黄梁木维管组织细胞激光显微切割技术体系建立

黄梁木是华南地区重要的速生用材树种,其快速的次生生长主要取决于包括形成层细胞在内的维管组织细胞的发育进程。为了精准获取维管组织形成层细胞与高质量RNA,该研究以黄梁木幼苗为材料,通过石蜡切片与激光显微切割(LMD)结合的方法,成功获得形成层、木质部和韧皮部细胞。结果表明:优化石蜡切片流程,以卡诺/丙酮作为固定剂(丙酮浓度10%~50%),100%正丁醇作为脱水剂和透明剂,之后经过梯度渗蜡、包埋、切片、脱蜡后可获得形态完整的组织切片且保证了组织细胞RNA的完整性。调试LMD参数,放大倍数为10×~20×;激光能量为42~44;切割孔径为4~13;切割速度为16~25;最终获得的这3种细胞的RNA完整性(RIN)均大于5.0,即形成层细胞RIN=5.0,韧皮部细胞RIN=6.6和木质部细胞RIN=7.9,满足后续RNA-seq分析。该研究通过优化石蜡切片和LMD参数,成功建立了黄梁木幼苗茎段维管组织细胞的激光显微切割技术体系,为进一步揭示林木树种维管组织分裂与分化的调控机制奠定基础。     

AtRabD2b共抑制植株死亡茎段的显微和超微结构观察

采用树脂包埋技术,以AtRabD2b共抑制植株死亡发生茎段为实验材料,制备半薄切片和超薄切片,观察突变体茎段死亡的细胞学特征。结果表明:(1)共抑制植株茎细胞的死亡首先在表皮细胞层中发现,然后向切片圆周两侧以及内侧细胞蔓延。(2)共抑制植株茎细胞出现染色质边缘化、叶绿体内囊体片层膜数目减少、细胞器成分被液泡吞噬等异常现象。这些细胞学特征暗示共抑制植株茎段发生了细胞程序性死亡,由此推断AtRabD基因对拟南芥茎顶端细胞的生长有重要的维持作用。     

基于改良石蜡切片技术的飞蝗胚胎浆膜表皮发育模式及形态变化观察

【目的】通过优化飞蝗Locusta migratoria胚胎石蜡切片制备技术,研究飞蝗胚胎发育时期浆膜表皮变化规律。【方法】通过优化飞蝗胚胎固定前处理、洗涤、脱水和透明等步骤,改良胚胎期石蜡切片制备技术;制备飞蝗胚胎发育第3-10天的石蜡切片,通过HE染色观察浆膜表皮发育规律;通过制备飞蝗发育第6-8天从卵孔端至卵尾端的纵切切片,观察飞蝗胚胎转旋时期的形态变化。【结果】飞蝗胚胎期石蜡切片技术优化后的操作步骤为:固定前Na Cl O预处理和打孔、洗涤30min、脱水30 min和透明30 min。通过此方法可获得表皮结构完整且清晰的胚胎期组织切片。在30℃条件下,飞蝗胚胎浆膜表皮及几丁质沉积形成于胚胎发育的第5天,在第8天时开始降解。飞蝗胚胎的转旋发生在第6-7天,伴随着浆膜和浆膜表皮的形态变化。【结论】本研究优化了飞蝗胚胎石蜡切片制备步骤,揭示了飞蝗胚胎浆膜表皮及胚胎转旋的发育模式,为昆虫胚胎发育的研究提供了基础资料。     

冰冻切片的H&E和LFB染色技术的优化及其在自身免疫疾病中的应用

该文旨在探讨基于冰冻切片样品进行苏木素–伊红(hematoxylin-eosin,HE)染色和快蓝(luxol fast blue,LFB)染色方法的优化及其在实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)组织病理分析中的应用。取正常及EAE建模后的C57BL/6小鼠脊髓样品各5份,每份脊髓样品一分为二,分别制成冰冻切片和石蜡切片样品,通过设置不同优化条件对这两组切片进行HE和LFB染色,比较两组切片染色的结果。石蜡切片经过HE染色和LFB染色后细胞的形态结构和组织结构清晰,冰冻切片HE染色和LFB染色后细胞结构清晰度几乎和石蜡切片一致,进一步将该方法应用于分析β-arrestin2基因敲除对于小鼠EAE发生中的病理变化,发现能够很好地重复出基因敲除加重EAE疾病这一现象。冰冻切片染色优化后,基于冰冻切片进行HE染色和LFB染色可以达到石蜡切片类似的效果,并且实验过程简洁快速,大大缩短了实验时间,提高了效率,可以较好地应用于分析自身免疫疾病病理变化,该优化方法将在自身免疫疾病特别是多发性硬化动物模型的研究中有广泛的应用。     

