烟草新驱动蛋白NtTKR过表达对酵母生长的影响

为了解烟草新驱动蛋白NtTKR功能信息,构建了EGFP融合的NtTKR基因酵母表达载体并转入酿酒酵母W303诱导表达,荧光显微镜观察结果表明,诱导条件下的转化细胞发出明亮的绿色荧光,表明转入的NtTKR得到了高效表达.转化株在诱导和非诱导条件下培养3d,可见NtTKR过表达菌落比对照小的多;但通过绘制8h内生长曲线发现二者细胞数目无显著差别;表明NtTKR过表达对酵母细胞生长的抑制可能更多的是因为抑制其体积增大,而非分裂速度减慢引起的细胞数目降低.已知NtTKR在烟草受精卵、发育各个时期的胚、幼叶、根尖及茎尖等细胞分裂旺盛部位均优势表达,推测该基因在烟草中可能与上述各部位新生细胞的体积增大相关.     

HCY-2在正常人胚呼吸系统中的表达

目的:研究同型半胱氨酸诱导基因(HCY-2)在发育不同时期的正常人胚气管和肺中的表达,揭示HCY-2基因在呼吸系统发育中的作用.方法:取正常人胚气管和肺,采用免疫组织化学ABC法对气管和肺内HCY-2表达变化进行分析.结果:HCY-2在人胚气管和肺发育的整个时期均表达,其表达部位主要在气管及其各级支气管的上皮和气管腺腺细胞内.结论:HCY-2表达蛋白可能与人胚气管及肺的发育密切相关.     

人参体细胞胚胎发生过程中的生理变化

采用考马斯亮蓝G-250染色法、蒽酮法、愈创木酚法等方法,研究了人参体细胞胚胎发生中可溶性蛋白、多糖及淀粉含量和POD及PPo活性的变化,并对体细胞胚发生的不同时期培养物内源LAA和ABA含量进行了测定.结果表明,多糖和淀粉含量在早期胚时较低,可溶性蛋白含量、POD及PPO活性和IAA含量在早期胚时最高;在成熟胚时期,ABA含量最高,且ABA与IAA的比值在成熟胚时也较高.说明多糖和淀粉参与调节胚性细胞的分化和发育过程,POD及PPO和可溶性蛋白与早期胚形成密切相关;ABA与IAA比值高有利于体细胞胚的发育成熟.     

榛子SBP基因家族鉴定及其系统进化和表达分析

SBP蛋白是植物所特有的一类转录因子,并广泛存在于植物中.研究发现,该基因家族成员参与响应包括植物生长、发育和抗逆等生物学过程,其功能研究具有重要意义.榛子是一种主要以果实为产品的重要经济林木,且该物种SBP基因家族的研究也未见报道.本研究以生物信息学手段为基础,结合转录组数据,从中筛选得到10个榛子SBP-like基因(SPL基因),并对其编码蛋白理化性质、保守区域和模体进行预测和分析,进而对基因家族进化关系和花发育不同时期表达水平进行了分析.结果表明:榛子的10个SPL基因可以分为5个大组群;均含有该基因家族所特有的C3H和C2CH结构域以及一个核定位信号;同时,基于花发育不同时期的转录组数据分析,部分成员在不同时期表达水平存在显著差异,预示该基因可能与花果发育有关.该研究将有助于榛子SPL基因参与其花果发育分子调控机制的深入研究.     

普通小麦的体细胞胚发生、发育相关蛋白质的毛细管电泳法鉴定

采用高效毛细管电泳技术对普通小麦不同类型的愈伤组织进行蛋白质电泳分析，发现不同类型的愈伤组织中含有多数相同的蛋白质和少数差异蛋白质．结合形态与结构分析，表明差异蛋白质可能与体细胞胚的发生和发育相关．与普通电泳及双向电泳相比，该技术具有快速可靠的特点，可望发展成为鉴定愈伤组织类型及体细胞胚发育时期特异蛋白质的有效方法．     

