硅对菜薹炭疽病防御反应中信号物质的影响

通过不同硅（Si）处理和接种炭疽病菌研究Si对菜薹肌醇磷脂信号系统和钙调素信号系统的影响。结果表明：Si对磷脂酰肌醇（PI）和磷脂酰肌醇-4-磷酸（PIP）的形成有明显的调节作用。感染炭疽病后,不同基因型的PI消长明显不同,适宜Si水平下感病后可诱导形成较高水平的PI。感染炭疽病菌后感病品种的中Si处理和抗病品种的低Si、中Si处理的PI呈明显增长趋势,而其它处理有消有长。感病品种在感病前期有抑制PIP产生的趋势,而后期转为促进作用,但抗病品种的变化规律与感病品种相反。在适宜Si水平下感染炭疽病菌后可明显提高抗、感病品种PIP含量,特别是在感病后期其诱导作用更明显,这可能与PI后期大量降解,传递抗病信息有关。Si影响钙调素（CaM）含量,其影响程度与炭疽病菌的作用有关。低Si水平明显促进了炭疽病菌对感病品种CaM的诱导作用,以中Si水平对抗病品种的诱导促进作用相对较强,而且感病品种的诱导促进作用比抗病品种明显。感病品种的中Si处理与抗病品种的低Si处理在感病后的CaM变化相对缓和。表明CaM参与炭疽病致病过程的调控。     

Verapamil和CPZ对金冠苹果衰老生理的影响

对金冠苹果经Ca2 通道阻塞剂Verapamil,CaM拮抗剂Chloropromaize（CPZ）处理后的一些生理生化变化做了研究。结果表明,Verapamil和CPZ促进果肉软化,增加膜透性和可溶性蛋白质含量,分别提早呼吸跃变、提高呼吸速率的峰值,并且加大了过氧化物酶和超氧化物歧化酶活性的变化幅度。     

Calmodulin is involved in the occurrence of extracellular Ca2+-dependent full-type hyperactivation in boar ejaculated spermatozoa incubated with cyclic AMP analogs

In mammals, hyperactivation is essential for sperm fertilization with oocytes in vivo. Two types of hyperactivation “full-type and nonfull-type patterns” can be observed in the spermatozoa from boars, bulls, and mice. We have a hypothesis that the full-type hyperactivation is a physiological (in vivo) pattern and are elucidating its molecular bases. The aims of this study were to detect calmodulin in boar sperm flagella by Western blotting and indirect immunofluorescence and to investigate effects of extracellular Ca²⁺ and calmodulin antagonists “W-7 and W-5 (W-5; a less potent antagonist)” on the occurrence of full-type hyperactivation in boar spermatozoa. Calmodulin was specifically detected as the 17-kDa antigen in the flagella and postacrosomal region of the heads. Full-type hyperactivation could be induced effectively in the samples incubated with 3.42 mM CaCl₂ for 120–180 min, and it was significantly reduced in the concentration-dependent manners of W-7 and W-5. Suppressing effects of W-7 on the full-type hyperactivation were stronger than those of W-5. These observations indicate that flagellar calmodulin is involved in the occurrence of extracellular Ca²⁺-dependent full-type hyperactivation in boar spermatozoa. This is the first indication of the intracellular Ca²⁺-sensing molecule which can function in the full-type hyperactivation.     

