Cyclin A2 is differentially expressed during oocyte maturation between gynogenetic silver crucian carp and gonochoristic color crucian carp

Silver crucian carp (Carassius auratus gibelio) is a unique gynogenetic fish. Because of its specific genetic background and reproduction mode, it is an intriguing model system for understanding regulatory mechanism of oocyte maturation division. It keeps its chromosomal integrity by inhibiting the first meiotic division (no extrusion of the first pole body). The spindle behavior during oocyte maturation is significantly different from that in gonochoristic fish. The chromosomes are first arranged in a tripolar spindle, and then they turn around and are reunited mutually to form a normal bipolar spindle. A new member of the fish A-type cyclin gene, cyclin A2, has been isolated by suppression of subtractive hybridization on the basis of its differential transcription in fully-grown oocytes between the gynogenetic silver crucian carp and gonochoristic color crucian carp. There are 18 differing amino acids in the total 428 residues of cyclin A2 between the two forms of crucian carps. In addition, cDNAs of cyclin A1 and cyclin B have also been cloned from them. Thus two members of A-type cyclins, cyclin A1 and cyclin A2, are demonstrated to exist in fish, just as in frog, humans, and mouse. Northern blotting reveals that cyclin A2 mRNA is more than 20-fold and cyclin A1 mRNA is about 2-fold in fully grown oocytes of gynogenetic silver crucian carp compared to gonochoristic color crucian carp. However, cyclin B does not show such a difference between them. Western blot analysis also shows that the cyclin A2 protein stockpiled in fully grown oocytes of gynogenetic crucian carp is much more abundant than in gonochoristic crucian carp. Moreover, two different cyclin A2 expression patterns during oocyte maturation have been revealed in the two closely related crucian carps. For color crucian carp, cyclin A2 protein is translated only after hormone stimulation. For silver crucian carp, cyclin A2 protein can be detected throughout the process of maturation division. The different expression of cyclin A2 may be a clue to understanding the special maturation division of gynogenetic silver crucian carp.

Comparative investigation on spindle behavior and MPF activity changes during oocyte maturation between gynogenetic and amphimictic crucian carp

The spindle behavior and MPF activity changes in the progression of oocyte maturation were investigated and compared with cytological observation and kinase assay between gynogenetic silver crucian carp and amphimictic colored crucian carp. MPF activity was measured by using histone I-Il as phosphorylation substrate. There were two similar oscillatory MPF kinase activity changes during oocyte maturation in two kinds of fishes with different reproductive modes, but there existed some subtle difference between them. The subtle difference was that the first peak of MPF kinase activity was kept to a longer-lasting time in the gynogenetic silver crucian carp than in the amphimictic colored crucian carp. It was suggested that the difference may be related to the spindle behavior changes, such as tripolar spindle formation and spindle rearrangement in the gynogenetic crucian carp.

MEIOTIC CHROMOSOME BEHAVIOR IN FEMALE INTERSEXES OF ARTIFICIAL TRIPLOID TRANSPARENT-COLORED CRUCIAN CARP

Chromosome behavior in meiosis was studied by air-drying, C-banding and surface-spreading methods in female intersexes of artificial triploid transparent-colored crucian carp (Carassius auratus). Chromosome pairing and contraction were obviously asynchronous. The preferential pairing of two homologous chromosomes was the major pattern of chromosome pairing, and a few triple pairing, repeated pairing, telomer or centromere associating and multiple pairing were also observed in the pachytene cells. The metaphase I cells were mainly composed of univalents, bivalents and trivalents, as well as few of other multivalents, such as tetravalents, pentavalents, hexavalents and heptavalents, were also found in some metaphase I cells. The chromosome elements including uni-, bi-, tri- and other multivalents varied considerably among the metaphase I cells, and the associating patterns of multivalents were also diverse. Some 6 n and 12 n cells, in which premeiotic endomitosis occurred once or twice, were found at the prophase and first metaphase of meiosis, and the pairing and associating patterns were basically similar to that of the triploid cells.

