RcAGL61基因调控雄蕊和花瓣之间转变影响月季花瓣数量

【目的】为了探究月季重瓣性状的调控机制,课题组前期筛选到一个与花发育相关的AG同源基因RcAGL61,本研究对该基因的功能进行了分析。【方法】利用荧光定量PCR（qRT-PCR）对该基因在‘窄叶藤本月季花’ב月月粉’杂交群体中重瓣株系和单瓣株系花芽5个发育时期的表达模式进行了分析,以重瓣株系和单瓣株系为材料,克隆RcAGL61,并进行生物信息学分析、亚细胞定位及VIGS实验。【结果】（1）该基因表达水平在单瓣株系的五个发育时期均显著高于重瓣株系,在单瓣株系花发育的S4-S5期比S1-S3期表达量明显升高。（2）RcAGL61编码区序列在单瓣株系和重瓣株系中一致,长度为495 bp,与RcAG基因序列相似度为30.75%,编码164个氨基酸,含有一个MADS-Box保守结构域,属于MADS-Box基因家族。（3）亚细胞定位发现RcAGL61蛋白定位于烟草表皮细胞的细胞核。（4）沉默该基因后,瓣化雄蕊数量增加,雄蕊数量减少,花瓣数量增加,萼片数量和雌蕊数量无显著变化。【结论】RcAGL61参与调控了雄蕊原基和花瓣原基间的转变,影响了月季的花瓣数量。     

不结球白菜BcLRK01基因对芜菁花叶病毒及胁迫诱导的响应

通过cDNA-AFLP技术,从芜菁花叶病毒(TuMV)侵染的不结球白菜幼叶中分离到一条差异表达的基因片段,克隆获得其cDNA全长为2 124bp,编码707个氨基酸的富亮氨酸重复类受体激酶,命名为BcLRK01。利用实时定量PCR研究了该基因在TuMV侵染及高盐、冷胁迫、水杨酸(SA)、茉莉酸(JA)、乙烯(ET)等处理下的表达情况,结果显示,TuMV侵染、高盐、冷胁迫、SA、JA和ET等均能诱导BcLRK01不同程度的表达,说明该基因可能是不结球白菜病毒病的病程相关基因,同时也参与高盐和冷胁迫以及SA、JA、ET等的信号途径。     

不结球白菜BcTuR1基因的克隆及表达

从不结球白菜抗芜菁花叶病毒(TuMV)品种‘短白梗'中克隆到一个抗TuMV相关基因,命名为BcTuR1(GenBank登录号FJ600374).该基因核苷酸序列全长1 019 bp,编码162个氨基酸.BcTuR1基因与芥菜抗病毒基因相似性最高为96%,其它没有与该基因相似性高于50%的序列.基因组DNA杂交表明,BcTuR1可能属于一个较小的多基因家族.实时定量PCR检测表明,芜菁花叶病毒能够诱导不结球白菜BcTuR1基因的转录表达,其在不结球白菜叶片中的表达特征说明它可能参与寄主对病毒的抗性.     

应用双分子荧光互补技术分析米曲霉Fus3与Ste12之间的蛋白互作

【背景】双分子荧光互补(Bimolecularfluorescencecomplementation,BiFC)在水稻恶苗病菌(Fusarium fujikuroi)等微生物蛋白互作中的应用已有报道,但在工业菌株米曲霉(Aspergillus oryzae)中还未见应用。【目的】探究米曲霉中Fus3和Ste12蛋白在生长发育中可能存在的相互作用关系,建立在米曲霉活细胞中检测蛋白互作的方法,即BiFC体系。该系统可用于特异性、可视化米曲霉目标蛋白在活细胞中的定位,并且可以更加直观地探究蛋白之间是否存在相互作用。【方法】利用MultisiteGateway复杂载体构建技术,使用切开的绿色荧光蛋白,将荧光蛋白分子的两个片段N端和C端分别与米曲霉Fus3和Ste12蛋白融合,对获得的转化株进行荧光观察。通过BiFC系统检测蛋白之间的相互作用。【结果】成功转化的米曲霉菌丝中观察到荧光,Fus3和Ste12在米曲霉中存在相互作用。【结论】通过BiFC技术证实蛋白质Fus3和Ste12在无性繁殖菌株米曲霉体内发生互作,暗示它们通过互作可能参与除了有性生殖之外的其他细胞功能,并为米曲霉蛋白互作功能研究提供一种新的检测技术和方法。     