利用 CRISPR/Cas9 技术定向编辑水稻 OsIQD1 基因

【目的】植物特异性 IQD 家族蛋白质是一类植物特有的钙调素结合靶蛋白,在植物的生物防 御和发育调节中发挥重要作用。前期酵母双杂筛选试验表明,Pik-H4 与 OsIQD1 存在互作关系,为进一步 揭示 OsIQD1 在水稻抗稻瘟病中的生物学功能,利用 CRISPR/Cas9 编辑技术对 OsIQD1 基因进行定点编辑。 【方法】通过序列比对获得水稻 OsIQD1。在 OsIQD1 第 1 外显子和第 5 外显子（DUF4005 结构域）区域设计 2 个 20 bp 的编辑靶点,将 2 个靶点核苷酸片段克隆至 pRGEB32 载体,获得 pRGEB32-OsIQD1-gRNA 载体,利用 农杆菌介导法转化水稻 Pik-H4 NIL 愈伤组织,经再生培养、潮霉素检测获得转基因阳性植株,并对 T0 代转基 因植株靶点区域序列进行 PCR 和测序,分析 osiqd1 的突变类型。【结果】转基因再生植株经过对抗潮霉素基因 HPT Ⅱ的 PCR 鉴定,获得 30 株阳性株系。对 T0 代植株靶点附近序列的测序结果表明,OsIQD1 基因被成功编 辑,两个位点的编辑效率分别为 21.67% 和 26.67%,其中,靶点 2 区域有 1 个纯合突变株系。【结论】研究结 果为进一步利用 osiqd1 突变体开展其参与钙离子 / 钙调素信号转导通路的机制研究提供了遗传材料,初步推定 OsIQD1 参与植物的基础免疫反应。     

New taxa in Aspergillus section Usti

Based on phylogenetic analysis of sequence data, Aspergillus section Usti includes 21 species, inclucing two teleomorphic species Aspergillus heterothallicus (= Emericella heterothallica) and Fennellia monodii. Aspergillus germanicus sp. nov. was isolated from indoor air in Germany. This species has identical ITS sequences with A. insuetusCBS 119.27, but is clearly distinct from that species based on β-tubulin and calmodulin sequence data. This species is unable to grow at 37 °C, similarly to A. keveii and A. insuetus. Aspergillus carlsbadensis sp. nov. was isolated from the Carlsbad Caverns National Park in New Mexico. This taxon is related to, but distinct from a clade including A. calidoustus, A. pseudodeflectus, A. insuetus and A. keveii on all trees. This species is also unable to grow at 37 °C, and acid production was not observed on CREA. Aspergillus californicus sp. nov. is proposed for an isolate from chamise chaparral (Adenostoma fasciculatum) in California. It is related to a clade including A. subsessilis and A. kassunensis on all trees. This species grew well at 37 °C, and acid production was not observed on CREA. The strain CBS 504.65 from soil in Turkey showed to be clearly distinct from the A. deflectus ex-type strain, indicating that this isolate represents a distinct species in this section. We propose the name A. turkensis sp. nov. for this taxon. This species grew, although rather restrictedly at 37 °C, and acid production was not observed on CREA. Isolates from stored maize, South Africa, as a culture contaminant of Bipolaris sorokiniana from indoor air in Finland proved to be related to, but different from A. ustus and A. puniceus. The taxon is proposed as the new species A. pseudoustus. Although supported only by low bootstrap values, F. monodii was found to belong to section Usti based on phylogenetic analysis of either loci BLAST searches to the GenBank database also resulted in closest hits from section Usti. This species obviously does not belong to the Fennellia genus, instead it is a member of the Emericella genus. However, in accordance with the guidelines of the Amsterdam Declaration on fungal nomenclature (Hawksworth et al. 2011), and based on phylogenetic and physiological evidence, we propose the new combination Aspergillus monodii comb. nov. for this taxon. Species assigned to section Usti can be assigned to three chemical groups based on the extrolites. Aspergillus ustus, A. granulosus and A. puniceus produced ustic acid, while A. ustus and A. puniceus also produced austocystins and versicolorins. In the second chemical group, A. pseudodeflectus produced drimans in common with the other species in this group, and also several unique unknown compounds. Aspergillus calidoustus isolates produced drimans and ophiobolins in common with A. insuetus and A. keveii, but also produced austins. Aspergillus insuetus isolates also produced pergillin while A. keveii isolates produced nidulol. In the third chemical group, E. heterothallica has been reported to produce emethallicins, 5'-hydroxyaveranthin, emeheterone, emesterones, 5'-hydroxyaveranthin.     

