基于rDNA ITS序列对绒泡菌目黏菌系统发育的探讨

绒泡菌目Physarida是黏菌纲Myxogastria最大的一个目,对其系统发育关系的研究一直是根据形态特征。为了从分子水平探讨绒泡菌目乃至黏菌纲的系统发育关系,以黏菌r DNA ITS通用引物对绒泡菌目5属8种黏菌的r DNA ITS进行扩增和测序,结合Gen Bank中已有的黏菌r DNA ITS序列,利用贝叶斯推断法(Bayesian inference,BI)和最大似然法(Maximum likelihood,ML)构建系统发育树。结果表明:绒泡菌目不同物种的r DNA ITS区在碱基组成和长度上差异明显,长度为777–1 445bp,G+C mol%在53.4%–61.9%之间。绒泡菌目与发网菌目Stemonitida聚类为两个明显的分支,在绒泡菌目分支上,绒泡菌科Physaraceae和钙皮菌科Didymiaceae各聚为一支,支持了形态学上以孢丝是否具有石灰质为依据区分这两个科的观点。由多份不同地理来源的鳞钙皮菌Didymium squamulosum材料组成的钙皮菌科又形成3个分支,证实了这个形态种是由地域来源广泛、繁殖亲和性各异和遗传变异较大的不同生物种组成的复合体。     

淡黄绒泡菌原质团的培养及其rDNA-ITS序列分析

首次实现了黏菌淡黄绒泡菌Physarum melleum原质团的燕麦琼脂培养,并通过ITS引物完成了淡黄绒泡菌rDNA的PCR扩增及序列分析。描述了其原质团的培养方法,提供了原质团rDNA的提取方法及ITS引物的扩增条件。利用邻近结合法构建系统发育树,对淡黄绒泡菌与相关分类单元的分子系统学关系进行了初步探讨。     

肌动蛋白基因和β-微管蛋白基因用于真黏菌系统发育的可行性研究

真黏菌是一类独特的菌物。目前对其进行的系统发育研究主要是基于形态特征,分子水平的系统发育研究上小亚基核糖体RNA基因和蛋白质合成延长因子基因研究相对较多。为了扩充能有效地进行真黏菌系统发育研究的基因资源,探讨了肌动蛋白基因和β-微管蛋白基因用于真黏菌系统发育的可行性。共获得14个基因序列,肌动蛋白基因和β-微管蛋白基因各7个。在GenBank中除多头绒泡菌Physarum polycephalum外并无其他真黏菌的肌动蛋白基因和β-微管蛋白基因序列,研究获得的14个基因序列为真黏菌基因的新序列。系统发育分析表明,肌动蛋白基因能够有效地将无丝菌目、团毛菌目、绒泡菌目和发网菌目区分为4个分支,其中发网菌目为一个独立的进化支,支持了根据子实体发育所认识的真黏菌纲内部具有两条进化路线的观点,因此显示出肌动蛋白基因对于真黏菌系统发育研究的重要价值。     

发网菌目黏菌子实体微量元素的初步分析

运用能散X-射线微区分析技术研究了发网菌目黏菌代表种的子实体的微量元素组成,结果表明:分属于7属的7种供试发网黏菌子实体囊被上的微量元素组成存在差异。尽管在显微镜下观察不到可见的如绒泡菌目黏菌子实体中存在的石灰质颗粒,但其中的钙元素所占比例均仍较高。同时,元素组成上的差异与子实体的颜色没有相关性。     

石斛内生炭角菌DNA提取方法优化及分子鉴定

本文对石斛内生炭角菌DNA提取方法进行优化,并基于ITS序列及其ITS2二级结构对其进行分子鉴定。比较试剂盒法及不同浓度（2%、3%、4%）CTAB法的DNA提取效果并对CTAB法进行优化;进行ITS序列的扩增及ITS2二级结构的预测。结果表明：4%的CTAB提取效果最佳,优化的CTAB法大大缩短了DNA提取时间;ITS序列与炭角菌属3个种的相似度均为99%,ITS2二级结构仅与Xylaria arbuscula的二级结构100%相似。本研究所优化的CTAB法能为后续相关研究提供便利;结合ITS序列及ITS2二级结构能对石斛内生炭角菌进行准确的分子鉴定。     

