一种小G蛋白TaRAN1在植物细胞周期和发育过程中的功能研究

　　小G蛋白作为信号转导中重要的分子开关， 进化相当保守，与许多不同的调控因子和效应器分子相互作用，产生细胞功能的多样性。近年来，人们不断发现植物中小G蛋白家族的新成员，也不断揭示小G蛋白的新功能，许多植物特有的信号途径和功能需要小G蛋白这个重要的分子开关来完成，使它越来越成为人们研究的热点问题。但是，有关植物中Ran GTPase及其编码基因的研究工作报道很少，对与之相互作用的调控蛋白研究进展也刚刚开始。 　　TaRAN1 (AF488730) 是小麦来源的Ran同源蛋白编码基因，全长1055 bp, 编码221个氨基酸，它在植物发育过程中的功能还没有任何报道。本论文在验证了它是小G蛋白Ran家族的成员后，从分子水平上还发现它在植物细胞周期调控、对生长素以及胁迫应答信号转导过程中都起着重要作用，这也说明了它可能作为信号转导过程中重要的转换因子，参与了很多细胞的基本生理过程。 　　利用原核表达系统及亲和色谱的方法纯化了TaRAN1融合蛋白，并用放射性标记的GTP和竞争实验证实了它具有特异的GTP结合活性。TaRAN1的转录产物在小麦幼茎和花芽等分生组织活动旺盛的器官表达较多，而在老叶中表达较少。利用洋葱表皮瞬时表达系统分析表现，TaRAN1蛋白主要定位于细胞核，但其没有典型的核定位信号。 　　细胞周期一直是生物学领域中的热门问题，人们虽然在动物细胞中取得了很大进展，但在植物细胞中的研究远落后于动物。裂殖酵母（Schizosaccharomyces pombe）是研究细胞形态和细胞周期的良好系统，利用此系统发现超表达TaRAN1的酵母细胞表现出许多新的细胞学表型，例如G2细胞周期延滞、染色体对紫外线敏感、细胞超长或多隔细胞的出现等;反义表达TaRAN1的酵母细胞呈近圆型、具有高度凝集的核并且生长速度缓慢、核质混合和无核细胞的数目明显增加。流式细胞仪检测实验也证实其细胞周期的异常。这些结果推测TaRAN1蛋白可能参与细胞周期的有丝分裂过程和发育的调控机制，并且在维持染色体结构稳定和完整性方面起着重要的作用。通过免疫荧光实验观察表明，超表达转基因酵母的微管多呈异常的狭小扇形结构，反义表达TaRAN1的酵母微管不能形成丝状结构，推测TaRAN1还可能参与微管（包括纺锤体）的结构形成过程。最后，我们用超表达TaRAN1的转基因拟南芥和水稻也证实了它的功能，其生长点表现出分生组织增多的原基、根生长点的有丝分裂指数有所改变、出现异常的细胞分裂时相等有关细胞周期异常的现象，更进一步说明了TaRAN1确实参与着细胞周期的调控过程，推测其与细胞周期从G2期进入M期的过程有关。 　　TaRAN1基因受IAA的诱导表达，且随着浓度的增加表达量增强。超表达的TaRAN1植株（包括拟南芥和水稻）的根表现出对外源生长素异常敏感，侧根显著变少，地上部分表现出生长素过量的表现型，顶端优势减弱，分蘖增多，生长周期延长等。HPLC测定转基因植物的IAA含量，明显高于对照。所以，TaRAN1可能还参与了复杂的生长素信号转导过程。TaRAN1基因还受各种胁迫处理的诱导表达，并且超表达植株对胁迫的忍受能力有明显提高，这说明TaRAN1还参与了胁迫信号应答的相应机制。Ran蛋白这些新功能目前还未见到其它报道。