家兔B细胞的最早来源部位

采用光镜和电镜技术对不同发育时期的家兔肝脏、骨髓和阑尾中的Ｂ细胞及其动态变化进行了系统观察，结果表明，家兔的胚肝可能是Ｂ细胞最早发生和分化的场所，可能具有与鸟类法氏囊类似的功能．家兔阑尾的淋巴组织在出生后逐渐发育形成，与鸟类法氏囊滤泡髓质部的形成不同，因此，阑尾可能不是Ｂ细胞的最早来源部位，而是外周淋巴器官．     

被子植物胚胎发育的分子遗传学研究

被子植物的胚胎发育有许多自身特点，它可分为形态发生，种子后熟两个阶段，前不仅产生了特定的组织器官或其原基，而且形成了根分生组织与茎端分生组织这两个主体模式元件，更重要的是决定了植物个体发育的程序与整体结构模式，体胚发生与合子胚发育经历相似的过程，体胚发生技术被广泛地用作检查合子胚发充早期子生物学事件的一种实验系统，但由于体胚发生与合子胚发育的基因表达也存在差异，对采用体胚发生技术研究合子胚发育机理的恰当性众说不一。除此之外，较有希望的研究方法还有遗传解剖法，人们已分离了大量与植物胚胎发育胶 有关的单基因突变体，对其研究的目的在于克隆与发育相关的那些基因，并进一步对之进行分析与鉴定以理解胚胎发育的分子机理，由于难以得珐足够量的材料用于分析，已分离到的大多数基因是胚发育较晚期特异性表达的基因，如种子储藏蛋白和LEA蛋白的基因，对它们的研究揭示了胚发育中基因表达调控的一些机理，如今已克隆到一些胚发育早期的基因，如与顶基轴向或辐射径向模式形成有关的基因，与细胞伸长，分裂，分化相关的基因等，本对被子植物中的胚胎发育及其研究进行了综合性阐述。     

拟南芥Patellin2相互作用蛋白的筛选及鉴定

Patellin 2(PATL2)蛋白是具有SEC14蛋白结构域的脂质转移蛋白,定位于细胞分裂后期的细胞板上,在细胞分裂过程中发挥重要的作用.同时,PATL2蛋白还受到植物激素和环境变化的调控,说明PATL2可能在植物激素和环境变化的调控下参与细胞分裂过程.为了深入了解PATL2蛋白的生物学功能,研究PATL2蛋白发挥作用的分子生物学机制,我们分析了它在植物体内的表达特点和与之相互作用的蛋白.我们通过免疫共沉淀与液相色谱-串联质谱法(LC-MS/MS)分析了过表达PATL2的转基因植株,并获得了多个可能与PATL2相互作用的蛋白,其中一个是细胞周期蛋白依赖性激酶CDKB2;2.通过观察PATL2启动子驱动的GUS报告基因在植物中的表达,发现PATL2基因在叶片、花瓣、花粉中广泛表达,但在角果中的表达非常有限,仅存在于角果的两端,这与已报道的CDKB2;2的组织分布部分相同.同时,烟草叶表皮细胞瞬时表达结果表明PATL2和CDKB2;2能够共定位于烟草叶表皮细胞的细胞膜和细胞质中.体外pull-down实验和双分子荧光互补实验(BiFC)实验分别确定了PATL2和CDKB2;2在植物体外和植物体内的相互作用.     

鸟类胚胎消化道原基细胞分化的研究

本文用不同发育时期鹌鹑胚与鸡胚的种间移植实验结果表明,相当于鸡胚H.＆ H.17—23期的鹌鹑胚后肠原基尚不具备自主分化的潜能,只能并入寄主大肠发育,但不同区域的消化道中胚层间质细胞对内胚层细胞具有不同的诱导能力.相当于鸡胚H.＆ H.28—23期的鹌鹑胚后肠原基已具有自主分化的潜能,在寄主鸡胚体腔内能定向发育成鹌鹑大肠.本文还对移植系统中消化道原基细胞的分化进行了分析.     

