玉米 rbcL 基因与兰藻 rbcS 基因定向融合及它们在宿主大肠杆菌中的表达

二磷酸核酮糖羧化酶/氧化酶(简称Rubisco, EC, 4.1.1.39)是绿色植物光合作用中参与固定CO2的关键酶。在高等植物，该酶是由8个分子量为55KD的大亚基(LSU)和8个分子量为14KD的小亚基(SSU)构成的16聚体。每个大亚基有四个活性中心，具有双向催化功能，其编码基因位于叶绿体基因组大单拷贝区;小亚基功能还不清楚，它由核基因组编码且有几个拷贝;未成熟小亚基N端有一段transit peptide，靠它的定向跨越叶绿体膜。迄今为止，取自几种植物材料的这二种亚基的氨基酸顺序和编码基因的核苷酸顺序分析业已完成。为该酶的遗传操作奠定了必要的基础。 由于Rubisco与人类利用太阳能和提高作物产量直接相关，所以成为通过生物技术进行改造的重大项目。 巢状假囊细菌(Anacyslis nidulans) R2是一种不含限制性内切酶的单细胞原核生物，能营光合作用，其Rubisco大亚基的氨基酸顺序与玉米的LSU同源性高达80%，但是第四个活性部位(Leu 456位)与玉米不同(Sys, 459位)，由此导致其对CO2的亲合力降低。另一方面，其rbcL与rbcS仅相隔93个bp，且同属一个操纵子。这意味着有可能用同源DNA片段等位交换的办法来改造其rbcL基因。 根据现有的资料，设计出玉米rbcL与兰藻rbcS定向重组于pUC119的兰图：先从pANP1155中切出0.7kb含蓝藻rbcS的PstI-HindIII片段，克隆进pUC119的lacZ启动子下游得pTAS28，采用Reverse primer作引物进行核苷酸顺序分析，确认蓝藻rbcS基因座落在pTAS28正链上。随后从pZmc460中切出包含玉米rbcL基因1.7kb的BglII-HincII片段，将它插入pTAS28的HincⅡ-BamHⅠ双酶切位点，得到pTMN3;为了比较，在另一个质粒pTMN7于1.7kb片段之前加进0.1kb的PstI-HaeIII蓝藻DNA。根据玉米rbcL基因核苷酸顺序（1218-1251）合成一个Oligonucleotide probe，对这三个质粒的总RNA抽提物进行Northern Blot，得到明显的杂交斑点;接着用菌体总蛋白冻干品进行了Western分析，并以新鲜的玉米和烟草叶片为对照，得到阳性结果。显然这二种基因重组之后仍能在宿主E. coli中正常表达。 真正的挑战应是下一步用上述二种质粒转化兰藻A. nidulans R2，考查其能否整合进基因组并表现出较低的氧化酶活性。

Probabilistic Modeling of Rosette Formation

英文摘要: Rosetting, or forming a cell aggregate between a single target nucleated cell and a number of red blood cells (RBCs), is a simple assay for cell adhesion-mediated by specific receptor-ligand interaction. For example, rosette formation between sheep RBC and human lymphocytes has been used to differentiate T cells from B cells. Rosetting assay is commonly used to determine the interaction of Fc gamma-receptors (Fc gamma R) expressed on inflammatory cells and IgG-coated on RBCs. Despite its wide use in measuring cell adhesion, the biophysical parameters of rosette formation have not been well characterized. Here we developed a probabilistic model to describe the distribution of rosette sizes, which is Poissonian. The average rosette size is predicted to be proportional to the apparent two-dimensional binding affinity of the interacting receptor-ligand pair and their site densities. The model has been supported by experiments of rosettes mediated by four molecular interactions: Fc gamma RIII interacting with IgG, T cell receptor and coreceptor CD8 interacting with antigen peptide presented by major histocompatibility molecule, P-selectin interacting with P-selectin glycoprotein ligand 1 (PSGL-1), and L-selectin interacting with PSGL-1. The latter two are structurally similar and are different from the former two. Fitting the model to data enabled us to evaluate the apparent effective two-dimensional binding affinity of the interacting molecular pairs: 7.19x10(-5) mu m(4) for Fc gamma RIII-IgG interaction, 4.66x10(-3) mu m(4) for P-selectin-PSGL-1 interaction, and 0.94x10(-3) mu m(4) for L-selectin-PSGL-1 interaction. These results elucidate the biophysical mechanism of rosette formation and enable it to become a semiquantitative assay that relates the rosette size to the effective affinity for receptor-ligand binding.