水稻RAD21/REC8家族基因的分离与功能分析

染色体黏着是有丝分裂和减数分裂的关键事件，是保证姊妹（或同源）染色体正确分离并分配到子细胞中的关键调控环节之一，它建立于细胞分裂前的S期将新复制的姊妹染色体紧密联系在一起。来自酵母的研究结果已经证明姊妹染色体之间的黏着是由多亚基的蛋白质复合体－黏着素所介导的。在芽殖酵母有丝分裂中，黏着素由Scc1，Scc3，Smc1和Smc3四个亚基组成。减数分裂黏着素的组成与有丝分裂中的相似，只是Scc1被其减数分裂特异的Rec8变体所替换。目前，已经从高等真核生物线虫，果蝇，人，鼠以及拟南芥中分离到了黏着素相关的基因，但是对于这些基因在高等真核生物特别是植物细胞分裂中的功能还知之甚少。即使在酵母中人们对于减数分裂和有丝分裂过程中有关染色体黏着与分离的许多基本问题仍然不清楚，而且许多现象表明减数分裂的详细机制在各种生物中存在重大差异。 我们通过同源克隆的方法证明水稻（和拟南芥）基因组编码4个RAD21/REC8-like基因。这4个基因均以单拷贝存在，在核苷酸水平上没有相似性。它们所编码的蛋白质的相似性主要局限于其N-末端结构域和C-末端结构域。这4个蛋白质的中间区域没有（或者仅有极低的）相似性，但是中间区域都含有潜在的核定位信号，PEST序列，分离酶的识别序列以及多个磷酸化位点。 半定量RT-PCR，原位杂交以及Western杂交结果显示这4个基因都在生殖器官中优势表达，但是它们在花发育过程中的表达动态是不同的。OsRAD21-1和OsRAD21-3都在减数分裂时期的颖花中表达量最高，但是OsRAD21-3还在成熟花粉中高表达;OsRAD21-4在减数分裂前的颖花中表达量最高;OsRAD21-2则在雌雄蕊形成时期表达最强，之后逐渐降低。这些结果暗示这4个基因的功能可能是不同的。 免疫荧光定位分析表明，OsRad21-1和OsRad21-3 特异地定位于有丝分裂的染色体上，其分布动态表明这两个蛋白可能都参与了有丝分裂姊妹染色体之间的黏着。由于水稻四个RAD21/REC8类基因中，只有OsRAD21-3在花粉发育过程中表达，同时水稻花粉的发育成熟要经过两次有丝分裂，推测OsRad21-3蛋白可能参与这两次有丝分裂过程姊妹染色体之间的黏着。OsRad21-4则特异地定位于减数分裂前间期到中期Ⅰ的染色体上，说明它可能特异地介导减数分裂过程姊妹染色体之间的黏着。与其它已知的Rad21/Rec8-like蛋白不同，不论在有丝分裂还是在减数分裂过程中，OsRad21-2蛋白都不定位于染色体上而是特异地定位在核仁中，并且它的动态变化与核仁重建和解体的动态规律在时间上也是相一致的，这说明OsRad21-2是一种新的核仁蛋白质而与染色体的黏着无关。 OsRAD21-4 RNAi转基因水稻植株的花粉活性受到严重影响，种子结实率降低。雄性减数分裂过程中染色体出现多种异常行为：前期Ⅰ染色体异常凝集;同源染色体提早分离;染色体出现片断化。进一步的FISH实验结果证明RNAi株系中同源染色体配对和姊妹染色体臂的黏着均发生异常。因此，OsRad21-4是酵母Rec8的同源蛋白，是正确的减数分裂所必需的。 与表达分析和功能分析所得的结果相一致，进化树分析可以将Rad21/Rec8-like蛋白质分为三个亚家族：（1）Rad21亚家族，参与有丝分裂姊妹染色体黏着;（2）Rec8亚家族，参与减数分裂染色体黏着;（3）Rno亚家族，目前仅发现于高等植物中，是一种核仁蛋白质而与其它的Rad21/Rec8-like蛋白的功能不同，可能不参与染色体之间的黏着。