脂肪组织石蜡切片制作方法探讨

目的探讨脂肪组织石蜡切片制作方法,改善和提高其切片质量。方法将送检的脂肪组织切开固定于中性甲醛液中;取材后将标本放入脱水机中,调整脱水机设置程序,在常规方法的基础上适当增加组织再固定及脱水、透明时间;包埋时采取挤压组织等改进方法。结果通过改进和调整后制作的脂肪组织蜡块平整无塌陷,石蜡切片优良,厚薄均匀,完整,无空洞、无挤压、镜下组织结构完整清晰,细胞无挤压、重叠等现象。细胞形态清晰,着色均匀艳丽。结论通过上述方法的改进,能尽可能多地减少脂肪组织的脂滴,增加脂肪组织的硬度,使组织和石蜡能很好的融合在一起,有利于切出完整的厚薄均匀的石蜡切片,提高制片质量。     

落葵薯粘液细胞分布及发育的解剖学研究

采用石蜡切片法对落葵薯(Anredera cordifolia(Tenore)Steenis)粘液细胞的分布及发育进行了研究。结果表明,粘液细胞普遍存在于落葵薯的茎、叶、叶柄中,粘液细胞单个散生分布于茎的髓及皮层组织中;叶的栅栏组织和海绵组织中都可见粘液细胞,且海绵组织中的数量明显多于栅栏组织中的;叶柄中的粘液细胞不多,主要分布在维管束四周的皮层组织中。粘液细胞的发育分为4个阶段:原始细胞阶段、液泡化阶段、成熟阶段和细胞质解体阶段。粘液细胞最早出现于第六叶原基,且其发育与茎、叶器官的组织分化不同步。     

江豚(Neophocaena asiaeorientalis)的消化器官Ⅱ. 肠、肝、胰

本篇为江豚(Neophocaena asiaeorientalis)消化器官研究的第二部分。 标本来源见李悦民(1984)。肠的长度和肝的重量都由新鲜标本测得。组织学材料用10%甲醛溶液固定,石蜡包埋,切片用苏木精曙红,PAS和Van Gieson法染色。     

3种桉树组培苗不定根发生发育过程的解剖学观察

以巨尾桉‘GL9’、尾巨桉‘DH32-29’和尾边桉‘XF35’3种桉树无性系组培生根苗为材料,采用常规石蜡切片技术,对石蜡切片制作过程中的固定环节进行了优化,观察了桉树不定根的发育过程。结果表明:(1)采用FAA固定液固定材料,可获得染色清晰,组织完整的桉树根系切片。(2)‘GL9’和‘DH32-29’在生根诱导8d后生出不定根,生根类型为皮部生根;‘XF35’在生根诱导12d后生出不定根,生根类型为愈伤组织生根;‘GL9’不定根的根尖和根基处均有细胞旺盛分裂,‘DH32-29’不定根只在根尖有细胞旺盛分裂,‘XF35’不定根则只在其根基处有大量旺盛分裂的细胞。     

野生大豆花发育相关基因GsLFY的功能研究

LEAFY/FLORICAULA (LFY/FLO)是植物特有的转录因子家族,在控制花器官的诱导与发育中起着重要的作用,但是与野生大豆花发育相关的LFY/FLO同源基因的研究尚未见报道。本研究从野生大豆中克隆获得1个LFY同源基因,命名为GsLFY,该基因CDS全长1224 bp,包含完整的开放阅读框,编码407个氨基酸。利用实时荧光定量PCR技术对GsLFY在不同组织中的表达情况进行了分析,结果显示GsLFY在根、花以及种子中表达,在茎、叶、茎尖中不表达; 在花发育的四轮不同器官中(萼片、花瓣、心皮和雄蕊)进行实时荧光定量PCR,结果显示GsLFY在花萼和雄蕊中表达,在花瓣和心皮中不表达。酵母单杂交实验结果显示,GsLFY具有转录激活活性。拟南芥原生质体瞬时表达结果表明,GsLFY定位于细胞核中。转GsLFY基因烟草植株开花期比对照提前约29 天,这为通过分子育种的方法获得花期改变的大豆新品种提供了基因资源和理论基础。     