Spindle-like Lanthanide Orthovanadate Nanoparticles: Facile Synthesis by Ultrasonic Irradiation, Characterization, and Luminescent Properties

A general and facile ultrasonic irradiation method has been established for the synthesis of the lanthanide orthovanadate LnVO(4) (Ln = La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu) nanoparticles from an aqueous solution of Ln(NO3)(3) and NH4VO3 without any surfactant or template. X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), selected area electron diffraction (SAED), and photoluminescence (PL) spectra as well as kinetic decays were employed to characterize the as-prepared products. Ultrasonic irradiation has a strong effect on the morphology of the LnVO(4) nanoparticles. The SEM and TEEM images illustrate that the as-formed LnVO(4) particles have a spindle-like shape with an equatorial diameter of 30-70 nm and a length of 100-200 am, which are the aggregates of even.

Genome constitutions of Hystrix patula, H-duthiei ssp duthiei and H-duthiei ssp longearistata (Poaceae : Triticeae) revealed by meiotic pairing behavior and genomic in-situ hybridization

Genomic constitutions of three taxa of Hystrix Moench, H. patula, H. duthiei ssp. duthiei and H. duthiei ssp. longearistata, were examined by meiotic pairing behavior and genomic in-situ hybridization. Meiotic pairing in hybrids of H. patula x Pseudoroegneria spicata (St), H. patula x Elymus wawawaiensis (StH), H. patula x H. duthiei ssp. longearistata, H. patula x Psathyrostachys huashanica (Ns(h)), H. duthiei ssp. duthiei x Psa. huashanica, H. duthiei ssp. longearistata x Psa. huashanica, Leymus multicaulis (NsXm) x H. duthiei ssp. longearistata averaged 6.53, 12.83, 1.32, 0.29, 5.18, 5.11 and 10.47 bivalents per cell, respectively. The results indicate that H. patula has the StH genome and H. duthiei ssp. duthiei and H. duthiei ssp. longearistata have the NsXm genome. Results of genomic in-situ hybridization analysis strongly supported the chromosome pairing data; therefore it is concluded that the type species of Hystrix, H. patula, should be included in Elymus, and that H. duthiei ssp. duthiei and H. duthiei ssp. longearistata should be transferred to Leymus.

固着磨料加工工艺对磷酸盐钕玻璃亚表面缺陷的影响

散粒磨料研磨与固着磨料研磨是光学研磨加工过程中的两种主要手段,但两者材料去除的机制不同。目前针对高功率固体激光装置中的主要工作物质——磷酸盐激光钕玻璃的亚表面缺陷(SSD)研究相对较少,因此在实验的基础上,通过系统地研究固着磨料对磷酸盐激光钕玻璃的研磨工艺过程,分析了多种因素,如磨料粒径、载荷大小、机床转速,以及结合剂材料与冷却液等对钕玻璃亚表面缺陷形成的影响,并与散粒磨料研磨工艺所产生的亚表面缺陷进行了比较,对关键工艺参数进行定量,为高质量钕玻璃制造工艺的选型以及进一步优化亚表面缺陷提供了重要的参考数据。

磷酸盐钕玻璃表面/亚表面损伤特性实验研究

系统地研究了光学研磨过程中,磨料粒径、载荷大小以及机床转速对钕玻璃表面及亚表面损伤的影响。结果表明,机床转速和载荷基本不改变材料表面粗糙度,而较大载荷或较低机床转速产生较大的亚表面缺陷,表面粗糙度和亚表面缺陷缺陷深度基本与最大磨料粒径呈正比,载荷增倍使亚表面缺陷与表面粗糙度的常数比值增加0.05。研究结果为钕玻璃加工工艺改进提供了参考依据。