水稻RAD21/REC8家族基因OsRAD21-3的功能研究

有丝分裂和减数分裂包含一系列协同作用的事件，姊妹染色体的黏着是其中非常重要的一个环节。通过对芽殖酵母的研究发现姊妹染色体的黏着是由一个多亚基的蛋白复合体——黏着素介导的，在有丝分裂过程中，黏着素由Scc1（裂殖酵母中为Rad21）、Scc3、Smc1和Smc3四个亚基组成，而在减数分裂中Scc1被其同源蛋白Rec8所取代。通过对其它真核生物包括线虫、果蝇、爪蟾、鼠、人以及拟南芥等进行的研究显示，黏着素介导的姊妹染色体黏着的基本机制在真核生物中具有保守性，但在不同的物种中，其具体的分子机制还存在着差异。开花植物的有性生殖过程包含了一个非常特殊的花粉发育阶段，其间发生了两次花粉的有丝分裂，对其我们还知之甚少。Rad21/Rec8作为黏着素的重要组分，对水稻中它的同源蛋白进行深入研究可以帮助我们对植物有丝分裂和减数分裂的染色体黏着机制有进一步的了解。 我们发现在水稻基因组中存在4个编码Rad21/Rec8家族蛋白的基因，其中OsRAD21-3位于水稻第8号染色体上，其编码的蛋白与其它的Rad21/Rec8家族蛋白一样，具有保守的N-和C-末端结构域。序列比对和进化分析的结果表明OsRad21-3蛋白属于Rad21亚家族，而免疫荧光定位分析显示OsRad21-3可以特异地定位于有丝分裂的染色体上，由此说明OsRAD21-3应当是酵母RAD21的直系同源基因。对OsRAD21-3进行半定量RT-PCR，Western杂交以及原位杂交的结果显示它在生殖器官花中优势表达，在营养器官中也能检测到表达。在花中它在从雌雄蕊形成期到成熟花粉期都有表达，其表达的位置主要位于减数分裂时期的小孢子母细胞以及减数分裂后的小孢子和花粉中。在水稻四个RAD21/REC8基因中，OsRAD21-3是唯一一个在花粉发育过程中表达的基因，说明它可能在减数分裂后的雄配子体发育过程中发挥了功能。 采用RNAi手段对OsRAD21-3的功能进行研究发现OsRAD21-3 RNAi转基因水稻植株的育性显著下降，进一步的分析表明其花粉活性受到严重影响。对OsRAD21-3i株系的雄性减数分裂过程及减数分裂后的小孢子和花粉发育过程进行观察发现，减数分裂过程中有少量细胞存在染色体的异常行为，但总体上没有对雄性减数分裂造成严重影响，而减数分裂后的小孢子和花粉发育过程则出现了显著异常，表现为花粉有丝分裂的停滞或有丝分裂染色体分离的扰乱，因此，OsRAD21-3应当主要是通过在花粉的有丝分裂过程中发挥功能而参与减数分裂后的雄配子体发育过程，而它在雄性减数分裂过程中可能也有一定作用。 利用原位杂交技术分析了水稻另外三个RAD21/REC8基因的表达特性，结果表明OsRAD21-1、OsRAD21-2和OsRAD21-4都在减数分裂前及减数分裂期的小孢子母细胞中表达，但在减数分裂后较晚时期的小孢子中则没有检测到表达。OsRAD21-4在小孢子母细胞中的表达为其参与减数分裂过程染色体的黏着提供了证据，而OsRAD21-1和OsRAD21-2在此过程中的功能还有待进一步研究。此外，OsRAD21-1和OsRAD21-2还在营养器官有丝分裂旺盛的区域表达，OsRAD21-1作为RAD21的同源基因可能与OsRAD21-3在参与有丝分裂染色体黏着方面存在功能上的冗余性，但OsRAD21-3参与花粉的发育过程则是其所特有的，这可能也在一定程度上说明了为什么水稻中会存在4个RAD21/REC8基因，并帮助我们更好地了解了它们在功能上的分化。