Improved method of obtaining micro-core paraffin sections in dendroecological research

树轮气候学及树轮生态学研究在国内外取得了长足进展, 但对树轮与气候的响应机制缺乏深入的了解, 迫切需要进行以微树芯石蜡切片为基础的树轮生态学研究。形成层活动监测从生理生态学角度出发, 广泛用于树轮对气候变化响应的机制探讨研究中, 然而目前还没有对微树芯石蜡切片制作的方法作详细的论述和探讨。该文通过对祁连圆柏(Sabina przewalskii)等5个不同树种微树芯石蜡切片的制作实践, 对已有的植物石蜡切片技术进行改进, 增加组织软化的步骤, 并对其他步骤作了相应的调整, 详细介绍了微树芯石蜡切片制作的方法, 为树轮生态学、解剖学, 以及木材解剖学关于木质化材料石蜡切片的制作方法提供参考。     

In vitro floral induction from thin longitudinal sections and micro-cuttings of juvenile cork oak material

In vitro flowers were obtained from thin longitudinal sections excised at the proximity of the apical region or nodal regions of the main stem and axillary shoots of in vitro Quercus suber stock shoots as well as from field-grown seedlings. In vitro floral induction was also achieved from nodal segments of 8- to 9-month-old seedlings or from micro-cuttings of embryonic main shoot and its axillary shoots of 2-month-old seedlings. The more juvenile the material the shorter the period required to achieve flowering. Under the described experimental conditions we have thus been able to induce the expression of neoteny in a woody, long-cycle species such as cork oak.     

Application of cryo-compatible antibodies to human placenta paraffin sections

The hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE) -fixation and paraffin embedding technique has been described to expand possibilities for immuno-labellings due to low denaturation of proteins. In this study, the issue was addressed as to whether the HOPE technique could be a useful tool in placenta tissue-based studies when only cryo-compatible antibodies are available. Such antibodies can be used on cryostat sections only, giving results of considerably inferior morphological detail as compared to routinely fixed paraffin embedded tissue sections. Commercially available, only cryo-compatible, monoclonal antibodies against a conformational epitope of HLA-G (clone MEM-G/9) and leukocyte differentiation antigens CD56, CD163 and CD34 III were selected and applied to frozen sections, routinely formalin-fixed and HOPE-fixed paraffin sections. All tested antibodies immunolocalized their antigen on cryo sections and on HOPE-fixed but not formalin-fixed paraffin sections. The HOPE technique provides an excellent preservation of protein antigenicity together with well presented morphological details in paraffin embedded placenta tissues. The detection of native or conformation-dependent epitopes in paraffin sections expands the immunolocalization possibilities in placenta research and reproductive immunology.     

Morphological plasticity and adaptation level of distylous Primula nivalis in a heterogeneous alpine environment

Plant populations at high elevation face extreme climatic conditions and resource limitations. The existence of distylous species at different elevations can help us investigate their adaptation to high altitudes, the evolution of their morphological characteristics, as well as their responses to limited resources. Here, 17 populations of Primula nivalis at different elevations were evaluated regarding variations in plant morphological characteristics, biomass allocation, and morphological plasticity in a heterogeneous environment. Our results demonstrate that heterogeneous environments can affect plant morphological characteristics and resource allocation in each sexual morph of these plants. Moreover, environmental variations reduced morphological plasticity in the two plant morphs, and the plasticity of long style (LS) plants was greater than that of short style (SS) plants. There were significant negative correlations between morphological characteristics and elevation, rainfall, temperature, and sunshine, and these are the main variables that affect morphological characteristics and resource allocation of both morphs of P. nivalis plants in heterogeneous environments. The morphological characteristics of P. nivalis plants transplanted from high to lower elevations were not significantly different in either population. LS plants had greater morphological plasticity and adaptability in heterogeneous environments than SS plants. Elevational gradients and heterogeneous environments differentiated both morphs of P. nivalis plants with regards to morphology as well as adaptations. LS plants showed a higher level of adaptability than SS plants.     

