Duplication and independent selection of cell-wall invertase genes GIF1 and OsCIN1 during rice evolution and domestication

Background: Various evolutionary models have been proposed to interpret the fate of paralogous duplicates, which provides substrates on which evolution selection could act. In particular, domestication, as a special selection, has played important role in crop cultivation with divergence of many genes controlling important agronomic traits. Recent studies have indicated that a pair of duplicate genes was often sub-functionalized from their ancestral functions held by the parental genes. We previously demonstrated that the rice cell-wall invertase (CWI) gene GIF1 that plays an important role in the grain-filling process was most likely subjected to domestication selection in the promoter region. Here, we report that GIF1 and another CWI gene OsCIN1 constitute a pair of duplicate genes with differentiated expression and function through independent selection. Results: Through synteny analysis, we show that GIF1 and another cell-wall invertase gene OsCIN1 were paralogues derived from a segmental duplication originated during genome duplication of grasses. Results based on analyses of population genetics and gene phylogenetic tree of 25 cultivars and 25 wild rice sequences demonstrated that OsCIN1 was also artificially selected during rice domestication with a fixed mutation in the coding region, in contrast to GIF1 that was selected in the promoter region. GIF1 and OsCIN1 have evolved into different expression patterns and probable different kinetics parameters of enzymatic activity with the latter displaying less enzymatic activity. Overexpression of GIF1 and OsCIN1 also resulted in different phenotypes, suggesting that OsCIN1 might regulate other unrecognized biological process. Conclusion: How gene duplication and divergence contribute to genetic novelty and morphological adaptation has been an interesting issue to geneticists and biologists. Our discovery that the duplicated pair of GIF1 and OsCIN1 has experiencedsub-functionalization implies that selection could act independently on each duplicate towards different functional specificity, which provides a vivid example for evolution of genetic novelties in a model crop. Our results also further support the established hypothesis that gene duplication with sub-functionalization could be one solution for genetic adaptive conflict.

Study on early-stage development of conchospore in Porphyra yezoensis Ueda

Porphyra yezoensis Ueda (Rhodophyta) is a seaweed of economic importance with a typical dimorphic life cycle consisting of a leafy gametophyte and a filamentous sporophyte. Recently, it has been recognized as a model system for fundamental and applied studies in marine biological sciences. Conchospore, a major spore linking the two distinct multicellular phases in the life cycle, is most widely used in the breeding of P. yezoensis. In this paper, the early-stage development of conchospore, including the attachment and the cell wall formation, was studied with fluorescent reagents staining and Scanning Electron Microscopy detection. Results displayed: (I) the cell wall began to be generated after culturing for 4 h in the attached conchospores; (2) the initially released conchospores were plastids with some filmy, amorphous substance on the surface, and they attached to the fibers firmly via the actively secreted mucilaginous substances after their touch to the fibers; (3) cellulase and pectolase prohibited the attachment of conchospores in the different ways; and (4) only attached conchospores generated cell walls and developed normally, while the suspending ones could not. It indicated that the cellulose played crucial roles in the permanent attachment as the pectin did in the initial attachment. The conchospore attachment seemed to trigger the cell wall formation and the further development. Affects of light on the development of conchospores were also discussed. The results showed that high intensity (200 mu mol.m(-2).s(-1)) and long-wave (>= 580 nm) light facilitated the division rate of conchospores. (C) 2008 Elsevier B.V. All rights reserved.

