巴西橡胶树CCaMK基因家族成员的鉴定和表达分析

钙/钙调素依赖型蛋白激酶(CCa MK,calcium-and calmodulin-dependent protein kinase)是一种钙离子结合蛋白,在钙信号传导过程中起重要作用。本研究以已发表的CCa MK序列作为检索序列,对橡胶树和其他5种植物(木薯、水稻、杨树、蓖麻和拟南芥)的基因组和转录组数据进行全面搜索,鉴定得到6个CCa MK基因,其中包括一个橡胶树CCa MK基因,命名为Hb CCa MK1。序列分析发现:5种植物的CCa MK氨基酸序列同源性较高,为73%~94%。基因结构分析发现,Hb CCa MK1和所分析的其他植物CCa MK一样均,含有6个内含子、1个蛋白激酶结构域和3个EF-hand结构域;从结构和进化上看,Hb CCa MK1和蓖麻、木薯2种植物的CCa MK具有较近的亲缘关系;在表达方面,Hb CCa MK1在橡胶树根中表达丰度最高,其次为树叶和花;另外,Hb CCa MK1的表达还与叶片发育、真菌侵染和低温胁迫有关。     

Medicago truncatula DMI1 required for bacterial and fungal symbioses in legumes

Legumes form symbiotic associations with both mycorrhizal fungi and nitrogen-fixing soil bacteria called rhizobia. Several of the plant genes required for transduction of rhizobial signals, the Nod factors, are also necessary for mycorrhizal symbiosis. Here, we describe the cloning and characterization of one such gene from the legume Medicago truncatula. The DMI1 (does not make infections) gene encodes a novel protein with low global similarity to a ligand-gated cation channel domain of archaea. The protein is highly conserved in angiosperms and ancestral to land plants. We suggest that DMI1 represents an ancient plant-specific innovation, potentially enabling mycorrhizal associations.     

Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators

Rhizobial bacteria enter a symbiotic interaction with legumes, activating diverse responses in roots through the lipochito oligosaccharide signaling molecule Nod factor. Here, we show that NSP2 from Medicago truncatula encodes a GRAS protein essential for Nod-factor signaling. NSP2 functions downstream of Nod-factor-induced calcium spiking and a calcium/calmodulin-dependent protein kinase. We show that NSP2-GFP expressed from a constitutive promoter is localized to the endoplasmic reticulum/nuclear envelope and relocalizes to the nucleus after Nod-factor elicitation. This work provides evidence that a GRAS protein transduces calcium signals in plants and provides a possible regulator of Nod-factor-inducible gene expression.     

Induction of pre-infection thread structures in the leguminous host plant by mitogenic lipo-oligosaccharides of Rhizobium

Root nodules of leguminous plants are symbiotic organs in which Rhizobium bacteria fix nitrogen. Their formation requires the induction of a nodule meristem and the formation of a tubular structure, the infection thread, through which the rhizobia reach the nodule primordium. In the Rhizobium host plants pea and vetch, pre-infection thread structures always preceded the formation of infection threads. These structures consisted of cytoplasmic bridges traversing the central vacuole of outer cortical root cells, aligned in radial rows. In vetch, the site of the infection thread was determined by the plant rather than by the invading rhizobia. Like nodule primordia, pre-infection thread structures could be induced in the absence of rhizobia provided that mitogenic lipo-oligosaccharides produced by Rhizobium leguminosarum biovar viciae were added to the plant. In this case, cells in the two outer cortical cell layers containing cytoplasmic bridges may have formed root hairs. A common morphogenetic pathway may be shared in the formation of root hairs and infection threads.     

百脉根4个共生基因在水稻中的共表达及其对水稻转录组的影响

为建立水稻-根瘤菌共生固氮体系,将百脉根共生信号转导途径中的4个功能基因(LHK1、NSP1、NSP2和NIN)转入水稻,获得稳定转化的转基因水稻,并初步检测转基因水稻的表型和转录组水平上的差异。根瘤菌感染试验结果显示,根瘤菌侵染对照(转入空载体)和转基因水稻的根毛和表皮的比率没有显著变化,但接种根瘤菌14d后,对照与转基因水稻的侧根原基数有显著差异,转共生基因水稻的侧根原基明显增多。利用水稻cDNA芯片,检测转基因水稻组与对照组在接种根瘤菌7d和不接种根瘤菌条件下根的转录组变化,结果显示,转基因组与对照组水稻在植物防御反应、激素信号转导途径相关基因均有差异性表达,表明豆科共生基因影响了水稻对根瘤菌的反应。     

Intracellular calcium release mediated by sphingosine derivatives generated in cells

Soluble and hydrophobic lipid breakdown products have a variety of important signaling roles in cells. Here sphingoid bases derived in cells from sphingolipid breakdown are shown to have a potent and direct effect in mediating calcium release from intracellular stores. Sphingosine must be enzymically converted within the cell to a product believed to be sphingosine-1-phosphate, which thereafter effects calcium release from a pool including the inositol 1,4,5-trisphosphate-sensitive calcium pool. The sensitivity, molecular specificity, and reversibility of the effect on calcium movements closely parallel sphingoid base-mediated inhibition of protein kinase C. Generation of sphingoid bases in cells may activate a dual signaling pathway involving regulation of calcium and protein kinase C, comparable perhaps to the phosphatidylinositol and calcium signaling pathway.     

Legumes symbioses: absence of Nod genes in photosynthetic bradyrhizobia

Leguminous plants (such as peas and soybeans) and rhizobial soil bacteria are symbiotic partners that communicate through molecular signaling pathways, resulting in the formation of nodules on legume roots and occasionally stems that house nitrogen-fixing bacteria. Nodule formation has been assumed to be exclusively initiated by the binding of bacterial, host-specific lipochito-oligosaccharidic Nod factors, encoded by the nodABC genes, to kinase-like receptors of the plant. Here we show by complete genome sequencing of two symbiotic, photosynthetic, Bradyrhizobium strains, BTAi1 and ORS278, that canonical nodABC genes and typical lipochito-oligosaccharidic Nod factors are not required for symbiosis in some legumes. Mutational analyses indicated that these unique rhizobia use an alternative pathway to initiate symbioses, where a purine derivative may play a key role in triggering nodule formation.     

Effect of phospholipase C-gamma overexpression on PDGF-induced second messengers and mitogenesis

Platelet-derived growth factor (PDGF) stimulates phospholipase C (PLC) activity and the phosphorylation of the gamma isozyme of PLC (PLC-gamma) in vitro and in living cells. The role of PLC-gamma in the phosphoinositide signaling pathway was addressed by examining the effect of overexpression of PLC-gamma on cellular responses to PDGF. Overexpression of PLC-gamma correlated with PDGF-induced tyrosine phosphorylation of PLC-gamma and with PDGF-induced breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2). However, neither bradykinin- nor lysophosphatidic acid-induced phosphoinositide metabolism was enhanced in the transfected cells, suggesting that the G protein-coupled phosphoinositide responses to these ligands are mediated by other PLC isozymes. The enhanced PDGF-induced generation of inositol trisphosphate (IP3) did not enhance intracellular calcium signaling or influence PDGF-induced DNA synthesis. Thus, enzymes other than PLC-gamma may limit PDGF-induced calcium signaling and DNA synthesis. Alternatively, PDGF-induced calcium signaling and DNA synthesis may use biochemical pathways other than phosphoinositide metabolism for signal transduction.     

Synaptic theory of working memory

It is usually assumed that enhanced spiking activity in the form of persistent reverberation for several seconds is the neural correlate of working memory. Here, we propose that working memory is sustained by calcium-mediated synaptic facilitation in the recurrent connections of neocortical networks. In this account, the presynaptic residual calcium is used as a buffer that is loaded, refreshed, and read out by spiking activity. Because of the long time constants of calcium kinetics, the refresh rate can be low, resulting in a mechanism that is metabolically efficient and robust. The duration and stability of working memory can be regulated by modulating the spontaneous activity in the network.     

独脚金内酯合成路径及抗逆性研究进展

独脚金内酯(strigolactone,SL)是一类萜内酯,来源于类胡萝卜素生物合成途径,是通过一系列的酶促反应合成的。但关于独脚金内酯的生物合成路径尚未研究透彻,目前已经了解到D27蛋白、CCD7蛋白、CCD8蛋白、P450蛋白在这一过程中发挥着不可忽视的作用。现已从豌豆、水稻、矮牵牛、拟南芥等多分枝突变体中克隆得到多个与SL合成、传导途径相关的基因。其中,D27、CCD7和CCD8是独脚金内酯合成途径的上游基因,经过双加氧和脱氢,最后在MAX1的作用下生成了5-脱氧独脚金醇。独脚金内酯是一类新型激素,能够诱导根寄生植物种子萌发并且加快丛枝真菌出现分枝,有着阻碍植物分枝生长的作用,从而抵御各种胁迫,对植物的生长具有重要意义。     

A cytokinin perception mutant colonized by Rhizobium in the absence of nodule organogenesis

In legumes, Nod-factor signaling by rhizobia initiates the development of the nitrogen-fixing nodule symbiosis, but the direct cell division stimulus that brings about nodule primordia inception in the root cortex remains obscure. We showed that Lotus japonicus plants homozygous for a mutation in the HYPERINFECTED 1 (HIT1) locus exhibit abundant infection-thread formation but fail to initiate timely cortical cell divisions in response to rhizobial signaling. We demonstrated that the corresponding gene encodes a cytokinin receptor that is required for the activation of the nodule inception regulator Nin and nodule organogenesis.     

供应不同形态氮肥VA菌根对三叶草苗期生长和磷素营养的影响

本试验采用三室盆栽系统研究两种不同形态的氮肥(铵态氮和硝态氮)接种泡囊——丛枝(VA)菌根真菌对三叶草苗期植株生长和磷素营养的影响。测定各处理植株的生长量和根系的总长度,分析植株 N,P,K 等养分的含量和收获后中室边室土壤 Olsen-P 浓度及 pH值。结果表明,施用两种不同形态的氮肥,接种菌根真菌显著增加了植株的干物重,提高了植株中 P 的含量,且在 NH_4-N 的处理中,菌根效应要大于 NO_3-N 的处理。施 NH_4-N,菌根处理的边室土壤 pH 值和 Olsen-P 的含量都显著低于未接种的处理,而施硝态氮的处理菌根对pH 值的影响不显著。说明菌根对寄主植物的营养效应与环境中氮素的形态有密切的关系。     

三种土壤中VA菌根真菌对豆类作物侵染率的研究

本试验结果表明:在黄棕壤、两合土、沙土3种土壤中均有 VA 菌根真菌的存在,都以球孢霉属(Glomus)为主,以 Gl.fecundisporum 和 Gl.mosseae 为优势种。VA 菌根真菌对绿豆的侵染随绿豆苗的生长而逐步上升,到苗龄30天左右达高峰,以后保持相对稳定。3种土壤中 VA 菌根真菌的数量依次为黄棕壤、两合土、沙土,与对绿豆的侵染程度呈现一定的相关性。在栽种一季绿豆后,各土壤中的 VA 菌孢子数增加4倍左右。生长40天的菌根化植株地上部的干重和含磷量均高于对照植株,表明了 VA 菌根有增进吸磷和促进生长的作用。     

蓝桉根际菌根真菌的分离鉴定及其对蓝桉生长和光合特性的影响

蓝桉是桉树中少有的油、材兼用树种。从云南蓝桉人工林中采集根际土,调查菌根真菌的种类和优势种,并通过控制育苗基质中菌根真菌菌群的数量,分析菌根真菌对蓝桉苗木生长和光合特性的影响,探讨影响其生长的主要菌根真菌种类,以期从菌根化育苗方面为蓝桉的壮苗培育提供指导。结果表明:从根际土中分离出3属6种菌根真菌,分别为聚丛球囊霉(Glomus aggregatum)、何氏球囊霉(Glomus hoi)、多梗球囊霉(Glomus multicaule)、摩西球囊霉(Glomus mosseae)、幼套近明球囊霉(Claroideoglomus etunicatum)、缩隔球囊霉(Septoglomus constrictum),其中,聚丛球囊霉、幼套近明球囊霉和摩西球囊霉为优势菌种(三者合计占总孢子密度的80.77%)。苗龄10个月时,随着育苗基质中菌群数量增多,蓝桉苗木的菌根侵染率增大,侵染强度增强,且接种适量的菌根真菌对苗高、地径都具有显著(P<0.05)促进作用,且能显著(P<0.05)提高苗木的叶绿素含量和表观量子效率,降低光补偿点和光饱和点。相关性分析和逐步回归分析结果显示,对苗木生长和光合能力起主要促进作用的是聚丛球囊霉、幼套近明球囊霉和缩隔球囊霉。     

半番鸭与番鸭精巢组织差异表达转录组测序分析

【目的】研究半番鸭与番鸭精巢组织转录组差异表达基因,为进一步阐明半番鸭不育的遗传机制奠定理论基础。【方法】利用转录组测序方法对半番鸭和番鸭的精巢组织进行研究,筛选其差异表达基因,并对其功能进行注释和荧光定量PCR(quantitative real-time PCR,QRT-PCR)验证。【结果】测序共获得43.84Gb Clean Data,组装后共获得193 535条Unigene。DESeq分析发现3 597个基因在两个鸭品种间差异表达,其中上调基因1 194个和下调基因2 403个,包括与生殖功能相关的基因,如成纤维细胞生长因子(fibroblast growth factor,FGF)、蛋白激酶A(protein kinase A,PKA)、丝裂原活化蛋白激酶7(mitogen-activated protein kinase 7-like,partial,BMK)、生长因子受体结合蛋白2(growth factor receptor-bound protein 2,GRB2)、c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)和肿瘤坏死因子受体超家族成员6(tumor necrosis factor receptor superfamily member 6,FAS)等。GO(gene Ontology)分析发现382个差异基因获得功能注释,其中97个基因涉及发育繁殖生物学过程。KEGG(kyoto Encyclopedia of Genes and Genomes)通路分析表明差异表达基因共富集到50条信号通路中,其中17个通路显著富集,包括丝裂原活化蛋白激酶信号转导通路(mitogen-activated protein kinase signaling pathway,MAPK)、甘油酯代谢(glycerolipid metabolism)以及钙信号途径(calcium signaling pathway)信号通路等,与生殖功能密切相关的有促性腺激素释放激素信号通路(gonadotropin releasing hormone(Gn RH)signaling pathway)和MAPK信号转导通路。经QRT-PCR验证,差异基因表达变化模式与转录组测序结果一致,测序结果可靠。【结论】在转录组水平上筛选出半番鸭和番鸭精巢组织差异表达基因,揭示了Gn RH和MAPK信号通路在鸭的生殖活动中发挥了重要作用,为进一步探索半番鸭生殖系统的分化机理提供可靠的参考依据。     

黄土高原5种造林树种菌根根际土壤微生物群落多样性研究

为了准确评估造林树种在黄土高原生态恢复中的作用,采用BIOLOG检测法,对黄土高原柠条、沙棘、狼牙刺、油松和刺槐5种造林树种菌根根际土壤微生物群落多样性进行了研究。结果显示,各树种根际土壤孔平均颜色变化率AWCD为油松＞沙棘＞狼牙刺＞柠条＞刺槐;不同树种菌根侵染率和菌根根际的微生物功能多样性均有显著差异;群落丰富度、多样性指数分析表明,油松外生菌根根际土壤微生物功能多样性和群落种群结构最丰富,其次为沙棘和柠条;碳源利用中与主成分1和主成分2相关的主要碳源分别是羧酸类、氨基酸类和糖类物质。相关性分析表明,菌根侵染率与土壤微生物功能多样性呈极显著正相关,随着菌根侵染率的提高,根际土壤微生物功能多样性增加。外生菌根树种有利于改善黄土高原退化土壤的微生物群落结构,优于泡囊丛枝菌根树种,油松作为黄土高原植被恢复的先锋造林树种,可以改善该区域干旱贫瘠土壤的微生物群落结构。      

大兴安岭地区野生蓝莓菌根真菌着生状态的初步观察

对我国大兴安岭地区野生蓝莓资源中菌根真菌进行了实地采样调查,通过压片法对野生蓝莓菌根真菌着生状况进行了研究.结果表明:菌根真菌对野生蓝莓的侵染率在75%左右.外生菌丝有隔,菌丝可以通过一个或多个侵入点侵入同一根段或不同根段;观察到2种内生菌丝体类型:穿梭于细胞间的菌丝和胞内菌丝团.另外,还观察到菌丝体侵入细胞形成的一种"扳口"状结构和根表皮细胞的缺失.研究认为,大兴安岭野生蓝莓中菌根真菌侵染率较高,菌丝侵染方式多样.     

三种丛枝菌根真菌对黄瓜幼苗生长的影响

为探索丛枝菌根(AM)真菌对培育黄瓜壮苗的潜力，通过盆钵试验方法研究了接种3种常见VA菌根真菌Gl.intraradice,Gl.mosseae和gl.vrsiforne对黄瓜幼苗生长的影响。结果表明，在3种供试菌种中，Gl.mosseae对黄瓜幼苗的生长表现出明显的促进作用，即成苗时接种处理根系的生物量比相应对照植株提高了12．5％，地上部生物量增加了21．42％。菌根对植株的促生作用与其改善植株的养分吸收能力有关，一方面3种菌根真菌都不同程度地提高了黄瓜幼苗根系从基质中吸收N和P的能力，其中又以Gl.mosseae的作用最大，与对照相比，接种Gl.mosseae处理的植株根系的含N量提高了11．69％，而含P量比对照增加了1．14倍。另一方面，供试菌根真菌对Mg、Cu和Zn的吸收上也表现出不同程度的促进作用。     

利用iTRAQ技术和转录组筛选芍药属远缘杂交不亲和基因

【目的】远缘杂交育种是目前牡丹、芍药品种改良和育种的主要方法,而远缘杂交不亲和一直是制约其快速发展的主要因素。本研究从牡丹、芍药远缘杂交授粉后不亲和应答相关的柱头差异蛋白与转录组方面深入研究,揭示牡丹、芍药远缘杂交不亲和的分子机理,为杂交育种提供理论依据。【方法】以芍药‘粉玉奴’自交、芍药‘粉玉奴’与牡丹‘凤丹白’杂交为供试材料,在授粉后24 h采取柱头,分别进行同位素标记相对定量（iTRAQ）和转录组技术分析。对所获得的蛋白和转录组数据进行生物信息学分析,并对其中可能与远缘杂交不亲和相关的基因进行定量PCR验证。【结果】利用iTRAQ技术分析牡丹、芍药远缘杂交后柱头中蛋白质的表达差异,共鉴定到685个差异蛋白,富集到了188条通路,其中显著富集的Pathway有18条。与不亲和授粉相关代谢通路有RNA降解、钙信号途径、丝裂原活化蛋白激酶（mitogen-activated protein kinase signaling pathway,MAPK）信号途径、磷脂酰肌醇信号系统。在RNA降解代谢通路中,烯醇酶（Enolase）、热休克蛋白DnaK（HSP70）及病菌抗原（GroEL）均表达下调。在钙信号途径中,钙调蛋白（CALM）表达下调,腺苷酸转运酶（adenine nucleotide translocase,ANT）表达量增加,表达上调。MAPK信号途径中,乙二醛酶Ⅰ（GloI）表达下调。磷脂酰肌醇信号系统中的钙调蛋白（CALM）表达下调。随机选取与差异蛋白相关的6个基因进行qRT-PCR验证,结果显示,6个基因的表达与蛋白质水平趋势相一致,均表达下调。通过转录组测序,共获得了52 998个有注释信息的Unigene,占所有Unigene的40.37%。基于6组样品的RPKM（Reads Per Kilobase per Million）值,共筛选到16 224个差异基因。其中上调基因13 361,下调基因2 863个。对差异基因进行Pathway显著富集分析,杂交与自交相比,不亲和差异表达的基因主要富集在氧化磷酸化代谢、ABC转运蛋白、次级代谢产物等通路。与远缘杂交不亲和相关且发生显著变化的基因有CalS-5、CalS-12（胼胝质酶）和SPL（squamosa promoter binding protein-like）表达上调,ABCF（ABC transporter family protein）表达下调。【结论】在转录组和蛋白数据共注释到6个蛋白、4个基因与植物不亲和性密切相关,这些蛋白与基因可能在远缘杂交不亲和方面发挥着重要作用。     

拟轮枝镰孢与玉米籽粒互作的差异基因筛选及代谢通路分析

【目的】拟轮枝镰孢(Fusarium verticillioides)引起的玉米穗腐病是我国玉米产区发生严重的病害之一，论文旨在了解病原菌与玉米籽粒互作过程中的基因表达差异，为揭示病原菌的致病机制和玉米抗病机制提供依据。【方法】对拟轮枝镰孢侵染玉米籽粒0、4、12和72 h的样品进行转录组测序，之后采用生物信息学分析，分别以玉米和拟轮枝镰孢基因组为参考，以|log₂FC|≥1,P-adjust<0.05为阈值筛选互作过程中玉米和拟轮枝镰孢的差异表达基因，利用GO和KEGG对其进行功能注释及富集分析。Goatools软件分析植物-病原互作、MAPK途径和植物激素信号转导通路相关差异基因的表达变化，采用实时荧光定量PCR(qRT-PCR)方法对测序差异基因进行验证。【结果】在互作4、12和72 h后拟轮枝镰孢分别有140、400和1 945个基因上调表达，有9、302和1 784个基因下调表达；玉米分别有293、692和1 426个基因上调表达，320、482和153个基因下调表达。GO和KEGG富集分析显示，侵染早期拟轮枝镰孢在细胞间隙生长，差异基因主要富集在...