水稻OsMYB3R-2 基因对逆境响应的功能研究

低温威胁水稻的生产，其中苗期和生殖阶段对寒害是最敏感的时期。在苗期，阶段性冷害使水稻幼苗生长延迟，甚至造成烂秧现象；在生殖阶段，无法预测的突然降温会导致水稻花粉不育，并致使水稻大幅减产。因此，对水稻逆境胁迫调控的分子机制的深入研究在理论和实践上具有重要的意义。本研究从东乡野生稻、栽培稻及其杂交后代的低温芯片中筛选对低温响应基因的分析着手，对其中一个受低温诱导上调的基因OsMYB3R-2 作进一步研究。生物信息学的分析表明OsMYB3R-2 编码一个R1R2R3 MYB 蛋白，利用基因枪瞬时转化法、酵母GAL4 系统和电泳迁移率变动分析发现OsMYB3R-2 蛋白能够定位在细胞核中、具有转录激活和DNA 结合特性，表现为MYB 转录因子的典型特征。 超表达OsMYB3R-2 的转基因水稻呈现幼苗的矮化和生长相对滞后的表型，对低温胁迫具有耐受性。盐抑制水稻种子的萌发，与野生型和反义的株系相比，OsMYB3R-2 超表达株系的萌发对盐敏感，表现为萌发过程及萌发之后幼苗的生长更加滞后。而OsMYB3R-2 转基因株系对干旱处理敏感。为了进一步寻找OsMYB3R-2 蛋白的靶序列及其调控的靶基因，我们利用电泳迁移率变动分析发现OsMYB3R-2 能够与有丝分裂特异的激活子（mitosis-specific activator）元件特异结合。在低温条件下，OsMYB3R-2 超表达能够激活水稻G2/M 期特异基因的表达，主要包括OsCycB1;1、OsCycB2;1、OsCycB2;2 和OsCDC20.1 等。另一方面，OsMYB3R-2 超表达能够增加根尖细胞的有丝分裂指数，这进一步说明OsMYB3R-2 参与了水稻细胞周期调控。EMSA、RT-PCR 和流式细胞仪分析的结果表明OsMYB3R-2 通过激活其靶基因OsCycB1;1 的表达参与水稻对低温胁迫的调控，该过程由细胞周期介导。 为了研究OsMYB3R-2 与水稻DREB/CBF 途径的关系，我们分析了转基因水稻中DREB/CBF 类基因及其可能调控的下游基因与OsMYB3R-2 的关系，RT-PCR 的结果表明超表达转基因植物中DREB 表达未见明显变化，而其下游基因OsCPT1 在低温条件下被激活表达。同时，转基因植物在低温条件下脯氨酸水平显著提高。这说明OsMYB3R-2 可能在水稻DREB/CBF 途径的下游参与调控。 总之，OsMYB3R-2 基因的超表达赋予转基因水稻在苗期对低温胁迫具有耐受性，并呈现矮化和生长滞后的表型。OsMYB3R-2 蛋白行使R1R2R3 MYB 转录因子的功能，在体外能够结合OsCycB1;1 和OsKNOLLE2 基因启动子中有丝分裂特异的激活子元件，在低温条件下激活了G2/M 期特异基因的表达，这些基因包括OsCycB1;1、OsCycB2;1、OsCycB2;2 和OsCDC20.1。低温条件下，在OsMYB3R-2 转基因超表达株系中OsCPT1 基因的转录被激活，细胞的游离脯氨酸的含量也显著增高。这些结果都表明OsMYB3R-2 基因在水稻的冷胁迫信号途径中起重要的作用，该过程受细胞周期及DREB/CBF 途径介导。 我们的实验结果暗示水稻对低温的耐受是通过分生组织细胞周期调控完成的，这个过程由OsMYB3R-2 等关键基因控制。

Identification and molecular evolution of cow CENP-A gene family

The centromere protein A (CENP-A), a histone H3-like protein, provides an essential role for chromosomal segregation during mitosis and meiosis. In this study we identified ten new CENP-A-like genes (excluding the original CENP-A gene) in cow by searching

A SERINE KINASE REGULATES INTRACELLULAR-LOCALIZATION OF SPLICING FACTORS IN THE CELL CYCLE

Small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP splicing factors containing a serine/arginine-rich domain (SR proteins) concentrate in 'speckles' in the nucleus of interphase cells(1). It is believed that nuclear speckles act as storage sites for splicing factors while splicing occurs on nascent transcripts(2). Splicing factors redistribute in response to transcription inhibition(3,4) or viral infection(5), and nuclear speckles break down and reform as cells progress through mitosis(6). We have now identified and cloned a kinase, SRPK1, which is regulated by the cell cycle and is specific for SR proteins; this kinase is related to a Caenorhabditis elegans kinase and to the fission yeast kinase Dsk1 (ref. 7). SRPK1 specifically induces the disassembly of nuclear speckles, and a high level of SRPK1 inhibits splicing in vitro. Our results indicate that SRPK1 mag have a central role in the regulatory network for splicing, controlling the intranuclear distribution of splicing factors in interphase cells, and the reorganization of nuclear speckles during mitosis.