烟草质体分裂相关基因NtFtsZ cDNAs的克隆及功能分析

通过RT-PCR和cDNA末端快速扩增(RACE)方法从烟草中克隆了两个质体分裂相关基因NtFtsZ1-1和NtFtsZ1-2的cDNA.推导的氨基酸序列分析表明,两者均具有FtsZ蛋白的典型特征和GTP结合位点;N端氨基酸序列分析也表明,NtFtsZ1-1和NtFtsZ1-2都具有质体导肽特征,发现了在高等植物中至少存在两个具有质体导肽的FtsZ;基于氨基酸序列的分子谱系分析也支持这一结果.核酸杂交表明两者在植物的不同发育时期具有相似的表达谱.将这两个基因转入E.coli中表达,它们能影响宿主菌的正常分裂,初步验证了其功能.这些研究提示在高等植物中FtsZ可能具有更多样化的功能.     

驱动蛋白研究进展

本文介绍了驱动蛋白结构及其生物物理和化学方面的最新进展，分析了其定向运动可能的物质基础．     

Tracking kinesin-driven movements with nanometre-scale precision

Several enzyme complexes drive cellular movements by coupling free energy-liberating chemical reactions to the production of mechanical work. A key goal in the study of these systems is to characterize at the molecular level mechanical events associated with individual reaction steps in the catalytic cycles of single enzyme molecules. Ideally, one would like to measure movements driven by single (or a few) enzyme molecules with sufficient temporal resolution and spatial precision that these events can be directly observed. Kinesin, a force-generating ATPase involved in microtubule-based intracellular organelle transport, will drive the unidirectional movement of microscopic plastic beads along microtubules in vitro. Under certain conditions, a few (less than or equal to 10) kinesin molecules may be sufficient to drive either bead movement or organelle transport. Here we describe a method for determining precise positional information from light-microscope images. The method is applied to measure kinesin-driven bead movements in vitro with a precision of 1-2 nm. Our measurements reveal basic mechanical features of kinesin-driven movements along the microtubule lattice, and place significant constraints on possible molecular mechanisms of movement.     

Movement of microtubules by single kinesin molecules

Kinesin is a motor protein that uses energy derived from ATP hydrolysis to move organelles along microtubules. Using a new technique for measuring the movement produced in vitro by individual kinesin molecules, it is shown that a single kinesin molecule can move a microtubule for several micrometers. New information about the mechanism of force generation by kinesin is presented.     

Switch-based mechanism of kinesin motors

Kinesin motors are specialized enzymes that use hydrolysis of ATP to generate force and movement along their cellular tracks, the microtubules. Although numerous biochemical and biophysical studies have accumulated much data that link microtubule-assisted ATP hydrolysis to kinesin motion, the structural view of kinesin movement remains unclear. This study of the monomeric kinesin motor KIF1A combines X-ray crystallography and cryo-electron microscopy, and allows analysis of force-generating conformational changes at atomic resolution. The motor is revealed in its two functionally critical states-complexed with ADP and with a non-hydrolysable analogue of ATP. The conformational change observed between the ADP-bound and the ATP-like structures of the KIF1A catalytic core is modular, extends to all kinesins and is similar to the conformational change used by myosin motors and G proteins. Docking of the ADP-bound and ATP-like crystallographic models of KIF1A into the corresponding cryo-electron microscopy maps suggests a rationale for the plus-end directional bias associated with the kinesin catalytic core.     

Mechanics of the kinesin step

Kinesin is a molecular walking machine that organizes cells by hauling packets of components directionally along microtubules. The physical mechanism that impels directional stepping is uncertain. We show here that, under very high backward loads, the intrinsic directional bias in kinesin stepping can be reversed such that the motor walks sustainedly backwards in a previously undescribed mode of ATP-dependent backward processivity. We find that both forward and backward 8-nm steps occur on the microsecond timescale and that both occur without mechanical substeps on this timescale. The data suggest an underlying mechanism in which, once ATP has bound to the microtubule-attached head, the other head undergoes a diffusional search for its next site, the outcome of which can be biased by an applied load.     

