of Amolops in Hongkong .We collected seven populations of A. hongkongensis,，one population of A.ricketti from Hong Kong and other seven populations of A.ricketti from East and South of Chinese mainland. As well as one population of A. daiyunensis and one population of A.wuyiensis Phylogenetic relationship were analyzed of four species. Discussed whether A.hongkongensis is an endemic species and how can we make the conservation and management decisions. 1. Conservation Genetics of A. hongkongensis A. hongkongensis has a low nucleotide diversity, the results of genetic diversity, haplotype network, neutrality test and the mismatch distributions indicate that A. hongkongensis experienced a recent expansion after a bottle neck. They had enough time to accumulated haplotype diversity, but it’s too short to have a high nucleotide diversity（Nei M et al，1975，Avise J C，2000；Li et al，2003）. The result of AMOVA reveals that it has much gene exchange among the populations of A. hongkongensis. The clades of the phylogenetic tree were mixed together, no significant genetic differentiation among 8 populations and they share the same ancestor from the network analysis, these indicate that they are monophyly and should be protected as one ESU. Combined with the information of relationships of interspecies, genetic distance and distribution investigate, We conclude that A. hongkongensis is an endemic species of Hong Kong. Considering on the status of low genetic diversity in A.hongkongensis, and this species was listed in the IUCN red list as near threatened, as well as listed in the . Furthermore, it’s habitat loss and degradation more rapidly as the human activity got higher and higher. So it’s urgent to protect them in Hong Kong. Our results suggest that Tai Lam Wu and TAI MO Shan -Shing Mun populations have the higher priority to be protected because their higher genetic diversity. 2．Phylogenetic relationships among populations of Amolops ricketti from the Southern and eastern China A. ricketti has the considerable genetic diversity of mitochondrial haplotypes within and among populations, and Mitochondrial DNA diversity was higher in populations at the central area of the present distribution range of the frog，i. e. the Longsheng population and Zhangjiajie population, than at the edges of their distribution range. They have no share haplotype among populations, and have a significant genetic differentiation. Genetic distance is high among the populations, especially the distance of Nankun and Sangang group with others, which suggested that they evolved independently. May be there is a cryptic species or a new species, a further study is needed. The results of gene tree and the genetic distance clearly demonstrate that the population from LanTau island is A. ricketti, so we agree with Chen et al(2005) . That means A.ricketti have a new distribution site: LanTau island, HongKong. Phylogenetic relationships were analyzed through NJ and Mrbayes methods and got a consistent topological structure, the structure indicated that the ingroup were comprised four groups. Populations Longsheng and Zhangjiajie were first clustered as clade A; Populations Fangcheng was clustered together (clade B) as a sister group to clade A；Populations Shenzhen and Lantau island were sister groups and clustered as clade C；Then the clade D included populations Nankunshan and Nanling in Guangdong province and Sangang in Fujian province. 3. Phylogenetic Relationships among these four specises Phylogenetic relationships based on 1503bp CO1 gene and the genetic distance show that A. hongkongensis close to A. daiyunensis whereas A.ricketti near to A.wuyiensis. Nevertheless, there are two haplotypes in A.daiyunensis and the genetic distance between them higher than the distance between DY1 with A. hongkongensis. A. hongkongensis is nested in the paraphyletic ancestral species A. daiyunensis. Without significant difference in the morphological characters, So, we considered both A.daiyunensis and A.hongkongensis are valid species, may be this represents a case of ‘budding speciation’ like Batrachuperus pinchonii(Fu and Zeng,2008) in the population of A. daiyunensis. Just like two species above A. wuyiensis and A. ricketti are not monophyly, instead, A.wuyiensis is nested in the paraphyletic ancestral species A.ricketti. We need do more research to make sure whether they are new species.

eta photoproduction on the proton in a chiral constituent quark approach via a one-gluon-exchange model

A formalism based on a chiral quark model (chi QM) approach complemented with a one-gluon-exchange model, to take into account the breakdown of the SU(6)circle times O(3) symmetry, is presented. The configuration mixing of wave functions for nucleon and resonances are derived. With few adjustable parameters, differential cross-section and polarized-beam asymmetry for the gamma p -> eta p process are calculated and successfully compared with the data in the center-of-mass energy range from threshold to 2 GeV. The known resonances S-11(1535), S-11(1650), P-13(1720), D-13(1520), and F-15(1680), as well as two new S-11 and D-15 resonances, are found to be dominant in the reaction mechanism. Moreover, connections among the scattering amplitudes of the chi QM approach and the helicity amplitudes, as well as decay widths of resonances, are established. Possible contributions from the so-called missing resonances are investigated and found to be negligible.

A correlation between radiation sensitivity and initial chromatid breaks in cancer cell lines revealed by Calyculin A-induced premature condensation

Three human malignancy cell lines were irradiated with Co-60 gamma-rays. Initial chromatid breaks were measured by using the chemically induced premature chromosome condensation technique. Survival curves of cells exposed to gamma rays was linear-quadratic while the efficiency of Calyculin A in inducing PCC of G(2) PCC was about five times more than G(1) PCC. A dose-dependent increase in radiation-induced chromatid/isochromatid breaks was observed in G(1) and G(2) phase PCC and a nearly positive linear correlation was found between cell survival and chromatin breaks. This study implies that low LET radiation-induced chromatid/isochromatid breaks can potentially be used to predict the radiosensitivity of tumor cells either in in vitro experimentation or in in vivo clinical radiotherapy.