不结球白菜BcTuRsO基因的克隆及表达

从不结球白菜抗病品种短白梗中克隆到一个抗芜菁花叶病毒(TuMV)相关基因,命名为BcTuRsO(Gen-Bank登录号FJ600376).该基因核苷酸序列全长650 bp,编码194个氨基酸,与已克隆的抗病基因有不同程度的同源性.系统进化树分析表明,该基因在不同物种之间具有保守性.基因组DNA杂交表明,BcTuRsO基因可能以单拷贝形式存在,其表达是组成型的.实时定量PCR检测表明,芜菁花叶病毒能够诱导不结球白菜BcTuRsO基因的转录表达,其在不结球白菜叶片中的表达特征说明它可能参与寄主对病毒的抗性.     

转单、双Bt基因741杨外源基因表达和抗虫性比较

【目的】研究联合使用两种或两种以上的抗虫基因的抗虫效果, 同时鉴定并筛选出转双Bt基因741杨对鳞翅目和鞘翅目害虫有较强抗性的株系。【方法】选取转三基因（Cry3Aa+Cry1Ac+API）741杨8个株系、 转双基因（Cry1Ac+API）741杨1个株系和转单基因（Cry3Aa）741杨3个株系为试材, 从外源基因PCR检测、 毒蛋白表达和抗虫性三方面对转基因株系进行对比分析。【结果】经PCR扩增后各转基因株系出现了预期的电泳条带。ELISA蛋白检测显示转基因株系中都有与所含基因相应的Bt杀虫蛋白表达。用转基因株系新鲜叶片进行柳蓝叶甲Plagiodera versicolora和美国白蛾Hyphantria cunea室内饲虫实验表明： 转入不同抗虫基因的杨树对昆虫的抗性具有选择性, 对非靶标昆虫没有毒杀作用。转双Bt基因741杨具有双抗性, 不同转基因株系表现出高中低的抗性水平： 在对柳蓝叶甲的抗性上, 筛选出的其中5个高抗株系（pCCA1, pCCA2, pCCA5, pCCA6和pCCA9）的抗性水平明显比含Cry3Aa单Bt基因的3个高抗株系（pCC11, pCC53和pCC84）高; 在对美国白蛾的抗性上, 有7个株系（pCCA2~pCCA7和pCCA9）的抗性水平与含Cry1Ac单Bt基因株系（pB29）表现一致, 只有1个株系（pCCA1）对美国白蛾表现出了极低的抗性。【结论】多个抗虫基因在741杨上的联合使用, 不仅扩大了抗虫谱, 其中的高抗株系还具有了更高的抗虫能力, 有效地发挥了基因的叠加效应。     

甘蓝型油菜BnaCIPK15基因的表达分析与功能鉴定

CBL-CIPK是高等植物中广泛存在的一类解析Ca~(2+)信号的蛋白。该研究在前期工作基础上,对甘蓝型油菜(Brassica napus L.)的BnaCIPK15基因进行了亚细胞定位、双分子荧光互补(BiFC)、酵母双杂交和qRT-PCR检测等一系列分析,以探究BnaCIPK15蛋白在ABA激素响应中的作用。结果显示:(1)亚细胞定位发现,BnaCIPK15蛋白定位于细胞质和细胞核中; BiFC分析发现,BnaCIPK15蛋白与BnaCBL1/3/4/9蛋白之间的互作较强,与BnaCBL10仅有微弱互作。(2)qRT-PCR检测发现,BnaCIPK15基因受ABA和冷胁迫的诱导极显著上调表达,而对百草枯(Paraquat)、活性氧(H_2O_2)和热胁迫的诱导较弱,表明BnaCIPK15基因很可能参与ABA和冷胁迫的调控过程。(3)酵母滴定实验结果显示,BnaCIPK15蛋白与脱落酸(ABA)信号通路中的BnaHAB1蛋白(属于蛋白磷酸酶PP2C家族)存在明显的互作,而与BnaABFs/AREB3/ABI5转录因子无明显互作;BiFC验证显示,BnaCIPK15与BnaHAB1蛋白之间存在互作信号,而BnaCIPK15与BnaHAB2组合没有观察到信号,证明BnaCIPK15与BnaHAB1磷酸酶具有特异互作特征,推测BnaCIPK15可能参与调控ABA信号转导。研究认为,甘蓝型油菜中可能存在基于BnaCIPK15-BnaHAB1的互作模块,并参与ABA的信号转导和网络调控。     