芍药属两物种染色体结构变异杂合性研究

芍药属由大约35个灌木和多年生草本种组成，分为三个组：牡丹组(Sect. Moutan)、北美芍药组(Sect. Onaepia)和芍药组(Sect. Paeonia)。四川牡丹(Paeonia decomposita Handel-Mazzetti)和块根芍药(P. intermedia Meyer)分别隶属于牡丹组和芍药组。在该属的所有种中，染色体基数均为 x = 5，最短的五号染色体是端部着丝粒染色体，很容易辨认。 本论文研究了块根芍药三个居群22个个体和四川牡丹两个居群13个个体的减数分裂。减数分裂异常广泛发生，以至于发现所有被研究的个体都有数量不等的桥、断片和单价体。结果表明在中期I，块根芍药第一个居群平均每个小孢子母细胞有2.17个棒状二价体和2.7个环形二价体，第二个居群平均每个细胞有2.04个棒状二价体和2.86个环形二价体，第三个居群平均每个细胞有2.21个棒状二价体和2.71个环形二价体。而在四川牡丹中，第一个居群平均每个小孢子母细胞有2.09个棒状二价体和2.81个环形二价体，第二个居群平均每个细胞有1.85个棒状二价体和3.08个环形二价体。 块根芍药第一个居群的平均减数分裂染色体构型是2n = 10 = 0.25 I + 4.87 II，第二个居群是2n = 10 = 0.20 I + 4.90 II，第三个居群是2n = 10 = 0.17 I + 4.92 II，在该种的平均构型是2n = 10 = 0.21 I + 4.89 II。四川牡丹第一个居群的平均减数分裂染色体构型是2n = 10 = 0.21 I + 4.90 II，第二个居群是2n = 10 = 0.14 I + 4.93 II，在该种的平均构型是2n = 10 = 0.20 I + 4.90 II。在块根芍药中，不同个体的配对系数变化范围在69.5%和81.07%之间，在四川牡丹中在72.97%和81.37%之间。 在后期I和末期I，出现了染色体桥、断片、落后染色体、不等分离等异常现象。最明显的减数分裂异常是后期I桥/断片。尽管在不同的居群中桥/断片异常出现的频率有所变化(块根芍药居群一是26.03%，居群二是11.67%，居群三是13.39%;四川牡丹居群一是7.59%，居群二是9%)，但是这种异常出现在所有个体中(块根芍药平均为18.67%，四川牡丹平均为7.69%)。结果表明，所有的个体都是染色体臂内倒位结构杂合体，广泛存在于野生自然居群中，可能存在某些选择优势。而且，桥的出现频率和断片的大小在个体之间是变化的，这因此表明在这两个种中存在不同的倒位。然而，这两个种在野生居群中是如何维持染色体结构杂合的，其维持机制还有待于进一步阐明，还需要更进一步的证据。 该研究还揭示了块根芍药和四川牡丹这两个种具有共同的第五号染色体减数分裂异常：与长臂相比，短臂在遗传距离和物理距离之间存在巨大的背离。短臂的遗传距离，通过交叉频率计算出来，约是长臂的三十分之一（块根芍药）。然而，物理距离用臂的比率表示，大约是长臂的三分之一，物理距离是遗传距离的十倍。 在四川牡丹红心桥居群和其它居群之间，臂比存在微小的差异，而且在芍药属不同的种内也发现了存在差异。在四川牡丹中，环形二价体(两个臂形成交叉)和棒状二价体(仅一个臂形成交叉)的比率是1.94 : 98.06，而在块根芍药中是3.42 : 96.58。在这两个种中，棒状二价体大大多于环形二价体。在第五号染色体的短臂上可能存在某些“搭车效应”，这表明第五号染色体的短臂上存在高度永久杂合，导致短臂高度保守、极为稳定。这与芍药属古老的分布格局、进化历史长可能存在某些联系。四川牡丹第五号染色体的后期I倒位桥出现频率非常低，仅为0.51 - 3.47%，平均为1.43%。而且断片长度是变化的，其变化范围在1.7 - 10.8 µm之间。