地形和土壤特性对亚热带常绿阔叶林内植物功能性状的影响

植物功能性状与环境之间的关系是功能性状研究的核心问题.为了探讨地形和土壤特性的差异对亚热带常绿阔叶林植物功能性状的影响,并找到影响古田山亚热带常绿阔叶林植物形态和生理性状的主要环境驱动因子,我们于2008年和2009年夏天在古田山国家级自然保护区内24 ha大型监测样地测定了147个样方中115种常见木本植物的功能性状.所测性状包括3个生理生态性状(叶绿素含量、叶绿素荧光参数Fv/Fm和PIABS和枝条比导率)和4个形态性状(气孔密度、叶厚度、比叶面积和叶长/宽比).结合地形数据(平均海拔、凹凸度、坡度和坡向)和土壤数据(含水量、全氮含量、全磷含量、全碳含量和pH值),分析影响这些功能性状的主要驱动因子.排序结果显示,叶绿素含量随海拔和凹凸度的上升而下降,但与土壤中的氮含量和水分含量旱现正相关关系.由于古田山土壤呈酸性,土壤磷素缺乏,叶绿素荧光参数Fv/Fm和PIABS与土壤氮、磷含量呈现显著的负相关.枝条比导率与土壤含水量具有较为显著的正相关关系.比叶面积与海拔呈现正相关关系.研究结果表明,在小尺度上,海拔和凹凸度是影响亚热带常绿阔叶林植物功能性状晟关键的两个地形因子,而土壤含水量和伞氮含量是影响该地植物功能性状最主要的土壤因子.然而,由于土壤中磷的缺乏,诸如植物光合作用等一些重要的生理过程受到影响,使得某些性状与环境因子之间呈现出不同寻常的相关关系.     

Observation on Flower Bud Differentiation of Crape Myrtle in Red Soil Environment

Flower bud differentiation is a key component of plant blooming biology and understanding how it works is vital for flowering regulation and plant genetic breeding, increasing the number and quality of flowering. Red soil is the most widely covered soil type in the world, and it is also the most suitable soil type for crape myrtle planting. The flower buds of crape myrtle (Lagerstroemia indica) planted in red soil were employed as experimental materials in this study, and the distinct periods of differentiation were identified using stereomicroscopy and paraffin sectioning. We optimized the steps of dehydration, transparency, embedding, sectioning and staining when employing paraffin sections. When seen under a microscope, this optimization can make the cell structure of paraffin sections obvious, the tissue structure complete, and the staining clear and natural. The flower bud differentiation process is divided into 7 periods based on anatomical observations of the external morphology and internal structure during flower bud differentiation: undifferentiated period, start of differentiation period, inflorescence differentiation period, calyx differentiation period, petal differentiation period, stamen differentiation period, and pistil differentiation period. The differentiation time is concentrated from the end of May to mid-June. Crape myrtle flower bud differentiation is a complicated process, and the specific regulatory mechanism and affecting elements need to be investigated further.     

Induced compression wood formation in Douglas fir (Pseudotsuga menziesii) in microgravity.

In the microgravity environment of the Space Shuttle Columbia (Life and Microgravity Mission STS-78), were grown 1-year-old Douglas fir and loblolly pine plants in a NASA plant growth facility. Several plants were harnessed (at 45 degrees ) to establish if compression wood biosynthesis, involving altered cellulose and lignin deposition and cell wall structure would occur under those conditions of induced mechanical stress. Selected plants were harnessed at day 2 in orbit, with stem sections of specific plants harvested and fixed for subsequent microscopic analyses on days 8, 10 and 15. At the end of the total space mission period (17 days), the remaining healthy harnessed plants and their vertical (upright) controls were harvested and fixed on earth. All harnessed (at 45 degrees ) plant specimens, whether grown at 1 g or in microgravity, formed compression wood. Moreover, not only the cambial cells but also the developing tracheid cells underwent significant morphological changes. This indicated that the developing tracheids from the primary cell wall expansion stage to the fully lignified maturation stage are involved in the perception and transduction of the stimuli stipulating the need for alteration of cell wall architecture. It is thus apparent that, even in a microgravity environment, woody plants can make appropriate corrections to compensate for stress gradients introduced by mechanical bending, thereby enabling compression wood to be formed. The evolutionary implications of these findings are discussed in terms of "variability" in cell wall biosynthesis.     