辣椒MADS-box转录因子CaRIN基因克隆、表达与功能分析

【目的】为探究MADS-box家族基因RIN的表达特征和功能,解析其对辣椒类胡萝卜素代谢的影响。【方法】基于辣椒果实发育转录组,通过RT-PCR克隆辣椒MADS-box转录因子CaRIN基因CDS全长,对其进行生物信息学、表达模式、亚细胞定位、转录活性等分析,并探讨VIGS诱导CaRIN基因沉默对类胡萝卜素代谢的影响。【结果】（1）CaRIN基因CDS全长732 bp,编码243个氨基酸,蛋白分子量为27.95 kD,等电点（pI）为7.06,其编码蛋白具有典型的MEF2_like MADS结构域,属MICK-type型转录因子;（2）CaRIN基因主要在花和果实中表达,具有组织特异性;CaRIN定位在细胞核,并且具有转录激活活性。（3）CaRIN基因启动子具有ABRE等多个激素应答元件,外源脱落酸和乙烯利均能加速果实转红,诱导CaRIN及相关基因高表达;（4）VIGS诱导沉默CaRIN基因后,类胡萝卜素代谢途径基因PSY1、CCS、PDS、CRTZ、LCYB和NCED1表达水平降低为对照组的0.27-0.59倍,且果实总类胡萝卜素含量（0.379 mg/g）较对照（0.650 mg/g）显著降低。【结论】CaRIN可能是辣椒果实类胡萝卜素代谢过程的重要调控因子。     

水稻条纹病毒p2与本氏烟柯浩体蛋白互作

水稻条纹病毒(Rice stripe virus,RSV)编码的p2蛋白能与纤维蛋白互作并协助病毒系统运动.纤维蛋白定位在核仁和柯浩体上,作为柯浩体指示蛋白的coilin也参与病毒侵染过程.本文通过亚细胞共定位和双荧光互补实验证明p2与本氏烟柯浩体蛋白Nbcoilin互作;病毒诱导基因沉默技术瞬时沉默基因Nbcoilin后不影响植株的生长发育和水稻条纹病毒的侵染.所获结果表明,Nbcoilin虽然能与p2互作,但不影响水稻条纹病毒的侵染.     

Wolbachia在山楂双叶螨中的感染及对寄主生殖的影响

【目的】共生菌Wolbachia在多种叶螨寄主中引起细胞质不亲和及适合度改变,影响寄主的生物学特性。山楂双叶螨Amphitetranychus viennensis是重要的果树害螨,常暴发成灾。本研究旨在明确Wolbachia在山楂双叶螨中的感染情况及对寄主生殖的影响。【方法】采集自然种群的山楂双叶螨,运用多位点序列分型技术（multilocus sequence typing, MLST）对其体内Wolbachia感染率及株系进行分析;通过杂交试验及生物学观察,分析感染Wolbachia对山楂双叶螨单雌产卵量、后代孵化率、性比及死亡率的影响。【结果】山楂双叶螨自然种群感染一种株系的Wolbachia （wVie）,该Wolbachia株系与小黑花椿象Orius strigicollis和丽蝇蛹集金小蜂Nasonia vitripennis中的Wolbachia株系亲缘关系较近,而与叶螨属Tetranychus叶螨感染的Wolbachia株系亲缘关系较远。Wolbachia与4种分化较小的线粒体单倍型相关联。Wolbachia感染雌虫与不感染雌虫产卵量没有显著差异（P>0.05）。不感染雌虫与感染雄虫交配,卵孵化率显著低于其他杂交组合 (P<0.05),但孵化率仍达近75%。各交配组合的后代性比及死亡率变化不明显（P>0.05）。【结论】Wolbachia在山楂双叶螨种群中的侵染历史较短,对山楂双叶螨的产卵力、后代性比、死亡率没有影响。Wolbachia在山楂双叶螨中诱导产生弱的CI表型。     

