黑麂Y染色体的鉴别和Sry基因的克隆及定位

以流式细胞仪分离小麂(Muntiacus reevesi)Y染色体和黑麂(M. crinifrons)Y_(1),Y_(2),X+4和1号染色体,利用DOP-PCR技术富集了分离的各单条染色体。然后,将小麂的Y染色体的DOP-PCR产物经Cy_(3)标记后直接作为涂染探针,应用染色体涂染技术与雌雄黑麂的核型标本进行杂交,确认了黑麂真正的Y染色体为Y_(2)染色体。再以黑麂的Y_(1),Y_(2),X+4和1号染色体的DOP-PCR产物为模板,用人的特异性的SRY基因引物对其进行扩增,结果表明黑麂只有Y_(2)染色体出现了SRY扩增片段。

SRY基因进化的研究现状

国家自然科学基金重点项目及中国科学院重大项目资助。

SRY基因在人猿超科和旧大陆猴中具有不同的进化规律

通过PCR扩增、测序,得到了白臀叶猴和红面猴的SRY基因全序列。结合现有的灵长类其他物种序列进行分析,验证了HMG盒的保守性。通过构建系统发育树,比较旧大陆猴和人猿超科两个类群内和类群间HMG盒侧翼序列Ka/Ks的比率。

Localization of Sry gene on Y chromosome of Muntjac munticus vaginalis

The chromosomes 1, Y-1, Y-2 of Muntjac munticus vaginalis were isolated by fluorescence activated chromosome sorting and amplified by degenerate oligonucleotide primed-polymerase chain reaction ( DOP-PCR). A primer pair within human Sry HMG box was design

赤麂Sty基因的定位及Y染色体的鉴别

应用流式细胞仪分离赤麂的1，Yl，Y2染色体，通过简并寡核苷酸引物聚合 酶链武反应(DOP-PCR)增加模板数量．用人的性别决定基因HMG框内设计1对引 物进行PCR扩增．在雄性赤麂Y2染色体DOP-PCR产物中扩增出与人SRY基因同源 的Sly基因片段，经克隆测序后，初步证明赤麂Y2染色体是真正的Y染色体，同时对 赤麂s珂基因进行了初步定位．

Cloning and characterization of the first amphibian bradykinin gene

More than ten bradykinin-related peptides and their cDNAs; have been identified from amphibians, but their genes are unknown. In present study, four cDNAs encoding one, two, four and six copies of bradykinin-related peptides were cloned from the frog (Odorrana grahami) skin cDNA library, respectively. Three bradykinin-related peptides (bradykinin, Thr6-bradykinin, Leu5Thr6-bradykinin) were deduced from these four cDNA sequences. Based on the cDNA sequence, the gene sequence encoding an amphibian bradykinin-related peptide from O. grahami was determined. It is composed of 7481 base pairs including two exons and two introns. The first exon codes signal peptide and the second exon codes acidic spacer peptide and Thr6-bradykinin. The promoter region of the bradykinin gene contains several putative recognition sites for nuclear factors, such as SRY, GATA-1, LYF-1, DeltaE, CDXA, NKX-2.5, MIF1 and S8. The current work may facilitate to understand the regulation and possible functions of amphibian skin bradykinin-related peptides. (C) 2009 Elsevier Masson SAS. All rights reserved.

Differential expression and dynamic changes of SOX3 during gametogenesis and sex reversal in protogynous hermaphroditic fish