白藜芦醇诱导HepG2细胞凋亡中线粒体差异蛋白鉴定

为研究线粒体在白藜芦醇诱导人肝癌细胞系HepG2细胞凋亡中的作用机制。分离提取白藜芦醇处理的HepG2细胞及对照组细胞的线粒体蛋白质,双向电泳分离差异蛋白质,飞行时间质谱鉴定差异蛋白点,摸索并建立了一种有效分离提取细胞线粒体蛋白质和双向电泳的方法。初步分析鉴定了四个显著性差异蛋白,着丝粒蛋白Kinesin protein和CENP-E降低,证明白藜芦醇对细胞周期及细胞骨架的调节作用,Peptidase (mitochondrial processing)表达降低,线粒体核糖体蛋白L7/L12（Mitochondrial ribosomal protein L7/L12, MRP L7/L12）表达升高,表明白藜芦醇诱导HepG2细胞与其对线粒体功能的影响有关。     

Retrograde transport by the microtubule-associated protein MAP 1C

Microtubules are involved in several forms of intracellular motility, including mitosis and organelle movement. Fast axonal transport is a highly ordered form of organelle motility that operates in both the anterograde (outwards from the cell body) and retrograde (from the periphery towards the cell body) direction. Similar microtubule-associated movement is observed in non-neuronal cells, and might be involved in secretion, endocytosis and the positioning of organelles within the cell. Kinesin is a mechanochemical protein that produces force along microtubules in an anterograde direction. We recently found that the brain microtubule-associated protein MAP 1C (ref. 7) is a microtubule-activated ATPase and, like kinesin, can translocate microtubules in an in vitro assay for microtubule-associated motility. MAP 1C seemed to be related to the ciliary and flagellar ATPase, dynein, which is thought to produce force in a direction opposite to that observed for kinesin. Here we report that MAP 1C, in fact, acts in a direction opposite to kinesin, and has the properties of a retrograde translocator.     

A novel genome-wide fulllength kinesin prediction analysis reveals additional mammalian kinesins

Kinesins are microtubule-based motor proteins that perform diverse functions[1―6], including the transloca- tion of vesicles, organelles, chromosomes, proteincomplexes, RNA-binding proteins (RNPs), etc. They also help to orchestrate microtubule dynamics …     

Localization of cytoplasmic dynein to mitotic spindles and kinetochores

What is the origin of the forces generating chromosome and spindle movements in mitosis? Both microtubule dynamics and microtubule-dependent motors have been proposed as the source of these motor forces. Cytoplasmic dynein and kinesin are two soluble proteins that power membranous organelle movements on microtubules. Kinesin directs movement of organelles to the 'plus' end of microtubules, and is found at the mitotic spindle in sea urchin embryos, but not in mammalian cells. Cytoplasmic dynein translocates organelles to the 'minus' end of microtubules, and is composed of two heavy chains and several light chains. We report here that monoclonal antibodies to two of these subunits and to another polypeptide that associates with dynein localize the protein to the mitotic spindle and to the kinetochores of isolated chromosomes, suggesting that cytoplasmic dynein is important in powering movements of the spindle and chromosomes in dividing cells.     

A structural change in the kinesin motor protein that drives motility

Kinesin motors power many motile processes by converting ATP energy into unidirectional motion along microtubules. The force-generating and enzymatic properties of conventional kinesin have been extensively studied; however, the structural basis of movement is unknown. Here we have detected and visualized a large conformational change of an approximately 15-amino-acid region (the neck linker) in kinesin using electron paramagnetic resonance, fluorescence resonance energy transfer, pre-steady state kinetics and cryo-electron microscopy. This region becomes immobilized and extended towards the microtubule 'plus' end when kinesin binds microtubules and ATP, and reverts to a more mobile conformation when gamma-phosphate is released after nucleotide hydrolysis. This conformational change explains both the direction of kinesin motion and processive movement by the kinesin dimer.