木本植物性别决定基因研究进展

雌雄异株植物是研究性别决定遗传机制及性染色体起源与进化的理想材料, 而克隆性别决定基因是解析性别决定遗传机制的关键。木本植物中有丰富的雌雄异株植物, 且包括2种相反的性别决定系统: XY型(雌株为同配型的XX, 雄株为异配型的XY)和ZW型(雌株为异配型的ZW, 雄株为同配型的ZZ)。此外, 不同性别植株的经济价值也有所不同。在木本植物中开展性别决定机制研究不仅具有重要的理论意义, 还具有较高的生产应用价值。随着大规模基因测序技术的快速发展, 越来越多的木本植物性别决定基因被鉴定和克隆, 为解析雌雄异株植物性别决定机制和性染色体的演化过程提供了有力的实验证据。该文详细总结了近年来木本植物性别决定基因研究的重要进展, 并展望了未来的研究方向及发展趋势。     

欧洲黑杨MARs的分离克隆及序列分析

为获得林木植物核基质附着区(MARs)序列,诱导欧洲黑杨(Populus nigra)愈伤组织并制备悬浮细胞,裂解释放细胞核,除去非MARs的游离DNA片段,得到核基质。碱性缓冲液分离残留DNA与结合蛋白,回收并克隆残留DNA,测序后得到两个具有MARs序列特征的DNA片段A7和A23。序列特征分析结果表明它们都含有MARs的特征基元,通过与其他已发表的MARs序列比较,认为是从杨树中分离到的两个MARs片段,并预测了其功能。该研究是首次从木本植物获得MARs序列。     

红芽芋球茎片两步法离体快繁及其再生苗生理和光合特性研究

以江西铅山红芽芋(Colocasia esculenta L.Schott var.cormosus‘Hongyayu’)试管苗为材料,建立了芋球茎片两步法离体快繁体系,并对其再生苗的形态指标、染色体数目、生理和光合特性以及叶绿素荧光特性进行了检测。结果表明:(1)红芽芋球茎片单芽诱导的最佳培养基为MS+KT 2 mg/L+6-BA 1 mg/L+NAA0.1mg/L,诱导培养30d后将单芽从球茎片上分离,再接种到生根培养基(MS+KT 2mg/L+NAA 0.1mg/L)上培养30d即可形成完整植株,移栽成活率高达98%;(2)由球茎片单芽、丛生芽、不定芽离体快繁获得的红芽芋再生苗在形态指标、叶下表皮气孔参数、染色体数目、生理生化指标以及叶片光合特性参数和叶绿素荧光特性方面均无显著差异。说明红芽芋球茎片两步法离体培养的再生苗繁殖系数高、染色体数目稳定,该离体快繁体系可应用于江西铅山红芽芋的工厂化生产。     

山东银莲花叶片形态结构对异质生境和海拔变化的响应

山东银莲花为一分布极其狭域的稀有物种,对海拔600 m以上的针阔混交林和山顶灌丛两种异质的生境都具有较高的适应性。为探索其适应策略,选择两种异质生境中的5个海拔梯度样带,采用常规石蜡切片法和显微观察技术,对叶片进行观察、分析和测量,通过比较叶片外部形态特征参数和内部解剖结构的差异,推测其叶片适应海拔和异质生境的响应策略。结果表明:为适应阴暗、潮湿的针阔混交林和干旱、强光照的山顶灌丛两种不同环境,山东银莲花分别表现出不同的适应策略。针阔混交林下,叶片的背腹表皮毛密度、比叶面积和气孔相对开度较山顶灌丛的大,而气孔密度、叶片厚度、栅栏组织和海绵组织的厚度较山顶灌丛的小;山顶灌丛植株叶片栅栏组织细胞排列较林下更加整齐紧密。两种生境中叶片腹面表皮毛的长度、气孔相对开度都随海拔的升高而减小,且差异明显;而叶片厚度、比叶面积、气孔指数等对600 m以上海拔变化未表现出明显的规律性。本研究将为山东银莲花的保护和利用提供理论基础及依据,为其他植物的相关研究提供参考。