同源四倍体水稻雌配子体发育特性及变异胚囊研究

通过秋水仙素诱导获得同源四倍体水稻10个株系，包括6个恢复系、3个保持系和1个不育系，这些株系具有加倍的染色体组。田间观察表明10个株系具有特殊的农艺性状：茎杆变粗壮、植株颜色加深、叶片变厚、叶宽适度增加、分蘖数减少、有效分蘖的比率下降等。根尖有丝分裂鉴定表明，同源四倍体水稻10个株系具有正常的有丝分裂，观察细胞的染色体数目皆为2n=48。花粉母细胞减数分裂鉴定表明10个株系具有比较理想的减数分裂行为，后期I染色体滞后、末期I微核生成和末期II异常小孢子比率较低，能较好的完成减数分裂过程，其中后期I染色体滞后比率约为10%-20%，末期I微核生成比率约为1%-6%，末期II异常小孢子比率约为1%-8%。这提示，染色体联合和分离不规则导致三价体、单价体 和落后染色体等产生，并进一步导致在后期和末期不均横分离产生异常小孢子，这可能是同源四倍体株系结实率不高的原因之一。 同源四倍体水稻正常胚囊为蓼型，变异胚囊具有多种类型，其比率显著高于二倍体对照，变化范围为39.62%－69.85%。按变异胚囊的结构特点和形成方式，分为四种类型：退化型，结构变异型，无融合生殖型和反足细胞增殖型。退化型胚囊的平均比率为29.17%，包括小胚囊（15.04%）和完全退化胚囊（14.13%），前者仍有较小胚囊腔而后者胚囊腔缺失。结构变异胚囊包括结构缺失、结构重复和位置异常，反映了蓼型胚囊八核七细胞结构的变异，其在各株系的平均比率为18.96%。无融合生殖胚囊发生比率极低，平均比率为1.77%，类型包括反足胚和卵细胞胚。反足细胞增殖胚囊是反足细胞团频繁增殖形成，伴随上述三种变异发生使异常胚囊的多样性进一步增加，其在各株系的平均比率为10.62%。相关分析表明，同源四倍体水稻结实可能主要来自三部分：正常胚囊、正常型小胚囊和反足细胞增殖型胚囊。这三种胚囊具有相对完整的蓼型结构，可能具有较好的育性，其对结实率的贡献程度估计值分别为72.44%、15.12%、12.44%。此外，完全退化型胚囊和位置异常型胚囊对结实率分别表现出显著（-0.66）和极显著（-0.92）的负相关，这表明二者可能是结实性的抑制因素。 Ten autotetraploid strains, which include six restoring lines, three maintaining lines and a sterile line, are derived from artificial induction by colchicine treatments. Variations of agronomical traits are observed which include large organs, sturdy plants, long panicle length and deep leaf color and so on. It has been confirmed that autotetraploid strains exhibit normal chromosome behaviors in mitosis and the chromosome numbers are all 48. Moreover, abnormal chromosome behaviors are investigated in meiosis including univalent, trivalent, quatrivalent, chromosome lagging and microkernel and so on. It evaluates that the percentage of chromosome lagging in anaphase I is about 10%-20%, the percentage of microkernel in telophase I is about 1%-6% and the percentage of abnormal microspore in telophase II is about 1%-8%. In all, abnormal behaviors of chromosomes could induce univalent, trivalent and et al. and subsequently induce infertile microspore. That may be one of the causes of low seed sets in autotetraploid strains. Embryo sacs of autotetraploid strains are formed according to the Polygonum type. However, these strains exhibit variations of abnormal embryo sacs at high frequencies (39.62% - 69.85%). The variations are frequently involved in the spikelets of the main panicles and the first tillers, leading to obvious decreases of the percentages of normal embryo sacs among the strains. Four types of abnormal embryo sacs are classified basing on their different structures and origins: degenerated embryo sac (DES), structure variation (SV), apomixis (Apo) and antipodal cell proliferation (ACP). Embryo sacs of DES (29.17%) exhibit small embryo sacs (15.04%) or no embryo sac (14.13%), the former showing embryo sacs with decreased size and the latter showing no sac. Embryo sacs of AS (18.96%) include three subtypes: structure deletion, structure duplication and location variation, which suggests abnormalities of the eight nuclei, seven celled pattern of the Polygonum type. Embryo sacs of Apo (only 1.77%) include two origins of apomictic embryos: antipodal embryo and egg embryo. Embryo sacs of ACP are observed frequently (10.62%) in autotetraploid strains which subsequently increase the variations of abnormal embryo sacs. It evaluates by the Pearson’s correlation analysis that seed set is probably contributed by three origins of embryo sacs: normal embryo sacs, small embryo sacs (normal pattern) and embryo sacs of ACP. These three origins exhibit comparatively good structure of the Polygonum type and could account for seed set at a percentage of 72.44%, 15.12%, 12.44%, respectively. Moreover, the subtype of no embryo sac (NES) negatively related to seed set at the P>0.01 level (-0.92) and the subtype of location variation (LV) negatively related to seed set at the P>0.05 level (-0.66). Which suggest the two subtypes may have strong stress on seed set.