Quantifying Cell Binding Kinetics Mediated By Surface-Bound Blood Type B Antigen To Immobilized Antibodies

Cell adhesion is crucial to many biological processes, such as inflammatory responses, tumor metastasis and thrombosis formation. Recently a commercial surface plasmon resonance (SPR)-based BIAcore biosensor has been extended to determine cell binding mediated by surface-bound biomolecular interactions. How such cell binding is quantitatively governed by kinetic rates and regulating factors, however, has been poorly understood. Here we developed a novel assay to determine the binding kinetics of surface-bound biomolecular interactions using a commercial BIAcore 3000 biosensor. Human red blood cells (RBCs) presenting blood group B antigen and CM5 chip bearing immobilized anti-B monoclonal antibody (mAb) were used to obtain the time courses of response unit, or sensorgrams, when flowing RBCs over the chip surface. A cellular kinetic model was proposed to correlate the sensorgrams with kinetic rates. Impacts of regulating factors, such as cell concentration, flow duration and rate, antibody-presenting level, as well as pH value and osmotic pressure of suspending medium were tested systematically, which imparted the confidence that the approach can be applied to kinetic measurements of cell adhesion mediated by surface-bound biomolecular interactions. These results provided a new insight into quantifying cell binding using a commercial SPR-based BIAcore biosensor.

Impact of carrier stiffness and microtopology on two-dimensional kinetics of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) interactions

Mechanics and surface microtopology of the molecular carrier influence cell adhesion, but the mechanisms underlying these effects are not well understood. We used a micropipette adhesion frequency assay to quantify how the carrier stiffness and microtopology affected two-dimensional kinetics of interacting adhesion molecules on two apposing surfaces. Interactions of P-selectin with P-selectin glycoprotein ligand-1 (PSGL-1) were used to demonstrate such effects by presenting the molecules on three carrier systems: human red blood cells (RBCs), human promyelocytic leukemia HL-60 cells, and polystyrene beads. Stiffening the carrier alone or in cooperation with roughing the surface lowered the two-dimensional affinity of interacting molecules by reducing the forward rate but not the reverse rate, whereas softening the carrier and roughing the surface had opposing effects in affecting two-dimensional kinetics. In contrast, the soluble antibody bound with similar three-dimensional affinity to surface-anchored P-selectin or PSGL-1 constructs regardless of carrier stiffness and microtopology. These results demonstrate that the carrier stiffness and microtopology of a receptor influences its rate of encountering and binding a surface ligand but does not subsequently affect the stability of binding. This provides new insights into understanding the rolling and tethering mechanism of leukocytes onto endothelium in both physiological and pathological processes.

Developing A Microfluidic-Based System To Quantify Cell Capture Efficiency

Micro-fabrication technology has substantial potential for identifying molecular markers expressed on the surfaces of tissue cells and viruses. It has been found in several conceptual prototypes that cells with such markers are able to be captured by their antibodies immobilized on microchannel substrates and unbound cells are flushed out by a driven flow. The feasibility and reliability of such a microfluidic-based assay, however, remains to be further tested. In the current work, we developed a microfluidic-based system consisting of a microfluidic chip, an image grabbing unit, data acquisition and analysis software, as well as a supporting base. Specific binding of CD59-expressed or BSA-coupled human red blood cells (RBCs) to anti-CD59 or anti-BSA antibody-immobilized chip surfaces was quantified by capture efficiency and by the fraction of bound cells. Impacts of respective flow rate, cell concentration, antibody concentration and site density were tested systematically. The measured data indicated that the assay was robust. The robustness was further confirmed by capture efficiencies measured from an independent ELISA-based cell binding assay. These results demonstrated that the system developed provided a new platform to effectively quantify cellular surface markers effectively, which promoted the potential applications in both biological studies and clinical diagnoses.