紫菜壳孢子萌发过程及其世代差异研究

紫菜是一类海洋藻类研究的模式系统，具有重要的经济价值和理论研究意义。本研究基于紫菜的世代交替生活史，对其壳孢子萌发过程进行了研究，并对丝状孢子体与叶状配子体的世代差异进行了分析，主要包括以下三部分： 1） 对紫菜丝状孢子体与叶状配子体的连接枢纽——壳孢子的发育过程及其光影响进行了研究与讨论，发现壳孢子只有在附着后才能形成细胞壁并发育，说明壳孢子的附着是触发了壳孢子细胞壁的形成以及后期发育的开关，亦即附着是触发紫菜世代交替过程中配子体转录组表达的开关；纤维素酶和果胶酶均能抑制壳孢子附着，但影响机制各不相同，推测果胶质主要介导壳孢子的初始附着，而纤维素则与永久附着相关；波长≥580 nm的高强度（200 μmol•m-2•s-1）可见光有利于壳孢子早期发育。 2） 结合现有藻类数据，对坛紫菜丝状孢子体阶段11000 EST数据进行了大规模的生物信息学分析，结果首次发现坛紫菜丝状孢子体中可能存在PCK型C4光合固碳途径，并筛选出44条在紫菜孢子体中表达上调的代表基因。 3） 结合坛紫菜、条斑紫菜、海带和红毛菜，对红藻和褐藻等大型海藻孢子体与配子体阶段代表基因Rubisco的表达与羧化酶活性差异进行了研究分析，结果表明Rubisco的表达量和初始羧化酶活在其配子体中均显著高于其孢子体世代，即与藻体不同世代的相对复杂度无关，而与染色体倍性相关，说明Rubisco的世代差异极有可能与染色体倍性相连锁，因而可能是海藻世代交替过程中的重要功能基因。

Deletion of yeast CWP genes enhances cell permeability to genotoxic agents

We have previously reported the development of a novel genotoxic testing system based on the transcriptional response of the yeast RNR3-lacZ reporter gene to DNA damage. This system appears to be more sensitive than other similar tests in microorganisms, and is comparable with the Ames test. In an effort to further enhance detection sensitivity, we examined the effects of altering major cell wall components on cell permeability and subsequent RNR3-lacZ sensitivity to genotoxic agents. Although inactivation of single CWP genes encoding cell wall mannoproteins had little effect, the simultaneous inactivation of both CWP1 and CWP2 had profound effects on the cell wall structure and permeability. Consequently, the RNR3-lacZ detection sensitivity is markedly enhanced, especially to high molecular weight compounds such as 4-nitroquinoline-N-oxide (> sevenfold) and phleomycin (> 13-fold). In contrast, deletion of genes encoding representative membrane components or membrane transporters had minor effects on cell permeability. We conclude that the yeast cell wall mannoproteins constitute the major barrier to environmental genotoxic agents and that their removal will significantly enhance the sensitivity of RNR-lacZ as well as other yeast-based genotoxic tests.

Study on the Cell Structure and Compressive Behavior of Biodegradable Poly(epsilon-caprolactone) Foam

Biodegradable poly(e-caprolactone) (PCL) foams with a series of controlled structures were prepared by using chemical foaming method. The cell morphology was detected by scanning electron microscope (SEM). The compressive behavior of the foams was investigated by uniaxial compression test. The effect of density and structural parameters on the foam compressive behavior was analyzed. It was found that the relative compressive modulus has a power law relationship with relative density. Increasing of both the cell wall thickness and the cell density lead to higher compressive modulus of the foam; however, the cell size has no distinct effect on compressive behavior.

Transient expression of lacZ in particle bombarded Gracilaria changii (Gracilariales, Rhodophyta)

Using a Biolistic PDS 1000/He system, healthy thalli of Gracilaria changii were bombarded with gold particles coated with plasmid DNA containing the lacZ reporter gene. Transient expression of lacZ was observed in bombarded thalli under the rupture-disc pressures of 4482, 6206, 7584 and 8963 KPa, two days after bombardment. Although G. changii exhibits a slight blue background, positive expression and the background colour can be clearly differentiated. The results indicate that lacZ could be a useful reporter gene and that SV40 promoter could be an effective promoter for Gracilaria transformation.