紫芝基因组rDNA序列特征及ITS2二级结构比较

紫芝栽培品种‘紫芝S2’(武芝2号)的ITS序列与NCBI数据库中5个紫芝菌株/分离株相似度高达99.79%-100%,在系统进化树上相聚成一类。本研究预测‘紫芝S2’基因组与参考基因组中的rRNA基因簇,分析rDNA结构及各构件序列间的多态性。从高质量‘紫芝S2’基因组中挖掘得到完整rDNA,序列全长40.377 kb,由4组串联重复的(18S、5.8S、28S、5S) rRNA基因簇组成,并含有完整的基因内间隔区(ITS1、ITS2)和基因间间隔区(IGS1、IGS2)。在紫芝S2的rDNA中,高度保守的28S rRNA基因间出现3个SNP和2个插入(1 bp,10 bp)位点;虽然第4条ITS2中有1个SNP位点,但紫芝S2的4条ITS2在二级结构上的分子形态高度一致,与ITS2数据库中其他紫芝菌株仅存在螺旋区间夹角的微小差异。由‘紫芝S2’基因组rDNA的ITS2生成的DNA条形码与二维码,可以作为该栽培品种鉴定与同源物种其他菌株鉴别的分子标记。     

绒泡黏菌属下分类等级的新安排

为了澄清黏菌纲绒泡菌属Physarum种级分类上存在的一些混乱,以便进一步澄清该属内分类单元的系统演化关系,对绒泡菌属81个种进行了比较形态学研究。结果表明,该属可划分成5组（section）。给出了绒泡菌属分组的检索表,并明确了每组所含的物种,通过分组对绒泡黏菌属下分类等级进行了新安排。     

松萝属的5.8S-ITS2二级结构比较分析

本研究以松萝属为研究对象,构建了该属核糖体5.8S和第二转录区间（ITS2）的二级结构模型,并对种间的结构差异进行了比较。结果显示,松萝属的结构与先前发表的真核生物ITS2二级结构模型非常相似。属内种间的结构差异主要集中在第二转录区间的臂上。核酸序列分析所产生的系统树与结构特征的比较结果相一致,表明ITS2的二级结构信息可以作为一种辅助的分类手段,适用于松萝属的种的鉴定和亲缘关系研究。     

荔枝nrDNA ITS序列多态性分析

为了解荔枝nrDNA ITS序列多态性,对荔枝4个品种的nrDNA ITS序列进行克隆测序,分析其序列特征、核苷酸多态性、遗传距离、5.8S保守基序、二级结构最小自由能、系统发育树及rRNA二级结构等。结果表明,荔枝nrDNA ITS序列在品种间、品种内均存在多态性,同时包含假基因序列,表明其ITS序列逃离了致同进化。分子方差分析（AMOVA）显示,荔枝nrDNA ITS序列遗传变异主要来源于品种内。自然杂交可能是荔枝ITS序列产生多态性的主要原因。     

实验室培养条件下绒泡菌科四种黏菌个体发育比较研究

本文应用悬滴培养、燕麦-琼脂培养等方法及显微成像技术对绒泡菌科Physaraceae 4种黏菌（黄头绒泡菌Physarum flavicomum、淡黄绒泡菌Physarum melleum、垂头绒泡菌Physarum album、针箍菌Physarella oblonga）的个体发育特征进行比较研究,首次实现了垂头绒泡菌在实验室条件下的完整生活循环。4种黏菌孢子萌发均为V-型开裂,但萌发时间有所不同,黄头绒泡菌萌发所需时间最短,仅需5h,而针箍菌萌发所需时间最长,需要2d;针箍菌和淡黄绒泡菌形成的黏变形体均可转变为游动胞,而相同条件下黄头绒泡菌和垂头绒泡菌并未有游动胞形成;4种黏菌原生质团的生长速率不同,在光照刺激下针箍菌、淡黄绒泡菌、黄头绒泡菌和垂头绒泡菌的原生质团分别经过2-3d、2-3d、7d和13d,发育成子实体;完成孢子到孢子的整个生活史,针箍菌和淡黄绒泡菌需要25-30d,黄头绒泡菌和垂头绒泡菌需要40-45d。     

Characteristics of the membranes of the pellicle of the ciliatePseudomicrothorax dubius