IL-8-induced L-selectin shedding regulates its binding kinetics to PSGL-1

L-selectin plays a crucial role in inflammation cascade by initiating the tethering and rolling of leukocytes on endothelium wall. While many L-selectin molecules are rapidly shed from the cell surface upon activation, the remaining membrane-anchored L-selectin may still play an important role in regulating leukocyte rolling and adhesion with different binding kinetics. Here we developed an in vitro model to activate Jurkat cells via interlukin-8 (IL-8) and quantified the two-dimensional (2D) binding kinetics, using a micropipette aspiration assay, of membrane-anchored L-selectin to P-selectin glycoprotein ligand 1 (PSGL-1) ligand coupled onto human red blood cells (RBCs). The data indicated that L-selectin shedding reduced the amount of membrane-anchored L-selectin and lowered both its reverse and forward rates. These results suggested that the rolling dynamics of activated leukocytes was determined by two opposite impacts: reducing the surface presentation would enhance the rolling but lowering the kinetic rates would decrease the rolling. This finding provides a new insight into understanding how L-selectin shedding regulates leukocyte rolling and adhesion.

Study of the effect of alcohol on single human red blood cells using near-infrared laser tweezers Raman spectroscopy

The effect of alcohol solution on single human red blood Cells (RBCs) was investigated using near-infrared laser tweezers Raman spectroscopy (LTRS). In our system, a low-power diode laser at 785 nm was applied for the trapping of a living cell and the excitation of its Raman spectrum. Such a design could simultaneously reduce the photo-damage to the cell and suppress the interference from the fluorescence on the Raman signal. The denaturation process of single RBCs in 20% alcohol solution was investigated by detecting the time evolution of the Raman spectra at the single-cell level. The vitality of RBCs was characterized by the Raman band at 752 cm(-1), which corresponds to the porphyrin breathing mode. We found that the intensity of this band decreased by 34.1% over a period of 25 min after the administration of alcohol. In a further study of the dependence of denaturation on alcohol concentration, we discovered that the decrease in the intensity of the 752 cm(-1) band became more rapid and more prominent as the alcohol concentration increased. The present LTRS technique may have several potential applications in cell biology and medicine, including probing dynamic cellular processes at the single cell level and diagnosing cell disorders in real time. Copyright (c) 2005 John Wiley T Sons, Ltd.

10KD Zein 基因克隆、序列分析以及对植物百脉根（Lotus corniculatus L.）转化

本文应用聚合酶链式反应技术(PCR)在体外从玉米基因组中扩增富于含硫氨基酸的10KD玉米醇溶蛋白基因、克隆及序列分析结果，并用该基因转化豆科牧草百脉根以期获得有良好营养平衡的转基因牧草，从玉米无菌苗提取大片段的DNA．根据目的基因两端的DNA顺序合成一对引物，利用PCR技术经30个活环DNA扩增，得到了一特异的0.57Kb片段，克隆后对该片段进行限制性内切酶物理图谱分析并测定了其全部编码区序列．结果表明，克隆到的完整的10 Kd玉米醇溶蛋白基因编码区与国外报道的相比，其核苷酸顺序及推测的氧基酸序列同源率分别为96%和90%．将基因分别置于质粒pKYLX5及pKYLX71表达戴体，构建了分别在rbcS及CaMV 35S启动子调控下的10 KD zein基因的嵌合质粒．列用农杆菌介导的叶盘法及茎切段法，选用百脉根( Lotus corniculatus)品种一名分别为“里澳”及“马库”，以5-10天的无菌苗子叶和茎切段为外植体，进行基因转化，在含50mg／L km的MS培养基上，2-3周获得抗性的芽，将芽转到无激素的MS培养基上诱导生根，获得抗性植株，捡测工作正在进行中。