Assembly of the protoplasm of Codium fragile (Bryopsidales, Chlorophyta) into new protoplasts

The cell organelles of the coenocytic alga Codium fragile (Sur.) Hariot aggregated rapidly and protoplasts were formed when its protoplasm was extruded out in seawater. Continuous observation showed that there were long and gelatinous threads connecting the cell organelles. The threads contracted, and thus the cell organelles aggregated into protoplasmic masses. The enzyme digestion experiments and Coomassie Brilliant Blue and Anthrone stainings showed that the long and gelatinous threads involved in the formation of the protoplasts might include protein and saccharides as structure components. Nile Red staining indicated that the protoplast primary envelope was non-lipid at first, and then lipid materials integrated into its surface gradually. The fluorescent brightener staining indicated that the cell wall did not regenerate in the newly formed protoplasts and they all disintegrated within 72 h after formation. Transmission electron microscopy of the cell wall of wild C. fragile showed electron-dense material embedded in the whole cell wall at regular intervals. The experiments indicated that C. fragile would be a suitable model alga for studying the formation of protoplasts.

Embryology of Swertia (Gentianaceae) relative to taxonomy

The embryological features of three species of Swertia (s.l.) - S. erythrosticta, S. franchetiana, and S. tetraptera were characterized, and the observations were used, together with previously gathered data on other species, to evaluate a recently proposed polyphyly, based on molecular data, of Swertia s.l. Comparisons of species within the genus showed that they have diversified embryologically, and there are significant between-species differences. Notable features that vary between species include the number of cell layers that form the anther locule wall, the construction of the wall of the mature anther, tapetum origin, the cell number in mature pollen grains, the structure of the fused margins of the two carpels, the ovule numbers in placental cross-sections, the shape of the mature embryo sac, the degree of ovule curvature, antipodal variation and the presence of a hypostase, and seed appendages. They share characters that are widely distributed in the tribe Gentianeae, such as a dicotyledonous type of anther wall formation, a glandular tapetum with uninucleate cells, simultaneous cytokinesis following the meiosis of the microsporocytes, tetrahedral microspore tetrads, superior, bicarpellary and unilocular ovaries, unitegmic and tenuinucellar ovules, Polygonum-type megagametophytes, progamous fertilization, nuclear endosperm, and Solanad-type embryogeny. The presence of variation in embryological characters amongst the species of Swertia s.l. strongly supports the view that Swertia s.l. is not a monophyletic group. Frasera is better separated from Swertia s.l. as an independent genus, and is only distantly related to Swertia s. s. judging from the numerous differences in embryology. Swertia tetraptera is very closely related to Halenia, as they show identical embryology. (C) 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 155, 383-400.

Fracture Mechanisms And Size Effects Of Brittle Metallic Foams: In Situ Compression Tests Inside Sem

In situ compressive tests on specially designed small samples made from brittle metallic foams were accomplished in a loading device equipped in the scanning electron microscopy (SEM). Each of the small samples comprises only several cells in the effective test zone (ETZ), with one major cell in the middle. In such a system one can not only obtain sequential collapse-process images of a single cell and its cell walls with high resolution, but also correlate the detailed failure behaviour of the cell walls with the stress-strain response, therefore reveal the mechanisms of energy absorption in the mesoscopic scale. Meanwhile, the stress-strain behaviour is quite different from that of bulk foams in dimensions of enough large, indicating a strong size effect. According to the in situ observations, four failure modes in the cell-wall level were summarized, and these modes account for the mesoscopic mechanisms of energy absorption. Paralleled compression tests on bulk samples were also carried out, and it is found that both fracturing of a single cell and developing of fracture bands are defect-directed or weakness-directed processes. The mechanical properties of the brittle aluminum foams obtained from the present tests agree well with the size effect model for ductile cellular solids proposed by Onck et al. (C) 2008 Elsevier Ltd. All rights reserved.