Summary The membranes of the pellicle of the ciliatePseudomicrothorax dubius are investigated using thin section electron microscopy and freeze-fracture replicas. The plasma membrane is covered by a surface coat and is connected to the outer alveolar membrane by short, sometimes branched, bridges. The inner alveolar membrane is coated on both sides. The epiplasm lies in intimate contact with the cytoplasmic surface of this membrane, and there is a corresponding deposit on the other surface. This deposit is regularly striated.The epiplasmic layer and the alveoli are interrupted at sites of cytotic activity,e.g., the attachment sites of trichocysts, the cytoproct, and the parasomal sacs. The striated deposit ends where the epiplasm ends, indicating a direct relationship between these two epimembranous layers.There is a deposit along the sides of the first part of the tip of the trichocysts, and in this region the trichocyst membrane is free of intramembranous particles.The membrane of the parasomal sacs has a coat on both surfaces. That on the extraplasmic surface is similar to the surface coat of the plasma membrane. The origin of the cytoplasmic coat is unknown. The cytotic activity of these sacs is indicated by their highly irregular profiles.     

The early development of the tail and the transformation of the shape of the nucleus of the spermatid of the domestic fowl,Gallus gallus

Summary The differentiation of the spermatid, especially in reference to the formation of the flagellum, and transformation of the shape of the nucleus was investigated in the domestic fowl.In the early stage of the spermatid, a prominent Golgi apparatus appears around the centrioles. The Golgi vesicles then surround the axial-filament complex which develops from the distal centriole. These vesicles fuse to form continuous membrane at the earliest stage of flagellar formation, and in the succeeding stage Golgi lamellae are attached to the plasma membrane of the developing flagellum. From these observations, it is assumed that Golgi apparatus may be a source of the membrane system of the flagellum.The microtubules distributed around the nucleus form the circular manchette. The anterior region of the nucleus with the manchette is cylindrical in shape and the posterior region without it remains irregular in shape. When the circular manchette has been completed, the whole nucleus acquires a slender cylindrical shape. The circular manchette then changes into the longitudinal manchette. The nuclei of spermatids without a longitudinal manchette are abnormal in shape. In view of these observations it is assumed that the nuclear shaping of the spermatid may be accomplished by circular manchette and the maintenance of shape of the elongated nucleus by longitudinal manchette.The authors wish to thank Mr. Takayuki Mori for his helpful suggestions and technical advices     

Characterization of the composition of the aqueous humor and the vitreous body of the eye of the frog Rana temporaria L

This study aimed to analyze the aqueous humor (AH) and the vitreous body (VB) of the eye of the adult frog Rana temporaria L. as a representative species of amphibians, which lead a semi-terrestrial life. The presence of collagen, albumin, uric acid and electron donors was shown in both media; however, there are slight differences in their concentrations. To determine collagen, a spectral-fluorescent probe, cyanine dye, was used. The presence of collagen in AH of the frog was found at the first time. The total content of electron donors (ascorbic and uric acids, tryptophan, and tyrosine) in VB and HA was roughly estimated at ~ 1.5 × 10^− 4 mol/L. Both VB and AH absorb light in similar UV regions. The total protein and albumin contents in AH were found to be somewhat higher than those in VB. The uric acid content was at an equally low level in both intraocular media. It is supposed that the similarity of VB and AH compositions shown in this work is due to some exchange between VB and AH contents in the course of accommodation. The role of intraocular fluids in physiological functions of the eye and in protecting the retina against UV light is discussed.     

Observations on the permeability of the choriocapillaris of the eye

Summary The choriocapillaris is a fenestrated capillary bed located posterior to the retinal pigment epithelium. It serves as the main source of supply to the photoreceptors, retinal pigment epithelium, and other cells of the outer retina. The permeability of these capillaries to intravenously injected ferritin (MW — approx. 480,000; mol. diam. 11 nm) was examined in the mouse, rabbit, and guinea pig, each of which is characterized by a different type of retinal vascularization. In all three species, the bulk of the ferritin remained in the capillary lumina, where it appeared to be blocked at the level of the diaphragmed fenestrae. Some ferritin was present in endothelial cell vacuoles. The results confirm previous work on the rat choriocapillaris and indicate that the barrier function of the choriocapillary endothelium is present even among species in which the retinal circulation differs significantly.Supported by NIH grant EY03418