转Rubisco 活化酶大型同工酶基因(rca)水稻的光合作用特性研究

Rubisco 是催化光合暗反应第一步反应的酶，是唯一能将CO2 转变成碳水化合物的酶，由它固定和最后转化成的碳水化合物提供了植物、动物和微生物的食物和能量。但是，Rubisco 催化该反应的效率十分低，使之成为光合作用的限速步骤。由于Rubisco 的合成和催化过程十分复杂，人们很难通过直接改造Rubisco 提高植物固定CO2 的能力。而Rubisco 活化酶能活化Rubisco，使植物在生理CO2 浓度下具有最大的CO2 同化速率，因此研究活化酶有重要意义。水稻活化酶有2 个同工酶，大型同工酶比小型同工酶C 端多37 个氨基酸，其中包括两个Cys 残基。这两个Cys 残基的存在使活化酶大型同工酶对ADP 的存在更加敏感，其活性在硫氧还蛋白的介导下能被基质中氧化还原状态的变化所调节。由于活化酶大型同工酶对调节Rubisco 的活性具有的这种特殊作用，在本研究中，将活化酶大型同工酶rca基因用正义和反义引入水稻基因组，获得了过量表达活化酶大型同工酶基因和反义抑制活化酶基因表达的转基因植株，对其光合作用进行了生理和生化分析。 本研究的主要结果如下： Rubisco 活化酶大型同工酶基因的克隆：从水稻镇恢249 中克隆了1525 bp 的活化酶大型同工酶cDNA 序列。经过测序，它与报道的粳稻品种活化酶大型同工酶cDNA 序列（rca）完全相同。 构建了4 个植物表达载体：3 个为过量表达rca的载体，分别是pCBUbirca，pCBSrca 和 pCBSUbirca ，其中rca分别在水稻中高效表达的玉米Ubiquitin 启动子、受光调控的Rubisco 小亚基基因启动子和由这两个启动子构成的双启动子控制下表达; 1 个在Ubiquitin 启动子控制的反义rca载体，即 pCBUbi-antirca。 获得了转化rca的水稻再生植株：用日本晴，台北309，武育梗7 号和籼稻品种培矮64S 水稻成熟种子诱导愈伤组织。用改良的农杆菌浸染法将rca基因转化这些愈伤组织，在潮霉素筛选压力下获得抗性愈伤组织，经过2 天的干燥处理后，转入到含山梨醇的高渗分化培养基上培养，能迅速获得大量的芽和转化体再生植株。 获得了转rca基因的水稻植株：抗性愈伤组织和再生水稻幼苗的叶片经GUS 染色呈蓝黑色。PCR 扩增转基因水稻基因组内的潮霉素基因和rca，大部分转基因水稻中含有841 bp 的潮霉素基因片段和1525 bp 长的rca cDNA 片段。251 粒T1 代转基因水稻种子中189粒呈现潮霉素抗性，抗性种子/非抗性种子的比率约为3：1，接近孟德尔分离规律。Southern杂交表明rca序列已整合到水稻基因组，一般含1－2个拷贝。Western 杂交显示Rubisco 活化酶含量在转pCBUbi －antirca 的水稻中和对照比，几乎看不出，被反义抑制;转pCBUbirca 的水稻与对照含量相差无几;转pCBSUbirca，pCBSrca 载体的水稻中活化酶的含量比对照有极显著的增加。 T1 代转rca水稻的光合作用发生显著变化：转pCBSrca 和pCBSUbirca 的水稻在饱和光强下的Rubisco 初始活性、羧化效率、光合速率都明显高于对照，但是表观量子效率、色素含量和Rubisco 总活性与对照相似。两者相比，前者比后者更高;转反义rca（pCBUbi-antirca）基因的水稻饱和光强下的光合速率、表观量子效率、羧化效率、Rubisco 初始活性明显降低，色素含量和Rubisco 总活性基本不变;转pCBUbirca 的水稻中，光合作用的各项参数与对照基本相似。 T1 代转rca水稻的叶绿素荧光明显改变：转pCBSrca 和pCBSUbirca 的水稻ΦPSII 的值明显高于对照，而且前者qP 的值明显高于对照。两者相比，前者的ΦPSII 和qP 的值比后者高;转反义rca的水稻ΦPSII，F′v/F′m，qP 值和对照比都明显降低，但qN 的值升高;转pCBUbirca 载体的水稻中，叶绿素荧光的各项参数与对照基本相似。 转rca基因的水稻生长发育的变化：转pCBUbirca 载体的水稻整个生长发育过程与对照相似;转化pCBSrca 和pCBSUbirca 载体的水稻和对照比，植株高大，生长发育速度加快，抽穗、开花和结籽的时间提前。两者本身相比，前者比后者明显;转反义rca（pCBUbi-antirca）基因的水稻生长发育延迟，植株矮小，种子败育。 由上可见，Rubisco 活化酶大型同工酶rca基因在Rubisco 小亚基基因启动子、Ubiquitin 基因启动子和Rubisco 小亚基基因启动子共同控制下正义转入水稻的转基因植物光合作用的参数最好，光合效率提高，植物表型最好，生长发育加快，提前开花结籽。这一研究可能为获得高光合效率和高产量的水稻奠定了基础。

Structure, organization and expression of common carp (Cyprinus carpio L.) NKEF-B gene

Natural killer (NK) cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. Its functions are to enhance NK cell cytotoxicity and to protect DNA and proteins from oxidative damage. In this study, a partial cDNA sequence of carp NKEF-B was isolated from thymus cDNA library. Subsequently, the full-length cDNA of carp NKEF-B was obtained by means of 3' and 5' RACE, respectively. The full-length cDNA of carp NKEF-B was 1022 bp, consisting of a 73 bp 5'-terminal untranslated region (UTR), a 355 bp T-terminal UTR, and a 594 bp open reading frame coding for a protein of 197 amino acids. Carp NKEF-B contained two consensus Val-Cys-Pro (VCP) motifs and three consensus cysteine (Cys-51, Cys-70 and Cys-172) residues. Sequence comparison showed that the deduced amino acid sequence of carp NKEF-B had an overall similarity of 74-96% to that of other species homologues. Phylogenetic analysis revealed that carp NKEF-B forms a cluster with other known teleost NKEF-Bs. Then, by PCR we obtained a 5.1 -k long genomic DNA of carp NKEF-B containing six exons and five introns. Realtime RT-PCR results showed that carp NKEF-B gene was predominantly detected in kidney and head kidney under un-infected conditions. Whereas under SVCV-infection condition, the expression of NKEF-B gene was significantly increased in blood cells, gill, intestine and spleen, but maintained in liver, and decreased significantly in kidney and head kidney. Finally, the rNKEF-B was constructed and expressed in Escherichia coli. By using an antibody against carp rNKEF-B, immunohistochemical study further indicated that NKEF-B positive cells are mainly some RBCs and a few epithelial cells in gill and intestine, and that under SVCV-infection condition, these positive cells or positive products in their cytoplasm were mainly increased in gill and spleen sections of carp. The results obtained in the present study will help to understand the function of NKEF-B in teleost innate immunity. (C) 2008 Elsevier Ltd. All rights reserved.

An improved PCR method for direct identification of Porphyra (Bangiales, Rhodophyta) using conchocelis based on a RUBISCO intergenic spacer

An improved method of PCR in which the small segment of conchocelis is amplified directly without DNA extraction was used to amplify a RUBISCO intergenic spacer DNA fragment from nine species of red algal genus Porphyra (Bangiales, Rhodophyta), including Porphyra yezoensis (Jiangsu, China), P. haitanensis (Fujian, China), P. oligospermatangia (Qingdao, China), P. katadai (Qingdao, China), P. tenera (Qingdao, China), P. suborboculata (Fujian, China), P. pseudolinearis (Kogendo, Korea), P. linearis (Devon, England), and P. fallax (Seattle, USA). Standard PCR and the method developed here were both conducted using primers specific for the RUBISCO spacer region, after which the two PCR products were sequenced. The sequencing data of the amplicons obtained using both methods were identical, suggesting that the improved PCR method was functional. These findings indicate that the method developed here may be useful for the rapid identification of species of Porphyra in a germplasm bank. In addition, a phylogenetic tree was constructed using the RUBISCO spacer and partial rbcS sequence, and the results were in concordant with possible alternative phylogenies based on traditional morphological taxonomic characteristics, indicating that the RUBISCO spacer is a useful region for phylogenetic studies.

Validation of RADARSAT-2 fully polarimetric SAR measurements of ocean surface waves

C band RADARSAT-2 fully polarimetric (fine quad-polarization mode, HH+VV+HV+VH) synthetic aperture radar (SAR) images are used to validate ocean surface waves measurements using the polarimetric SAR wave retrieval algorithm, without estimating the complex hydrodynamic modulation transfer function, even under large radar incidence angles. The linearly polarized radar backscatter cross sections (RBCS) are first calculated with the copolarization (HH, VV) and cross-polarization (HV, VH) RBCS and the polarization orientation angle. Subsequently, in the azimuth direction, the vertically and linearly polarized RBCS are used to measure the wave slopes. In the range direction, we combine horizontally and vertically polarized RBCS to estimate wave slopes. Taken together, wave slope spectra can be derived using estimated wave slopes in azimuth and range directions. Wave parameters extracted from the resultant wave slope spectra are validated with colocated National Data Buoy Center (NDBC) buoy measurements (wave periods, wavelengths, wave directions, and significant wave heights) and are shown to be in good agreement.

红毛菜发育过程及其生理基础

红毛菜（Bangia Lyngb.）属于红藻门，与紫菜属同属红毛菜科，其味道和营养都优于紫菜。目前红毛菜栽培产业已在我国福建莆田展开，但栽培技术还有待提高。海藻栽培技术的发展和成熟依赖于对其生长发育过程的认识。本研究针对红毛菜发育过程及相关光合生理展开，并初步探讨了一采自山西娘子关泉淡水红毛菜群体（FWB）的系统地位。 色素突变标记的壳孢子萌发特征表明最初两次分裂产生的4细胞决定了完整植株的形态建成。成熟植株，为雌雄异体。雌性生殖器果胞的标志性分化结构为原始受精丝，环境因子是促发原始受精丝发展的外部因素，其膨大程度随受精的延迟而增大。原孢子是主要的无性生殖孢子类型，在不良环境中，藻体也会形成内生孢子或休眠包囊，或者藻体断裂后重新形成完整的植株。 红毛菜的生长发育很大程度上受环境因子的控制。高温不利于配子体的发育，15-20 ºC比较适宜。红毛菜无性繁殖的最适温度-光照组合为20 ºC-8 h，有性繁殖为15 ºC-12h。 不同发育阶段，PSII实际光合效率（Y(II)）与细胞的健康状况以及光合器官完整性及其在细胞内的分布有关，而与细胞的类型关系不大。健康的假根细胞、已分化未成熟的精子以及果孢子细胞均具有很高的Y(II)。色素体由中间位变为围周位，中央大液泡（营养藻丝）和大小纤维囊泡（成熟孢子与精子）的产生，使得细胞Y(II)降低。刚放散的壳孢子Y(II)很低，说明在壳孢子由贝壳基质释放到自由水体过程，光合作用受到一定程度抑制；而2h后，Y(II)开始恢复，rbcL的转录水平非常高，为孢子的萌发储备物质和能量需求。 在失水和低盐胁迫下，藻体均维持较高的Y(II)。干出处理至藻体重量不再变化，复水后Y(II)可回复初始水平。海生红毛菜在100％淡水培养基中（约20ºC）培养7天后，部分雄性藻体依然活着。从而体现了红毛菜位居高潮带的生理优势。 FWB终生行无性繁殖，藻体形态与发生以及染色体数目（4条）与海生群体没有区别。而rbcL-rbcS Spacer序列显示，红毛菜海生群体（无性和有性）具有完全相同的序列，而FWB与它们有5bp差异，但是与欧洲、北美地区的淡水群体仅1bp不同，初步说明所有淡水红毛菜群体具有共同的原始起源。