SOX3 has been suggested to play significant roles in gametogenesis and gonad differentiation of vertebrates, but the exact cellular localization evidence is insufficient and controversial. In this study, a protogynous hermaphrodite fish Epinephelus coioides is selected to analyze EcSox3 differential expression and the expression pattern in both processes of oogenesis and spermatogenesis by utilizing the advantages that gonad development undergoes transition from ovary to intersexual gonad and then to testis, and primordial germ cells and different stage cells during oogenesis and spermatogenesis are synchronously observed in the transitional gonads. The detailed and clear immunofluoresence localization indicates that significantly differential expression and dynamic changes of Sox3 occur in the progresses of gametogenesis and sex reversal, and EcSOX3 protein exists in the differentiating primordial germ cells, oogonia, and different stage oocytes of ovaries, and also in the differentiating primordial germ cells and the Sertoli cells of testis. One important finding is that the EcSox3 expression is a significant time point for enterable gametogenesis of primordial germ cells because EcSOX3 is obviously expressed and localized in primordial germ cells. As EcSox3 continues to express, the EcSOX3-positive primordial germ cells develop toward oogonia and then oocytes, whereas when EcSox3 expression is ceased, the EcSOX3-positive primordial germ cells develop toward spermatogonia. Therefore, the current finding of EcSOX3 in the differentiating primordial germ cells again confirms the potential regulatory role in oogenesis and germ cell differentiation. The data further suggest that SOX3, as a transcription factor, might have more important roles in oogenesis than in spermatogenesis.

A conserved family of doublesex-related genes from fishes

The sex-determining gene Mab-3 of C. elegans and the doublesex gene of Drosophila each contain a common DM domain and share a similar role. Human doublesex-related gene DMRT1 also encodes a conserved DM-related DNA-binding domain. We present here the amplification of a broad range of DM domain sequences from three fish species using degenerate PCR. Our results reveal unexpected complexity of the DM domain gene family in vertebrates. (C) 2002 Wiley-Liss, Inc.

Preparation and optical properties of trivalent europium-doped bulk and nanocrystalline SrY2O4

The nanocrystalline Sry(2)O(4):Eu3+ was prepared by a poly(vinyl alcohol) (PVA)+glycine-assist combustion method. The results of x-ray diffraction indicate that the resulting Sry(2)O(4):Eu3+ nanocrystals have much broader and less intense peaks compared with those in bulk material. The charge-transfer bands in Sry(2)O(4):Eu3+ nanocrystals shift to higher energies in contrast to those in bulk material. The spectral results revealed that in bulk SrY2O4: Eu3+ the Eu3+ ions occupied three nonequivalent sites, with one at the Sr site: one at the Y(1) site and another at the Y(2) site, while in nanocrystalline SrY2O4: Eu3+, the Eu3+ ions occupied only two nonequivalent sites; one at the Y(1) site and the other at the Y(2) site. Finally, by theoretical calculation and analysis, the analyzed results are in reasonable agreement with our experimental results.

Luminescence of Pb2+ and energy transfer from Pb2+ to rare earth ions in silicate oxyapatites

The Pb2+ luminescence in a series of silicate oxyapatites Me(2)(Y, Gd)(8)(SiO4)(6)O-2, Me(4)Y(6)(SiO4)(6)O (Me = Mg: Ca, Sr) is reported and discussed in relation to the crystal structure. The maximum wavelengths of the excitation (S-1(0)-P-3(1)) and emission (P-3(1)-S-1(0)) bands of Pb2+ are independent of the Mc:Y ratio (2:8 or 4:6) but they have lower energies in MgY-oxyapatites than in CaY- and SrY-oxyapatites. The Stokes shift of Pb2+ luminescence amounts to 11 100 to 11 400 cm(-1): which does not depend strongly on the host composition. There exists a mutual energy transfer between Pb2+ and Gd3+ in Sr2Gd8(SiO4)(6)O-2. At last, the dependence of the energy transfer efficiency of Pb2+-Sm3+, Tb3+: Dy3+ in Sr-2(La: Gd)(8)(SiO4)(6)O-2 and Ca-2(Y, Gd)(8)(SiO4)(6)O-2 on their doping concentrations was studied in more detail.

Expression pattern of dmrt4 from olive flounder (Paralichthys olivaceus) in adult gonads and during embryogenesis