水稻同源四倍体高代自交系农艺性状分析及遗传差异研究

课题组在不断地创制新的同源四倍体材料的同时，连续多年以提高结实率为目的培育、筛选自交系材料，已获得自交繁殖十二年的高代自交系材料。相对于诱导创制初期，材料表现出的结实率低，同种系单株间的差异较大；高代材料已表现出较显著的结实率提升和较一致的农艺性状表型。 本实验选取课题组多年培育的同源四倍体水稻高代自交系材料，通过形态学、农艺性状和细胞遗传学比较，研究水稻同源四倍体与二倍体之间的异同。结果显示，所有同源四倍体材料的染色体组成均为2N=4X=48，花粉母细胞(PMC)减数分裂行为较正常，99%以上的染色体都能在减数分裂中期I（MI）发生联会、配对，形成四价体和二价体，这与理论染色体组构成相符。在减数分裂过程中，结实率较高的材料染色体异常现象较少而结实率较低的材料染色体异常现象较严重。统计分析表明，二价体和四价体的比例对结实率没有显著影响，但是三价体的数目对结实率有一定影响。这一结果表明了结实率和细胞减数分裂行为可能存在相关性，同源四倍体的减数分裂行为为筛选高结实率的同源四倍体种系提供了依据。 然后，对同源四倍体水稻高代自交系材料进行农艺性状和品质性状的统计与分析。主要针对结实率、每穗实粒数、有效分蘖和穗长等主要农艺性状，以及直链淀粉含量这一重要的品质性状进行统计。将统计结果与1996年诱导加倍的初代材料的数据相对比分析，结果显示所有材料经过多代选育培养，其农艺性状已经有了较显著的提高，同时同源四倍体材料的农艺性状稳定性也有了较显著的提升。如结实率的提高幅度较大，所有材料的平均结实率均显著高于加倍初代，而同种材料不同单株间的结实率差异也显著地减少，变异系数（CV）的平均值由1996年的41.15%减少到了2008年的28.81%。其他重要农艺性状也有不同程度的改良，种内变异系数也相应地降低。此外，实验测量了同源四倍体材料和来源二倍体材料的直链淀粉含量。分析结果显示，部分材料的直链淀粉含量与二倍体亲本产生了较显著的差异，这可能是诱导加倍过程中的遗传变异造成的；同源四倍体材料的种内变异系数（CV）平均值由1996年的6%下降到了2008年的3.88%，显示出在品质性状方面，同源四倍体材料的遗传稳定性也有较显著的增加。同源四倍体材料农艺性状经过多年的选育，表现出一定的提升，同时，经过多年自交纯化，所有材料种系内的性状差异逐渐缩小，说明同源四倍体水稻的遗传稳定性随着自交纯化而增强，这为同源四倍体水稻的进一步选育打下了良好的基础。 最后，通过测量连续两年的自交系材料的遗传多态性，分析材料间遗传差异和种群遗传结构，深入研究连续两代材料间的遗传差异，研究同源四倍体水稻与二倍体材料遗传稳定性之间的差异。实验采用18对SSR微卫星标记对连续两代15个材料，共94份样本进行差异分析。通过扩增条带长度多态性分析，计算不同材料以及同种材料不同世代间的遗传距离，构建同源四倍体和二倍体水稻的分子指纹库，并绘制聚类图。结果显示，同源四倍体和二倍体不同材料间的遗传差异比较大，遗传距离处于0.4757至0.2816之间；而相同品种不同世代材料间的遗传差异较小，但也表现出一定的遗传差异。同种同源四倍体材料不同世代间的遗传差异比二倍体材料更大，两代四倍体材料间遗传距离处于0.1359至0.0485之间；而两代二倍体材料间的遗传距离处于均小于0.0388。结果表明，同源四倍体水稻高代材料具有一定的遗传稳定性，但与来源二倍体材料相比，其世代间的遗传变异性仍然较强。这种结果说明，经过多代的自交纯化培育，同源四倍体水稻材料能够建立起相对稳定的遗传结构，同时，其强于二倍体亲本的变异性有能够为新品种的选育，农艺性状、品质性状的改良提供一定的遗传基础。此外，分析结果表明通过分子标记辅助检验，水稻材料间的遗传多态性能够有效地区分不同的品种，这为水稻品种的分子鉴定提供了一定的依据。 本研究从细胞学鉴定，农艺性状统计分析以及分子标记辅助聚类分析多方面地对同源四倍体水稻高代系进行了研究，对探究同源四倍体水稻的遗传规律，进一步揭示其遗传特性、农艺性状的遗传构成，为进一步选育优质的多倍体水稻提供了一定的理论依据。 This group insists on creating new Autotetraploid Rice (Oryza sativa L.) materials, while improving the seed-setting of them for many years, cultivated and selected the inbred line materials, has obtained the high generation inbred lines after twelve years cultivation. Compared to the early induced materials, which shown the low seed setting, and the large difference between the different plants in the same germ-line; the high generation materials have shown significant improvement in seed setting and more uniform phenotype agronomic traits. The autotetraploid rice high generation inbred lines material, which has been cultivated for more than 12 years, was chose in this experiment. The similarities and differences between autotetraploid and diploid rice was studied through morphological, agronomic and cytogenetic ways. The results showed that all the chromosome of autotetraploid materials are composed of 2N=4X=48, the pollen mother cells (PMC) meiosis behavior is normal, more than 99% chromosomes in metaphase I(MI) were federated and paired to form tetravalents or bivalents, which constitutes a consistent theory of genome. In the meiosis process, the material with a higher seed setting showed less chromosome abnormal than the material whose seed setting is lower. However, statistical analysis showed that the bivalent and tetravalent rate had no significant impact on seed setting, but the number of trivalent had a certain impact on seed setting. The result shows that the seed setting may be related to the meiosis behavior, which provides a basis to cultivate new autotetraploid germ line with high seed setting through the meiotic behavior. Furthermore, the agronomic and quality traits of autotetraploid rice high generation inbred material were statistically analyzed. The statistically analysis was focused on major agronomic traits such as: seed setting, grains per panicle, effective tillers and panicle length, as well as the important quality trait amylose content. The statistic data was compared with the data in 1996, when the first induced generation of autotetraploid material, and the result shows that after a multi-generation breeding, the agronomic traits has been significantly improved