CaM与ZmCCaMK相互作用参与BR诱导的玉米叶片抗氧化防护

以玉米(Zea mays L.)为材料,通过酶联免疫法、实时荧光定量PCR技术、免疫共沉淀激酶反应以及原生质体瞬时表达体系等方法研究了在油菜素内酯(Brassinosteroids,BR)诱导的玉米叶片抗氧化防护过程中钙调素(Calmodulin,Ca M)的作用以及Ca M和Ca2+/Ca M依赖的蛋白激酶(Zm CCa MK)之间的关系。结果显示:外源BR处理显著提高了玉米叶片叶肉细胞原生质体中的Ca M含量,Ca M拮抗剂的预处理几乎完全抑制了BR诱导的抗氧化防护酶活性,表明Ca M参与了BR诱导的抗氧化防护。而且Zm CCa MK原生质体瞬时表达及瞬时沉默结果显示Zm CCa MK影响BR诱导的Ca M含量,Ca M拮抗剂的预处理也显著抑制了BR诱导的H2O2的产生、Zm CCa MK基因表达以及激酶活性。说明BR诱导产生的Ca M增强H2O2的积累,进一步诱导抗氧化防护酶活性增加,并且Ca M和Zm CCa MK之间存在互动机制。     

镉对玉米苗中钙调蛋白含量和Ca2+-ATPase活性的影响

试验采用营养液培养的方法,以玉米为试材,研究了不同供镉浓度（0﹑5﹑20和100 µmol/L）和处理时间（12﹑24﹑48﹑96、168 h）对植株体内钙调蛋白（CaM）含量及生物膜上的Ca²⁺-ATPase活性的影响。结果表明,植株可溶性Ca²⁺含量在镉胁迫后较不加镉处理增加,镉处理在叶和根中分别在48和24 h后达最高,然后随镉处理浓度和处理时间的增加逐步下降;同时镉诱导了植株CaM的合成,其含量随镉处理浓度和处理时间增加逐步增加,但20 µmol/L和100 µmol/L镉处理在168 h后有所下降;与不加镉处理相比,镉胁迫导致植株生物膜上的Ca²⁺-ATPase活性迅速升高,但随镉处理浓度提高和时间延长,镉胁迫植株的Ca²⁺-ATPase活性在48 h（质膜、液泡膜和内质网膜）和24 h（线粒体膜）后逐步降低。各膜上的Ca²⁺-ATPase活性依次为质膜> 液泡膜> 内质网膜> 线粒体膜,且同一微囊膜,根中的活性大于叶中。     

平欧杂种榛钙调蛋白基因ChaCaM的克隆与表达分析

以平欧杂种榛（Corylus heterophylla × C. avellana）品种‘达维’为材料,基于转录组测序数据,利用RACE技术克隆钙调蛋白基因,通过生物信息学进行序列预测分析,并利用实时荧光定量PCR技术分析其在不同组织和自交不亲和授粉后不同时间雌蕊中的表达模式。克隆获得一个钙调蛋白基因序列,命名为ChaCaM（GenBank登录号：KY211037）,其开放阅读框为450 bp,编码149个氨基酸,分子量16.8476 kD。生物信息学分析表明,ChaCaM为酸性稳定蛋白,属于疏水性蛋白,无跨膜域和信号肽存在,主要定位于细胞质基质中,存在糖基化和磷酸化位点。序列分析表明,ChaCaM包含两个EFh结构域,与其他物种的CaM高度相似。系统进化树表明,ChaCaM与烟草、可可和萝卜等的CaM亲缘关系极近,聚类在同一分支下。基因表达分析表明,ChaCaM在平欧杂种榛不同组织中的表达具有差异,且在自交不亲和授粉后的雌蕊中具有明显的表达量变化。ChaCaM具有典型的钙调蛋白基因特征,主要定位于细胞质基质中;表现为组成型表达,推测其参与了榛属植物的自交不亲和反应。     

兔抗小麦钙调素抗血清的制备及其纯化

为制备兔抗小麦钙调素抗血清并对其进行纯化,首先从小麦叶片中提取钙调素蛋白,对其初步纯化后免疫家兔,制备得到兔抗小麦钙调素抗血清,再以大肠杆菌中诱导表达的拟南芥钙调素蛋白作为抗原,利用PVDF膜结合法纯化得到了兔抗小麦钙调素抗血清,并通过Western Blotting对所纯化的多克隆抗体进行了鉴定。