Chromatid-type aberrations following irradiation in G0 lymphocytes with heavy ions

We describe a low level of chromatid-type aberrations which included the relatively rare isochromatid/chromatid triradial in peripheral blood lymphocytes that were irradiated, ostensibly in GO, with accelerated heavy C-12 ions. These were produced only at the energies of 69 MeV/n (34.6 keV/ mu m), almost absent at the energy of either 58.6 McV/n (46.07 keV/mu m) or 19.3 MeV/n (97 keV/mu m), nor were they found after low-LET X-rays. Mechanisms potentially responsible for their formation are discussed.

Correlation between initial chromatid damage and survival of various cell lines exposed to heavy charged particles

The biophysical characteristics of heavy ions make them a rational source of radiation for use in radiotherapy of malignant tumours. Prior to radiotherapy treatment, a therapeutic regimen must be precisely defined, and during this stage information on individual patient radiosensitivity would be of very great medical value. There are various methods to predict radiosensitivity, but some shortfalls are difficult to avoid. The present study investigated the induction of chromatid breaks in five different cell lines, including one normal liver cell line (L02), exposed to carbon ions accelerated by the heavy ion research facility in Lanzhou (HIRFL), using chemically induced premature chromosome condensation (PCC). Previous studies have reported the number of chromatid breaks to be linearly related to the radiation dose, but the relationship between cell survival and chromatid breaks is not clear. The major result of the present study is that cellular radiosensitivity, as measured by D-0, is linearly correlated with the frequency of chromatid breaks per Gy in these five cell lines. We propose that PCC may be applied to predict radiosensitivity of tumour cells exposed to heavy ions.

Survival and initial chromatid breakage in normal and tumour cells exposed in vitro to gamma rays and carbon ions at the HIRFL

Human hepatoma and normal liver cells were irradiated with C-12(6+) ion beams (linear energy transfer (LET) = 96 keV mu m(-1)) and gamma-rays at the Heavy Ion Research Facility in Lanzhou (HIRFL). The numbers and types of chromatid breaks were detected using the premature chromosome condensation technique. Irradiation with C-12(6+) ions produced a majority of isochromatid break types, while chromatid breaks were dominant for irradiation with gamma-rays. Experimental results showed that the initial level of chromatid breaks is clearly related to the absorbed dose from C-12(6+), ions and gamma-rays. The (12)C(6+)ions are relatively more effective at inducing initial chromatid breaks when compared with the gamma-rays. A relative biological effectiveness (RBE) of about 2.5 resulted for the induction of initial chromatid breaks by C-12(6+) ions relative to gamma-rays in both cell lines.

Nucleon form factors in a light-cone quark model with two-photon exchange

We estimate the two-photon exchange corrections to both proton and neutron electromagnetic physical observables in a relativistic light cone quark model At a fixed Q(2) the corrections are found to be small in magnitudes. but strongly dependent oil scattering angle Our results are comparable to those obtained from simple hadronic model in the medium momentum transfer region (C) 2009 Elsevier B V All rights reserved

Composite electrode for unitized regenerative proton exchange membrane fuel cell with improved cycle life

To improve the cycle life of unitized regenerative fuel cells (URFCs), an electrode with a composite structure has been developed. The cycle life and polarization curves for both fuel cell and electrolysis modes of URFC operation were investigated. The cycle life of URFCs was improved considerably and the performance was fairly constant during 25 cycles, which illustrates that the composite electrode is effective in sustaining the cyclic performance of URFCs. It shows the URFCs with such an electrode structure are promising for practical applications. (C) 2004 The Electrochemical Society.

Capillary electrochromatographic separation of ionizable compounds with a molecular imprinted monolithic cationic exchange column

A polymer-based monolithic capillary column imprinted with 4-aminopyridine (4-AP) was prepared by a thermally-initiated polymerization process; and its performance as a capillary electrochromatographic medium was evaluated in separating 4-AP and 2-AP isomers. The effects of experimental parameters, such as pH value and ionic strength of the buffer, the acetonitrile content in the mobile phase, and the applied voltage, on the resolution of these isomers had been carefully investigated. It was found that in the retention process there were interplays of multiple mechanisms of ion-exchange, molecular imprinting, and electrophoresis. These mechanisms allowed more sophisticated control of experimental parameters in the separation of ionizable compounds.

Separation of peptides on mixed mode of reversed-phase and ion-exchange capillary electrochromatography with a monolithic column

The mixed mode of reversed phase (RP) and strong canon-exchange (SCX) capillary electrochromatography (CEC) based on a monolithic capillary column has been developed. The capillary monolithic column was prepared by in situ copolymerization of 2-(sulfooxy)ethyl methacrylate (SEMA) and ethylene dimethacrylate (EDMA) in the presence of porogens. The sulfate group provided by the monomer SEMA on the monolithic bed is used for the generation of the electroosmotic flow (EOF) from the anode to the cathode, but at the same time serves as a SCX stationary phase. A mixed-mode (RP/SCX) mechanism for separation of peptides was observed in the monolithic column, comprising hydrophobic and electrostatic interaction as well as electrophoretic migration at a low pH value of mobile phase. A column efficiency of more than 280000 plates/m for the unretained compound has been obtained on the prepared monoliths. The relative standard deviations observed for to and retention factors of peptides were about 0.32% and less than 0.71% for ten consecutive runs, respectively. Effects of mobile phase compositions on the EOF of the monolithic column and on the separation of peptides were investigated. The selectivity on separation of peptides in the monolithic capillary column could be easily manipulated by varying the mobile phase composition.