Ectogenesis and the case against the right to the death of the foetus

Ectogenesis, or the use of an artificial womb to allow a foetus to develop, will likely become a reality within a few decades, and could significantly affect the abortion debate. We first examine the implications for Judith Jarvis Thomson’s violinist analogy, which argues for a woman’s right to withdraw life support from the foetus and so terminate her pregnancy, even if the foetus is granted full moral status. We show that on Thomson’s reasoning, there is no right to the death of the foetus, and abortion is not permissible if ectogenesis is available, provided it is safe and inexpensive. This raises the question of whether there are persuasive reasons for the right to the death of the foetus that could be exercised in the context of ectogenesis. Eric Mathison and Jeremy Davis have examined several arguments for this right, doubting that it exists, while Joona Räsänen has recently criticized their reasoning. We respond to Räsänen’s analysis, concluding that his arguments are unsuccessful, and that there is no right to the death of the foetus in these circumstances.     

On the diversity of the Cladocera in the tropics

The mythical concept of an impoverished tropical cladoceran fauna is refuted. On a planetary scale, around half of the cladoceran species presently known occur exclusively in the tropics-subtropics, often with considerable restriction to particular geographical subzones. On a regional (political) scale, the situation is often unclear because of the continued fragmentary nature of studies, and because political units are not a good basis for biogeographical comparisons. At the finest level of resolution (lake-perlake comparisons), there appears to be an upper limit of c. 50 cladoceran species per individual lake. No significant difference between lakes in the temperate zone and in the tropics could be established here. Daphnia is largely absent from the tropics, but is replaced by more Sidids, Moinids, and Bosminids, such that the average cladoceran community in the limnetic zone of a tropical lake is not characterized by less species but rather by lower population densities. This, in turn, is considered a consequence of higher prevalent predation levels in the tropics.     

Tubular networks in the terminal endings of the visual receptor cells in the human,the monkey,the cat and the dog

Summary A tubular network was found in the terminal endings of the visual receptor cells in the human, the monkey (Macaca mulatta), the cat and the dog. These tubules are arranged in close groups in the vicinity of the synaptic lamellae and the invaginated dendrites. According to the form, diameter, density of the tubules and to the consistence of the network formed by them one can distinguish at these places an initial type (type I), a transitory (type II) and a vesicular one (type III). In the the type III branching, bizarre forms are frequent. The diameter of all the tubules reaches 500–600 Å, their density and walls being the same as in the synaptic vesicles.Similar networks also occur in the axons of the visual receptor cells of the monkey.

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Zusammenfassung In den Endigungen der Photorezeptorzellen von Mensch, Affe (Macaca mulatta), Katze und Hund kommen aus Tubuli bestehende Komplexe vor. Organellenartig in geschlossenen Gruppen angeordnet, liegen sie in Nähe der synaptischen Lamellen und der invaginierten Dendriten. An diesen Stellen kann man nach Form, Durchmesser, Dichte und Konsistenz der von den Tubuli gebildeten Komplexe drei Typen unterscheiden: 1. einen initialen (Typus I), 2. einen Übergangstypus (Typus II) und 3. einen vesiculären Typus (Typus III). In letzterem kommen häufig verzweigte, bizarre Formen vor. Der Durchmesser sämtlicher Tubuli erreicht 500–600 Å. Ihre Dichte und ihre Wand gleicht denen der synaptischen Vesikel.Ähnliche Komplexe fanden wir auch in den Axonen der Photorezeptorzellen vom Affen.