The dmrt (doublesex and mab-3 related transcription factor) gene family comprises several transcription factors that share a conserved DM domain. Dmrt1 is considered to be involved in sexual development, but the precise function of other family members is unclear. In this study, we isolated genomic DNA and cDNA sequences of dmrt4, a member of the dmrt gene family, from olive flounder, Paralichthys olivaceus, through genome walking and real-time reverse transcriptase (RT)-PCR. Sequence analysis indicated that its genomic DNA contains two exons and one intron. A transcriptional factor binding sites prediction program identified a sexual development-related protein, Sox9 (Sry-like HMG box containing 9) in its 5' promoter. Protein alignment and phylogenetic analysis suggested that flounder Dmrt4 is closely related to tilapia Dmo (DM domain gene in ovary). The expression of dmrt4 in adult flounder was sexually dimorphic, as shown by real-time RT-PCR analysis, with strong expression in the testis but very weak expression in the ovary. Its expression was also strong in the brain and gill, but there was only weak or no expression at all in some of the other tissues tested of both sexes. During embryogenesis, its expression was detected in most developmental stages, although the level of expression was distinctive of the various stages. Whole mount in situ hybridization revealed that the dmrt4 was expressed in the otic placodes, forebrain, telencephalon and olfactory placodes of embryos at different developmental stages. These results will improve our understanding of the possible role of flounder dmrt4 in the development of the gonads, nervous system and sense organs.

牙鲆雌核发育分析鉴定与性别决定机制研究

本研究应用同工酶和随机扩增多态性DNA（RAPD）方法，分析了牙鲆野生种群和雌核发育群体的遗传多样性和遗传分化水平；应用RAPD方法对比分析了雌核发育牙鲆的亲鱼和子代DNA样品；采用PCR扩增牙鲆SRY同源片段并进行了序列分析；将RAPD扩增产物中性别连锁DNA片克隆与测序。主要结果有：1.牙鲆雌核发育群体同工酶的Ldh-C，Cat两个基因座位发生了重组，基因座位与着丝点之间的重组率分别为52.6％和29.8％；雌核发育群体RAPD分析发现136个DNA片段中有22个发生基因分离，但由于研究方法的局限，不能确定是否发生基因重组。2.牙鲆雌核发育群体的同工酶多态座位比例和平均杂合度分别为6.90%和0.0350；研究中提出了RAPD遗传多样参数修正算法；野生种群RAPD多态片段比例37.57％，多态座位比例20.88％，平均杂合度0.0852；雌核发育群体RAPD多态片段比例16.18％，多态座位比例8.98％，平均杂合度0.03898。同工酶与RAPD方法得出结果基本一致。3.牙鲆雌核发育群体与野生群体之间的RAPD遗传相似度I＝0.9036，遗传距离D＝0.1014，已超过一般鱼类地理种群之间的遗传距离值。雌核发育群体的遗传多样性指数（H＿O＝7.1982）远低于野生群体（H＿O＝28.0986）。雌核发育与野生群体H_(pop)／H_(sp)＝0.9716，（H_(sp)－H_(pop)）／H_(sp)＝0.0284，表明97.16％的遗传多样性是由群体内不同个体间的差异造成的，只有2.84％遗传多样性与群体间分化有关。4.分析过同工酶多态座位比例、同工酶平均杂合度、RAPD多态片段比例、RAPD多态座位比例、RAPD平均杂合度、群体遗传多样性指数H＿O等遗传多样性参数，牙鲆雌核发育群体各参数比野生群体减少55.39％－77.74％，说明它的遗传多样性严重损失。5.采用RAPD对照分析亲本与雌核发育子代胚胎的DNA样品。结果表明，雌鱼有、雄鱼无的基因，雌核发育子代表达；雌鱼无、雄鱼有的基因，雌核发育子代不表达，正常受精二倍体对照组表达正常。证明牙鲆雌核发育子代的遗传物质来自母本，雌核发育诱导使用的精子经过紫外线灭活后，遗传物质已被破坏，其携带的遗传信息没有传给子代，在DNA水平证明了雌核发育诱导方法和结果的可靠性。6.进行了牙鲆性别决定机制研究。PCR分析了哺乳动物性别决定基因SRY同源片段在牙鲆的表达情况。扩产物均无个体差异，也没有性别差异。将扩增产物中信号最强的sry-2片段测序，其648bp序列与人SRY基因相应的419bp片段进行了同源性分析，两者同源性为35％，同源性很低，只有随机的碱基重合。由于牙鲆SRY PCR 扩增带没有全部分析，不能肯定牙鲆没有SRY同源片段。RAPD分析确定3个引物的4条扩增片段与性别相关，分别克隆、测序，S134和S145－S两个片段得到了完整序列。