in all the materials, while the stability of agronomic traits also significant upgraded. For instant, the seed setting increased significantly, the average seed setting of all materials was significantly higher than the first induced generation, and the differences between different plants in the same species also significantly reduced, the average of the coefficient of variation (CV) was reduced from 41.15% in 1996 to 28.81% in 2008. Other important agronomic traits had improved in different degrees; the coefficient of variation within species is also reduced accordingly. In addition, the amylose content of autotetraploid and diploid materials was measured in this experiment. The results shows that the amylose content of some of the material differed from diploid parents significantly, it may caused by the genetic change during the inducing, autotetraploid materials intra-specific coefficient of variation (CV) average reduced from 6% in 1996 to 3.88% in 2008, shows that this is a significant increase of quality traits stability in autotetraploid rice. Agronomic traits of autotetraploid material shows some improvement after years of breeding, at the same time, after years of purification, all material within the germ-line gradually narrow the differences in traits indicates that autotetraploid rice genetic stability was enhanced, which laid a good foundation for the further autotetraploid rice breeding. Finally, this experiment studied the genetic differences between materials of two generations and researched the difference of genetic stability between diploid and autotetraploid rice materials through investigating the genetic polymorphism, genetic differences between materials and population genetic structure of inbred line materials of two consecutive years.18 pairs of SSR microsatellite markers for 15 materials of two generations were used in this experiment, and the total of 94 samples were analyzed. Through the amplification length polymorphism analysis of different materials and materials in different generations, the genetic distance between materials and generations was analyzed, a diploid and autotetraploid rice molecular fingerprint database and map rendering cluster were constructed. The result shows that the genetic distance is between 0.4757 to 0.2816 among different autotetraploid and diploid materials; the genetic distance between different generations of same species was less, but also shows a certain degree of genetic differences. The inter-generational genetic differences of autotetraploid materials were greater than of the diploid materials, which are 0.1359 to 0.0485 as the genetic distance; comparing with the 0.0388 of diploid materials. The result shows that high generation inbred autotetraploid rice material has a certain genetic stability, but the genetic variation between generations is still strong comparing with the source diploid materials. It indicates that, after many generations of purification cultivation, autotetraploid rice materials established a relatively stable genetic structure, at the same time, stronger variability than its diploid parents are useful in the breeding of new varieties, provides a genetic foundation to the agronomic and quality traits improvement. In addition, the analysis result shows that the through the molecular marker-assisted testing, rice genetic polymorphism between materials can effectively distinguish different species, provides a certain basis for molecular identification of varieties of rice. A series of investigation such as cytological identification, statistical analysis of agronomic traits, molecular marker-assisted cluster analysis was applied in this experiment to research genetic pattern of autotetraploid rice high generation inbred lines, revealed the genetic characteristics and the genetic composition of agronomic traits, provides a theoretical basis for the further selection of high quality autotetraploid rice.

Effects of hydrostatic pressure on microtubule organization and cell cycle in gynogenetically activated eggs of olive flounder (Paralichthys olivaceus)

Indirect immunofluorescence staining was used to detect cytological changes of isolated blastodisks during mitosis of flounder haploid eggs treated with hydrostatic pressure. Changes in microtubule structure and expected cleavage suppression were observed from blastodisk formation to the third cell cycle, with obvious differences between treated and control eggs. In most eggs, microtubules were disassembled and the nucleation capacity of the centrosome was temporarily inhibited after pressure treatment. Within 15-20 min after treatment, the nucleation capacity of the centrosome began to gradually recover, with slow regeneration of microtubules; approximately 25 min after treatment, the nucleation capacity of the centrosome recovered completely, regenerated distinct bipolar spindles, and the first mitosis ensued. During the second cell cycle, approximately 61% of the embryos were at the two-cell stage, with a monopolar spindle in each blastomere; that treatment was effective was based on second cleavage blockage. Approximately 15% of the eggs still remained at the one-cell stage and had a monopolar spindle (treatment was effective, according to the general model of first cleavage blockage). However, treatment was ineffective in approximately 15% of the embryos (bipolar spindle in each blastomeres) and in another 8% (bipolar spindle in one of the two blastomeres and a monopolar spindle in the other; both mechanisms operating in different parts of the embryo). This is the first report elucidating mitotic gynogenetic diploid induction by hydrostatic pressure in marine fishes and provides a cytological basis for developing an efficient method of inducing mitotic gynogenesis in olive flounder. (C) 2007 Elsevier Inc. All rights reserved.

Effects of cold shock on microtubule organization and cell cycle in gynogenetically activated eggs of olive flounder (Paralichthys olivaceus)

Cytological changes and subsequent mitotic processes were studied in gynogenetically activated eggs of olive flounder subjected to cold-shock treatment using indirect immunofluorescence staining of isolated blastodisks. Obvious differences between controls and treated eggs were detected during early cell division. The developmental process of haploid control was similar to that of the diploid control except several minutes delayed. Spindles disassembled by the cold-shock treatment regenerated soon after treatment, resulting in the occurrence of the first mitosis. The immature daughter centriole was easily depolymerized by cold-shock treatment, leading to the formation of the bipolar spindle in the first cell cycle and the formation of the monopolar spindle in the second cell cycle, resulting in chromosome set doubling. Some two-cell stage eggs had a monopolar spindle in one blastomere and a bipolar spindle in another during the second mitosis. These eggs had a high potency developing into haploid-diploid mosaics. To the best of our knowledge, this study is the first to clarify the mechanism of chromosome set doubling in marine fishes and provides a preliminary cytological basis for developing a reliable and efficient protocol for mitotic gynogenesis induction by cold-shock treatment in olive flounder.