     

Assessing the Physiological State of the Fucoids from the Barents Sea Littoral by the End of the Polar Night

Methods of amperometry and potentiometric titration were used to follow dark respiration (DR) and apparent photosynthesis (AP) in the fucoids Ascophyllum nodosum (L.) Le Jol, Fucus vesiculosus L., and F. serratus L. from the Barents Sea littoral by the end of the 40-day-long polar night. The macroalgae were shown to manifest species-specific low rates of photosynthesis and respiration. However, in spite of their low photosynthetic status due to the effects of subzero temperature and prolonged low or zero illumination, the macroalgae have been able to restore DR and AP to the initial level already by the day 9; the ability to restore AP depended on the level of illumination. The study of the changes in the carbonate–bicarbonate system in the light and darkness demonstrated that the macroalgae grown in darkness, in contrast to those grown in twilight, could absorb bicarbonate in darkness; however, they lost this capacity after two-day-long illumination at an irradiance of 7 mol/(m² s). Bicarbonate uptake in darkness and the capacity to restore the systems of photosynthesis and respiration in fucoid cells are discussed in the context of algal energy metabolism under the polar night conditions.     

Arranging the elements of the potassium channel: the T1 domain occludes the cytoplasmic face of the channel

The voltage-gated potassium channel is currently one of the few membrane proteins where functional roles have been mapped onto specific segments of sequence. Although high-resolution structures of the transmembrane portions of three bacterial potassium channels, the tetramerization domain and the cytoplasmic ball are available, their relative spatial arrangement in mammalian channels remains a matter of ongoing debate. Cryo-electron microscopic images of the six transmembrane voltage-gated Kv channel have been reconstructed at up to 18 Å resolution, revealing that the T1 domain tetramerizes and is suspended below the transmembrane segments. However, the resolution of these images is insufficient to reveal the location of the third piece of the puzzle, the inactivating ball domain. We have used the aberrant interactions observed in a series of chimæric channels to establish that an assembled T1 domain restricts access to the cytoplasmic face of the channel, suggesting that the N-terminal ball and chain may be confined in the space between the T1 domain and the transmembrane portion of the channel.     

Feeding the vertebrate retina from the Cambrian to the Tertiary

The retina of the vertebrate eye is metabolically active and requires nutritive support. During the last 540 million years it has evolved into forms as complicated and nutritionally demanding as those found in avian or primate eyes. Diffusion from the choroid is generally able to supply the metabolic needs of thin retinae. However, when the thickness exceeds the limits of diffusion, structures are needed to supplement the vascular supply from the choroid. These supplemental nutritive devices include the choroidal gland, the falciform process and preretinal vascular plexus of fish, the conus papillaris of lizards, the pecten oculi of birds, the intraretinal vessels of mammals and a few novel systems that remain difficult to classify. These vascular systems are among the most variable features of the vertebrate eye. Here, we review classical and recent findings regarding such retinal nutrition systems, propose a three category classification for them based on histologic origins and speculate on the evolutionary forces which drove their development.     

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     

Separation of the pineal vesicle from the wall of the third ventricle during the post-hatching development of the chicken

Summary According to light- and electron-microscopic observations the pineal organ of the 3-day-old chicken consists of a prominent end vesicle and a tapering parenchymal stalk. During this stage the pineal lumen is in open communication with the third ventricle. However, in the 40-day-old chicken, which still possesses a well-developed end vesicle, the proximal portion of the pineal stalk displays regressive changes leading to local fragmentation. At this stage the pineal stalk is reduced, and the pineal lumen is missing. In 1-year-old chickens the parenchyma of the proximal portion of the stalk is further diminished, and in 3-year-old domestic fowl is completely displaced by bundles of collagenous fibers, only some nerve fibers being present. This post-hatching pineal development may reflect the sequence of changes leading from pineal sense organs to pineal glands.This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan