白芷细胞外钙调素结合蛋白(ECBP21)免疫组化及金标定位分析

ECBP21是我们从白芷悬浮培养细胞外纯化及cDNA克隆的一种钙调素结合蛋白(CaMBP),亦是植物中首次报道的细胞外CaMBP。本实验以大肠杆菌表达的重组ECBP21蛋白为抗原,制备了高效价特异性抗体;随后,利用免疫组化及金标定位技术,研究了ECBP21蛋白组织特异性分布及亚细胞定位。免疫组化结果表明:ECBP21在白芷各组织中均有分布,但在叶、花、花序轴中较多,而在根中较少,并且ECBP21在细胞中多分布于细胞壁区域;免疫胶体金电镜定位结果显示:在白芷花序轴细胞中金颗粒主要分布在细胞壁,表明ECBP21蛋白主要定位于细胞壁区域,从而首次为细胞外CaMBP(ECBP21)的胞外存在提供了直观证据,并为进一步研究其在植物生长发育中的功能提供了初步信息。     

白芷培养细胞外钙调素结合蛋白的胶体金电镜定位研究

利用胶体金标记技术制备了具有生物活性的植物CaM-BSA-gold探针,并用此探针建立了白芷愈伤组织培养细胞胞外钙调素结合蛋白(CaMBPs)的透射电镜标记方法。标记结果显示,在1mmol/L Ca~(2 )存在下,用EGTA洗涤过的白芷愈伤组织培养细胞壁表面有金颗粒分布,而分别在含有EGTA、TFP、过量未标记的CaM、CaM抗体存在的情况下,用金标CaM探针进行标记.以及用金标羊抗兔抗体替代金标CaM探针进行标记的各对照组,细胞壁表面金颗粒则消失,说明白芷愈伤组织培养细胞壁表面存在着CaM结合位点或CaMBPs。     

番茄斑萎病毒运动蛋白NSm在植物细胞和昆虫细胞内的定位

[目的]研究番茄斑萎病毒(Tomato spotted wilt virus,TSWV)运动蛋白NSm的细胞定位.[方法]将NSm基因融合GFP后构建到植物双元表达载体pCHF3中,农杆菌介导浸润本氏烟叶片,同时将融合GFP的NSm基因利用Bac-to-Bac杆状病毒表达体系转染昆虫Tn细胞.在激光共聚焦显微镜下观察NSm-GFP在烟草表皮细胞和昆虫Tn细胞中的定位情况.[结果]观察发现与单独表达GFP在细胞壁周围和细胞核处均匀分布不同,NSm-GFP融合蛋白会在植物细胞内扩散,能够在细胞壁边缘定位,并且在胞间连丝处呈不连续的绿色荧光小点,偶尔成对出现在相邻的两个细胞之间;NSm蛋白在昆虫Tn细胞表面产生数量众多的管状结构并向外延伸.[结论]研究结果表明NSm能够特异性定位在植物细胞的胞间连丝处,并能在昆虫Tn细胞内表达,在细胞表面产生运动蛋白小管.     

植物钙调素结合蛋白研究进展

钙调素(CaM)作为最重要的一类Ca2 传感蛋白可以通过与其下游CaM结合蛋白(CaMBP)作用而调节细胞的生理功能.因此,对CaMBP的研究是揭示CaM作用机制的重要内容,是探明Ca2 -CaM信号转导系统的关键.该文从CaMBP和CaM的结合特性、植物CaMBP的分布以及植物CaMBP的生物学功能等方面综述了植物CaMBP的研究现状和最新进展.     

植物水孔蛋白的亚细胞分布与生理功能研究浅析

水孔蛋白(aquaporin,AQP)因具有水转运活性而得名,然而随着研究的深入,水孔蛋白转运活性的多样性与生理功能的多样性不断被报道．本文综合分析了植物水孔蛋白亚细胞定位与功能多样性的研究进展,重点综述了植物水孔蛋白广泛的亚细胞分布特点,以及亚细胞上的再分布现象与植物水孔蛋白生理功能多样性间的关系,并对植物水孔蛋白研究中存在的 问题及研究方向进行了分析,认为水孔蛋白多样化的生理功能的作用机制需要结合其组织定位与亚细胞定位进行分析才能 揭示．     

转脂蛋白CaMBP10的胶体金标记及对白菜中CaMBP10结合蛋白的鉴定

植物转脂蛋白 (LTP)是一类广泛存在于高等植物中的空间结构高度保守的碱性小分子蛋白,其确切功能和调节机制至今仍不清楚.本室从白菜中分离的钙调素结合 蛋白10 (CaMBP10),经序列分析被鉴定为植物转脂蛋白家族成员.近期研究结果表明 ,CaMBP10 参与了植物的生物与非生物胁迫反应.为了深入探讨CaMBP10的抗性机制,确定植物中与其相互作用的蛋白质,本文拟建立胶体金标记CaMBP10 的方法,通过凝胶覆盖分析,检测植物样品中的CaMBP10 结合蛋白为此,对标记反应的最适条件进行了优化,确定最佳条件为：交联剂戊二醛用量为0.034%,交联反应pH值为7 .0,交联反应时间为40 min,胶体金颗粒度为10 nm,胶体金溶液的pH为7.0. 本文确定建立了植物样品中CaMBP10结合蛋白的分析与鉴定方法.     

蚕豆叶片细胞中IAA的胶体金免疫电镜定位

利用胶体金免疫电镜技术对蚕豆(Vicia faba L.)叶片细胞中的IAA定位进行了研究。幼嫩叶片的叶肉细胞中金颗粒主要分布在细胞核和叶绿体中,细胞质及细胞壁也有金颗粒标记。成熟叶片的叶肉细胞中金颗粒主要分布在叶绿体和细胞质,细胞壁也有少量金颗粒标记,液泡中没有发现金颗粒标记。成熟叶片小叶脉的韧皮细胞发现有大量的金颗粒标记,金颗粒主要标记在传递细胞的细胞壁中。小叶脉的维管束鞘细胞中也有很多的金颗粒标记,金颗粒主要分布在叶绿体、细胞质及细胞壁中。幼嫩叶片组织不进行IAA的固定或用正常兔IgG代替IAA抗体染色的对照,很难发现金颗粒标记。对IAA在组织及亚细胞中的定位及其生理意义进行了讨论。     

普通烟草CESA基因家族成员的鉴定、亚细胞定位及表达分析

纤维素合成酶蛋白(cellulose-synthase proteins, CESA)是一类质膜定位蛋白,以蛋白复合体的形式存在于质膜上合成纤维素,在细胞壁建成和植物生长发育过程中起着非常重要的作用。本研究利用CESA蛋白保守域序列PF03552检索普通烟草(Nicotiana tabacum L.)蛋白序列,并通过拟南芥(Arabidopsis thaliana)10个CESA蛋白序列在普通烟草基因组数据库中利用TBLASTN程序进行比对,共获得21条NtCESA基因候选序列,对这些序列进行蛋白序列理化性质分析、系统进化树构建、基因结构分析、保守结构域及跨膜区分析和组织表达模式分析,并对NtCESA9和NtCESA14两个蛋白进行了亚细胞定位实验。结果表明：获得的21条NtCESA蛋白序列的理化性质相似;系统进化分析将21个NtCESA基因和10个AtCESA基因分成5个分支,每一个分支各成员之间的进化相对保守,基因结构类似,不同分支之间的基因结构差异也较小;NtCESA蛋白结构域相对保守,都含有CESA蛋白典型的N端锌指结构、C端跨膜区和DDD-QXXRW保守功能域;组织表达分析结果表明,大部分NtCESA基因在幼苗和成熟期烟草的根、叶、胚芽和愈伤组织中都有表达,同一个分支中的基因表达模式基本一致,并且NtCESA基因参与初/次生细胞壁纤维素的合成与该基因编码蛋白的跨膜区数目存在关联,表明NtCESA基因家族成员功能上的复杂性;亚细胞定位结果证实NtCESA9和NtCESA14为质膜定位蛋白。本研究为烟草CESA基因家族功能的深入研究奠定了基础。     

植物细胞外源蛋白亚细胞定位的机制及其应用

为将不同的生理功能区隔化,植物细胞分化出具有特异性结构特征的细胞器.分化的细胞内膜系统和分子水平的蛋白质转运调控机制为外源蛋白亚细胞定位表达提供了显著的有利条件.当蛋白质被加载适当的定位信号或启动子时,蛋白质的分选途径便确定下来,同时决定蛋白质的表达终点.本文根据相关研究主题分析了蛋白质亚细胞定位机制,重点阐述包括ER腔、质外体、液泡、蛋白体等细胞器和内膜结构在内的蛋白定位因素,同时探讨了目前蛋白定位因素的应用情况.本文确定亚细胞定位因素将成为植物基因工程领域控制亚细胞水平表达的重要技术策略.     

杭白芷根中分泌道发生方式、分布及其挥发油积累过程研究

为探明杭白芷(Angelica dahurica var.formosana)根中分泌道发生方式、分布及其挥发油转运积累特征,该研究利用光镜及透射电子显微镜技术观察分泌道发生过程及挥发油转运特征,结合组织化学定位确定挥发油的主要积累部位。结果表明:杭白芷根中分泌道由中柱鞘细胞最先发生,次生结构中分泌道主要分布在韧皮部和皮层中;挥发油的合成不仅与分泌细胞中质体及细胞质有关,而且还与周围细胞关系密切;分泌细胞内高尔基体和内质网丰富,可能先通过形成小泡参与转运,再经由细胞壁向腔道内转移;相邻分泌细胞靠近角隅处的细胞壁分泌活动活跃,腔道内积累大量电子致密物质;成熟分泌道中分泌细胞及其腔道内积累大量油滴,因此挥发油主要积累场所为分泌细胞及其腔道。该研究明确了杭白芷根中分泌道的发生方式、分布及其挥发油积累部位,揭示了分泌道发育过程中挥发油的转运积累特征,为进一步阐明分泌组织生长发育与有效成分积累关系提供了理论依据。     

Characterization of a cDNA coding for an extracellular calmodulin-binding protein from suspension-cultured cells of <Emphasis Type="Italic">Angelica dahurica</Emphasis>

In order to characterize a specific extracellular 21-kDa calmodulin-binding protein (named: ECBP21) from Angelica dahurica L. suspension-cultured cells, the cDNA coding for the protein has been cloned. Here, Southern blot analysis shows that there are at least two copies of ECBP21 gene in Angelica genome. Using truncated versions of ECBP21 and synthetic peptide in CaM binding assays, we mapped the calmodulin-binding domain to a 16-amino acid stretch (residues 200–215) at the C-terminal region. The ECBP21 was localized in the cell wall area by the immunogold electron microscopy and by GFP labeling method. These results define ECBP21 as a kind of an extracellular calmodulin-binding protein (CaMBP). Furthermore, using Northern blot analysis, we examined the expression dynamics of ecbp21 during the incubation of Angelica suspension-cultured cells and the treatments with some growth regulators. The above studies further provide the molecular evidence for the existence of the gene coding for extracellular CaMBPs and imply a possible role for ECBP21.G.-H. Mao and L.-X. Hou contributed equally to this work.     

白芷细胞外21kD钙调素结合蛋白的生理功能初探

利用Ｓｅｐｈａｄｅｘ Ｇ１００ 及ＣＭＳｅｐｈａｒｏｓｅ层析法,从白芷悬浮培养细胞的胞外盐提液中纯化了２１ ｋＤ 钙调素结合蛋白（２１ ｋＤＣａＭＢＰ） ,测其等电点的ｐＨ 为８ ．９ ,紫外薄层扫描显示其纯度达９４ ％ 以上。以此蛋白为抗原,用免疫小鼠腹水法制备了特异性抗体。由纯化的２１ ｋＤＣａＭＢＰ及其特异性抗体研究其对白芷悬浮培养细胞增殖的影响,结果表明：加入外源纯化的２１ ｋＤＣａＭＢＰ 抑制细胞增殖,半抑制浓度约８ μｇ／ｍｌ;而加入２１ ｋＤＣａＭＢＰ特异性抗体却促进细胞增殖效应。推测胞外内源存在的２１ ｋＤＣａＭＢＰ在生理条件下可能具有参与调节胞外活化ＣａＭ 信号分子的浓度,从而调节胞外ＣａＭ 的生物学功能。     

Authentication of Angelica anomala Avé-Lall cultivars through DNA barcodes

Angelica anomala Avé-Lall (Chuanbaizhi in Chinese) is an important medicinal plant which can be used in traditional Chinese medicines; however, there are no authentic and universal methods to differentiate this Sichuan famous-region drug of A. anomala from a large number of non-famous-region and false drugs. It has been demonstrated that DNA barcoding is a molecular diagnostic method for species identification, which uses a single standardized DNA fragment. In this study, we tested five DNA barcoding candidates (matK, ITS, ITS2, rbcL, and psbA-trnH), and we found that ITS was the best candidate to authenticate the famous-region drug of A. anomala. Moreover, through comparative analysis of these five DNA barcodes between A. anomala and Angelica dahurica, we found that ITS had the most and ITS2 had more variable regions, but the psbA-trnH, rbcL, and matK regions were identical. Hence, we suggest ITS as the DNA barcoding to identify A. anomala and A. dahurica. Moreover, we are determined to adopt the A. anomala as the accurate Latin name of Chuanbaizhi.     

白芷有效成分提取工艺的优化及其中药材质量研究

目的：优化白芷中药材有效成分的提取工艺;全面系统地考察与研究市场上白芷中药材的质量。方法：以白芷中欧前胡素和异欧前胡素的含量为指标,采用高效液相色谱法测定,利用正交试验,对渗漉法提取白芷有效成分的工艺条件进行优选;按照《中国药典》2005年版一部白芷项下[含量测定]方法检验了62批次市场上销售的白芷药材中香豆素类物质的含量。结果：乙醇渗漉提取法：药材浸泡24h,加80%乙醇8倍量渗漉,欧前胡素和异欧前胡素的平均提取率为78%;市场上销售的白芷药材中香豆素类物质的含量符合药典的要求。结论：应用渗漉法工艺提取白芷中药材有效成分效率高,稳定性好,适合工业化生产;目前市场上流通的白芷中药材的质量可靠。     

Roles of the plasma membrane and the cell wall in the responses of plant cells to freezing

In an effort to clarify the responses of a wide range of plant cells to freezing, we examined the responses to freezing of the cells of chilling-sensitive and chilling-resistant tropical and subtropical plants. Among the cells of the plants that we examined, those of African violet ( Saintpaulia grotei Engl.) leaves were most chilling-sensitive, those of hypocotyls in mungbean [ Vigna radiata (L.) R. Wilcz.] seedlings were moderately chilling-sensitive, and those of orchid [ Paphiopedilum insigne (Wallich ex Lindl.) Pfitz.] leaves were chilling-resistant, when all were chilled at -2 degrees C. By contrast, all these plant cells were freezing-sensitive and suffered extensive damage when they were frozen at -2 degrees C. Cryo-scanning electron microscopy (Cryo-SEM) confirmed that, upon chilling at -2 degrees C, both chilling-sensitive and chilling-resistant plant cells were supercooled. Upon freezing at -2 degrees C, by contrast, intracellular freezing occurred in Saintpaulia leaf cells, frost plasmolysis followed by intracellular freezing occurred in mungbean seedling cells, and extracellular freezing (cytorrhysis) occurred in orchid leaf cells. We postulate that chilling-related destabilization of membranes might result in the loss of the ability of the plasma membrane to act as a barrier against the propagation of extracellular ice in chilling-sensitive plant cells. We also examined the role of cell walls in the response to freezing using cells in which the plasma membrane had been disrupted by repeated freezing and thawing. In chilling-sensitive Saintpaulia and mungbean cells, the cells with a disrupted plasma membrane responded to freezing at -2 degrees C by intracellular freezing. By contrast, in chilling-resistant orchid cells, as well as in other cells of chilling-resistant and freezing-resistant plant tissues, including leaves of orchard grass ( Dactylis glomerata L.), leaves of Arabidopsis thaliana (L.) Heynh. and cortical tissues of mulberry ( Morus bombycis Koids.), cells with a disrupted plasma membrane responded to freezing by extracellular freezing. Our results indicate that, in the chilling-sensitive plants cells that we examined, not only the plasma membrane but also the cell wall lacked the ability to serve as a barrier against the propagation of extracellular ice, whereas in the chilling-resistant plant cells that we examined, not only the plasma membrane but also the cell wall acted as a barrier against the propagation of extracellular ice. It appears, therefore, that not only the plasma membrane but also the cell wall greatly influences the freezing behavior of plant cells.     

Identification of Ca2+-dependent calmodulin-binding proteins in rat spermatogenic cells as complexes of the heat-shock proteins

Ca2+-calmodulin (CaM)-binding proteins in rat testes were characterized by assays for CaM-binding activity using the CaM-overlay method on transblots of electrophoresed gels and purification by gel-filtration, ion exchange, and adsorption chromatographies. A major CaM-binding protein complex (CaMBP) was identified and found to be comprised of three proteins with molecular masses 110, 100, and 70 kDa. Amino acid sequence analyses of lysylendopeptidase digests from these proteins indicated that all of the constituents of CaMBP are very similar to the members of the heat-shock protein family, i.e., the 110-kDa protein is similar to the APG-2/94 kDa rat ischemia-responsive protein, the 100-kDa protein is similar to the rat counterpart of the mouse APG-1/94 kDa osmotic stress protein, and the 70-kDa protein is similar to the rat testis-specific major heat-shock protein (HSP70). Immunohistochemistry using anti-CaMBP and anti-CaM antibodies demonstrated that CaMBP was co-localized with CaM in the cytoplasm of pachytene spermatocytes and nuclei of round spermatids. In addition, CaMBP, but not CaM, was localized at a high level in the residual bodies of elongated spermatids. The possible relevance of CaMBP to regulation of cell cycle progression and spermatogenesis is discussed in this paper.     

CaMBP-10的cDNA克隆和表达及钙调素结合活性分析

采用RT PCR法 ,从中国大白菜中分离了编码CaMBP 1 0的cDNA克隆 .该cDNA全长 4 96bp ,编码 92个氨基酸 ,3′端含有 2 1 6bp的非编码区和poly A尾 .将此BP 1 0cDNA的成熟蛋白序列导入表达质粒pET1 5b并转化至大肠杆菌E .coliBL2 1 (DE3)condonplus RIL进行表达 .以免疫印迹和钙调素结合分析法对重组BP 1 0进行鉴定 ,证明其保持了与天然BP 1 0相同的钙调素结合活性 .氨基酸和核苷酸序列分析结果显示 ,它与植物转脂蛋白高度同源 ,特别是含有 8个保守半胱氨酸 .BP 1 0与转脂蛋白之间具极为相似的理化性质如分子量、等电点、热稳定性等 .据此认为 ,CaMBP 1 0是转脂蛋白家族的新成员 ,Ca2 + CaM信号系统可能参与植物转脂蛋白功能的调节     

Plasma membrane-cell wall adhesion is required for expression of plant defense responses during fungal penetration

Fungal pathogens almost invariably trigger cell wall-associated defense responses, such as extracellular hydrogen peroxide generation and callose deposition, when they attempt to penetrate either resistant or susceptible plant cells. In the current study, we provide evidence that the expression of these defenses is dependent on adhesion between the plant cell wall and the plasma membrane. Peptides containing an Arg-Gly-Asp (RGD) motif, which interfered with plasma membrane-cell wall adhesion as shown by the loss of the thin plasma membrane-cell wall connections known as Hechtian strands, reduced the expression of cell wall-associated defense responses during the penetration of nonhost plants by biotrophic fungal pathogens. This reduction was associated with increased fungal penetration efficiency. Neither of these effects was seen after treatment with similar peptides lacking the RGD motif. Disruption of plant microfilaments had no effect on Hechtian strands but mimicked the effect of RGD peptides on wall defenses, suggesting that the expression of cell wall-associated defenses involves communication between the plant cell wall and the cytosol across the plasma membrane. To visualize the state of the plasma membrane-cell wall interaction during fungal penetration, we observed living cells during sucrose-induced plasmolysis. In interactions that were characterized by the early expression of cell wall-associated defenses, there was no change, or an increase, in plasma membrane-cell wall adhesion under the penetration point as the fungus grew through the plant cell wall. In contrast, for rust fungus interactions with host plants, there was a strong correlation between a lack of cell wall-associated defenses and a localized decrease in plasma membrane-cell wall adhesion under the penetration point. Abolition of this localized decreased adhesion by previous inoculation with a fungus that increased plasma membrane-cell wall adhesion resulted in reduced penetration by the rust fungus and induction of cell wall-associated defenses. These results suggest that rust fungi may induce a decrease in plasma membrane-cell wall adhesion as a means of disrupting the expression of nonspecific defense responses during penetration of host cells.