植物细胞壁富含羟脯氨酸糖蛋白生化特性及基因克隆的研究

植物细胞壁富含羟脯氨酸糖蛋白生化特性及基因克隆的研究分成三部分： 第一部分，首先从胡萝卜愈伤组织的细胞壁中获得0.2M氯气钙可溶性蛋白质组分, 经10% TCA沉淀及羧甲基纤维素层析，分离得到一个伸展蛋白，并以SDS-聚丙烯酰胺凝胶电泳，氨基酸组成分析，分子电镜观察对所得的伸展蛋白进行了鉴定，结果表明，在胡萝卜愈伤组织中只存在一种伸展蛋白;SDS-聚丙烯酰胺凝胶电泳的考马斯蓝染色和PAS反应均为阳性，表明伸展蛋白除蛋白质组分还含有糖基;氨基酸组成分析表明，羟脯氨酸的克分子含量约占全部氨基酸的40%，丝氨酸的克分子含量约为羟脯氨酸的四分之一，含有大量的碱性和中性氨基酸，而只含有非常少量的酸性氨基酸，并且这些氨基酸克分子百分数也接近于胡萝卜富含羟脯氨酸糖蚤白基因推测出的克分子百分数。这些结果表明已经得到了一个电泳纯的伸展蛋白。伸展蛋白分子电镜观察证明它是一个棒状分子，长度为87纳米。 第二部分，用得到的伸展蛋白免疫家兔和大鼠，得到的抗体的免疫双扩散效价分别为1:2和1:1，用酶联免疫吸附分析测定的兔抗体效价为1:12，800，同时还建立了竞争性酶联免疫吸附分析测定伸展蛋白含量的标准曲线，线性范围为10-0.00001微克。利用得到的抗体对大豆下胚轴伸展蛋白的合成进行了研究。免疫荧光定位表明，大豆下胚轴表皮细胞及表皮下几层薄壁细胞有大量的荧光标记，并且这些荧光标记大部分分布在细胞质内。大豆下胚轴O.lM Tris-HCl pH7.4和0.2M氯化钙的提取物Western Blotting分析证明0.2M氯化钙提取物有与胡萝卜伸展蛋白电流性质相似的组分，并且这个组分在真菌诱导物处理的大豆下胚轴中的积累明显高于受伤处理的下胚轴。受伤和真菌诱导物处理大豆下胚轴中伸展蛋白积累的变化已经用Western Blotting分析和斑点酶联免疫吸附分析来观察，发现真菌诱导物处理的对灰斑病抗性的大豆下胚轴能够较快地积累伸展蛋白（24 - 48小时），而敏感品系的大豆下胚轴则合成伸展蛋白较晚（43-72小时）。在观察大豆下胚轴免疫荧光定位也发现类似的结果。对伸展蛋白基因的转录活性的初步研究认为抗性品系的大豆下胚轴同源mRNA转录可能早于24小时，而敏感品系大豆下胚轴同源mRNA转录可能在24小时后。以上结果认为大豆下胚轴含同源的mRNA和蛋白质组分，因此推测大豆基因组DNA有伸展蛋白基因。 第三部分，根据第二部分得到的结果，用已经得到的编码胡萝卜伸展蛋白的基因（克隆于pUC8质粒载体中）作探针，与大豆基因组DNA的EcoRI部分酶解片段杂交寻找大豆伸展蛋白基因，已经发现四个片段与探针DNA有同源性。它们的分子量分别约为23kb，8kb，5kb和2.8kb，并且23kb片段可能有更高的同源性。将23kb片段插入pUC9质粒载体上进行可隆，并用菌落原位杂交筛选获得5个克隆，对其中两个克隆用ECoRI酶解并进行分子杂交分析，重组质粒被EcoRI切成四个片段。2.8kb片段为pUC9栽体，2kb片段为与pDC5AI质粒伸展蛋白同源性较好的片段。对于23kb片段的重组质粒有待于进一步的分析。

白皮松花粉管发育过程中RNA、蛋白质和多糖类物质的合成及其动态变化

花粉管是有花植物受精过程中雄性生殖单位的载体，它同根毛、真菌菌丝一样，具有典型的极性顶端生长模式。裸子植物花粉与被子植物相比，具有萌发时间较长，生长缓慢等特点。但是目前人们对于裸子植物花粉萌发和花粉管生长的机理还不清楚。本文以裸子植物白皮松（Pinus bungeana）的花粉为材料，采用细胞学和生理生化方法，包括应用普通光学显微镜、荧光显微镜、激光扫描共聚焦显微镜、显微红外光谱（FTIR）和透射电镜（TEM）等技术，对其花粉萌发和花粉管生长过程进行了较为系统的研究，旨在进一步揭示裸子植物花粉管发育的调控机制。 本论文首先研究了外源Ca2+ 和3种调钙药物（A23187、EGTA、TMB8）对白皮松花粉萌发和花粉管生长的影响。结果表明，在离体培养条件下，高浓度的Ca2+（1%）能完全抑制白皮松花粉的萌发，低浓度的Ca2+ 则影响不大，而花粉萌发和花粉管生长的最适Ca2+ 浓度为0.01%。用Ca2+ 载体A23187、Ca2+ 螯合剂EGTA和钙通道阻滞剂TMB8分别处理花粉后，花粉萌发和花粉管生长均受到抑制。另外，用 Ca2+ 荧光探针 Fluo-3AM标记，对Ca2+ 的分布变化进行了观察，发现在花粉萌发的初期，Ca2+ 向萌发孔聚集。在正常生长的花粉管中Ca2+ 呈梯度分布，顶端荧光最强。与对照相比，A23187处理后花粉粒中荧光增强，而EGTA和TMB8处理的花粉粒中荧光强度均减弱。并且这3种调钙药物还破坏了花粉管顶端的Ca2+ 浓度梯度，最终导致花粉管的生长受阻。 花粉萌发和花粉管的生长依赖于RNA和蛋白质的不断合成。在放线菌素D的存在下，花粉萌发基本不受影响，但花粉管的生长速度下降，花粉管中RNA含量也减少。而经过放线菌酮处理后，花粉萌发和花粉管生长均受到抑制，花粉管中蛋白质含量降低，同时花粉管顶端显著膨大。通过SDS-PAGE的结果表明，花粉粒萌发前后蛋白质图谱有明显差异。FTIR光谱分析表明，两种抑制剂处理均导致花粉管壁的化学组成发生了变化，例如蛋白质和饱和酯含量减少，而羧酸的含量增加。此外，由放线菌酮和放线菌素D处理后，花粉管的超微结构也发生了明显变化，其中特别是花粉管顶端的分泌系统遭到严重破坏。 纤维素的正常合成对于白皮松花粉管的生长是必需的。在正常培养基中添加纤维素生物合成抑制剂2，6-二氯苯腈（DCB）后，花粉萌发几乎不受影响，但是花粉管的形态发生异常，生长速率降低。DCB处理还导致花粉管壁中纤维素含量下降，而胼胝质在花粉管顶端积累。用识别酯化果胶的JIM7和识别酸性果胶的JIM5对离体培养的白皮松花粉管进行标记后，发现果胶成分呈异常分布图式。FTIR光谱分析结果表明花粉管细胞壁中蛋白质、羧酸以及饱和酯含量增加。同时，在电镜下观察发现，花粉管细胞壁顶端呈现不均匀加厚，其中主要的细胞器，如高尔基体和线粒体等膜结构均遭到破坏。 上述结果说明，白皮松成熟花粉粒中已含有花粉萌发和花粉管早期生长所必需的Ca2+ 和RNA，但是在花粉管的后续伸长过程中仍需要外源Ca2+ 的参与以及新RNA、蛋白质的不断合成。与被子植物不同，裸子植物花粉萌发的启动也需要新蛋白的合成。尽管在花粉管中纤维素的含量很低，但是对于细胞壁的构建、花粉管的正常形态的维持起着关键作用。