水稻花药绒毡层特异基因及减数分裂相关基因的分离与功能分析

　利用RNA减法杂交、差异筛选和5’-RACE等方法从水稻分离到了一花药绒毡层特异表达的基因RA39。Southern 杂交表明，RA39在水稻基因组中是以单拷贝的形式存在的。RT-PCR 结果初步表明，RA39是一水稻花药特异表达的基因。RNA原位杂交进一步表明，RA39主要在水稻花药的绒毡层中表达，而且在小孢子母细胞减数分裂期和四分体时期表达量最高。RA39 cDNA全长1013bp，编码298个氨基酸残基。 RA39 cDNA与数据库中的已知序列没有明显的相似性，由其推测的多肽与核糖体失活蛋白（ribosome-inactivating protein, RIP）的序列相似在19-34%之间。多重序列排列分析结果表明构成RIPs活性位点的5个关键氨基酸残基在RA39中是保守的，在蓖麻毒蛋白中分别为Tyr80、 Tyr123、 Glu177、 Arg180 and Trp211 。利用原核表达系统，通过蛋白质分离和纯化获得了在SDS电泳图谱上为单一条带的纯的RA39蛋白，用兔rRNA作底物进行的酶活性分析证明该蛋白有N-糖基化作用，是一种类型I的核糖体失活蛋白。反义转基因植株的花粉用TTC进行活性染色结果显示其活性明显减弱，成熟的T0代反义转基因植株的结实率明显降低，只有对照的20-60%。这说明，RA39蛋白可能和小孢子母细胞的发育相关。 　　酵母DMC1是减数分裂过程中同源染色体配对和重组修复所必需的减数分裂特异基因。根据酵母Dmc1和拟南芥AtDmc1的保守区设计简并性引物，通过RT-PCR和RACE等方法，从水稻中分离出了酵母DMC1的同源基因OsDMC1。RT-PCR分析表明，OsDMC1在花中表达量最高，在根中表达量较低，在叶片和幼芽几乎不表达。水稻基因组中有两个拷贝的OsDMC1。OsDmc1蛋白与酵母Dmc1和拟南芥AtDmc1氨基酸一致性分别为53%和81%。 　　酵母Spo11在减数分裂过程中具有催化DNA双链断裂从而起始同源重组的功能。以酵母Spo11氨基酸序列为探针和现有的数据库通过数据分析，结合RACE技术，克隆了水稻SPO11同源基因OsSPO11-1， OsSPO11-1是一个单拷贝基因，有3个外显子和2个内含子，在转录过程中通过内含子的可变剪切产生4个不同的转录本（OsSPO11-1A、OsSPO11-1B、OsSPO11-1C和OsSPO11-1），其中，OsSPO11-1A是一个未剪切的转录本，OsSPO11-1B包含内含子2，OsSPO11-1C包含内含子1，OsSPO11-1D是一个完全剪切的转录本。这些转录本编码的蛋白有一致的246氨基酸残基的C-端，包含了Spo11/TopVIA家族蛋白共有的5个功能基元，是该家族的新成员。OsSPO11-1A和 OsSPO11-1C在花中优势积累，OsSPO11-1B是花特异的，而OsSPO11-1D在营养器官中优势积累。在花中该基因主要在减数分裂的花粉母细胞和胚曩中表达，在减数分裂期的绒毡层细胞和不同花器官的微管束细胞中也表达。这些结果说明内含子涉及到了OsSPO11-1表达的器官特异性调节，该基因除了参与减数分裂的调节外，在体细胞的发育中可能起重要作用。

水稻减数分裂基因OsDMC1的功能研究

减数分裂是有性生殖物种世代交替的转折点;减数分裂不仅维持了基因组的稳定性，而且通过重组创造了遗传多样性。在减数分裂过程中，DNA复制一次后进行两次连续的细胞分裂。第二次减数分裂与有丝分裂类似，涉及到姊妹染色单体的分离;而第一次减数分裂是独一无二的，在这次分裂中同源染色体分离。为保证同源染色体的准确分离，同源染色体在减数分裂前期I通过一系列复杂的过程结合在一起形成稳定的二价体。这些复杂的前期I事件包括同源配对、联会和重组。分子遗传学、细胞学和生物化学研究已经表明酵母DMC1基因在减数分裂重组、配对和联会过程中起了重要作用。OsDMC1基因是酵母DMC1基因在水稻中的同源基因。本课题应用RNA干扰技术分析了该基因在水稻生长发育过程中的功能。利用本实验室设计的RNAi工具载体pWTC605构建OsDMC1-RNAi载体;并通过农杆菌介导的水稻愈伤组织转化法获得了转基因株系;进而通过PCR和Southern杂交鉴定筛选出阳性的OsDMC1-RNAi株系。OsDMC1-RNAi株系的营养生长正常，但结实率显著降低;成熟花粉的Alexander染色显示这些OsDMC1-RNAi株系的花粉是败育的。通过内源OsDMC1基因表达量的半定量RT-PCR、Western杂交分析以及OsDMC1特异性small RNA的Northern杂交鉴定，证实OsDMC1-RNAi株系的不育表型与RNA干扰介导的内源OsDMC1 mRNA和蛋白水平的降低是相关的。进一步深入的细胞学观察显示，OsDMC1基因的knockdown导致OsDMC1-RNAi株系的雄性减数分裂异常，表现为二价体形成缺陷、染色体不等分分离和异常四分体的产生;OsDMC1基因的knockdown同时还诱导了雄性减数分裂进程的改变。荧光原位杂交实验揭示OsDMC1-RNAi株系的减数分裂同源配对过程是缺陷的。这些研究结果表明OsDMC1基因是水稻减数分裂的必需基因，该基因在减数分裂同源配对过程中起了重要作用。