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。

Evolution of the rbcS gene family in Solanaceae: concerted evolution and gain and loss of introns, with a description of new statistical guidelines for determining the number of unique gene copies.

Thesis (Ph.D.)--University of Washington, 2014

Divergent calcium signaling in RBCs from Tropidurus torquatus (Squamata – Tropiduridae) strengthen classification in lizard evolution

Abstract Background We have previously reported that a Teiid lizard red blood cells (RBCs) such as Ameiva ameiva and Tupinambis merianae controls intracellular calcium levels by displaying multiple mechanisms. In these cells, calcium stores could be discharged not only by: thapsigargin, but also by the Na+/H+ ionophore monensin, K+/H+ ionophore nigericin and the H+ pump inhibitor bafilomycin as well as ionomycin. Moreover, these lizards possess a P2Y-type purinoceptors that mobilize Ca2+ from intracellular stores upon ATP addition. Results Here we report, that RBCs from the tropidurid lizard Tropidurus torquatus store Ca2+ in endoplasmic reticulum (ER) pool but unlike in the referred Teiidae, these cells do not store calcium in monensin-nigericin sensitive pools. Moreover, mitochondria from T. torquatus RBCs accumulate Ca2+. Addition of ATP to a calcium-free medium does not increase the [Ca2+]c levels, however in a calcium medium we observe an increase in cytosolic calcium. This is an indication that purinergic receptors in these cells are P2X-like. Conclusion T. torquatus RBCs present different mechanisms from Teiid lizard red blood cells (RBCs), for controlling its intracellular calcium levels. At T. torquatus the ion is only stored at endoplasmic reticulum and mitochondria. Moreover activation of purinergic receptor, P2X type, was able to induce an influx of calcium from extracelullar medium. These studies contribute to the understanding of the evolution of calcium homeostasis and signaling in nucleated RBCs.

TRM1, a YY1-like suppressor of rbcS-m3 expression in maize mesophyll cells

The genes rbcS and rbcL encode, respectively, the small and large subunits of the photosynthetic carbon dioxide fixation enzyme ribulose bisphosphate carboxylase/oxygenase. There is a single rbcL gene in each chloroplast chromosome; a family of rbcS genes is located in the nuclear genome. These two genes are not expressed in mesophyll cells but are in adjacent bundle-sheath cells of leaves of the C4 plant Zea mays. Two regions of the maize gene rbcS-m3 are required for suppressing expression in mesophyll cells. One region is just beyond the translation termination site in the 3′ region, and the other is several hundred base pairs upstream of the transcription start site. A binding site for a protein with limited homology to the viral, yeast, and mammalian transcription repressor-activator YY1 (Yin-Yang I), has now been identified in the 3′ region. A maize gene for a protein with zinc fingers homologous to those of YY1 has been isolated, characterized, and expressed in Escherichia coli. The gene is designated trm1 (transcription repressor-maize 1). The protein TRM1 binds to the YY1-like site and, in addition, TRM1 binds to two sequence regions in the 5′ region of the gene that have no homology to the YY1 site. Mutagenesis or deletion of any of these three sequences eliminates repression of rbcS-m3 reporter genes in mesophyll cells.

Red/far-red and blue light-responsive regions of maize rbcS-m3 are active in bundle sheath and mesophyll cells, respectively.

Leaves of the C4 plant maize have two major types of photosynthetic cells: a ring of five large bundle sheath cells (BSC) surrounds each vascular bundle and smaller mesophyll cells (MC) lie between the cylinders of bundle sheath cells. The enzyme ribulose bisphosphate carboxylase/oxygenase is encoded by nuclear rbcS and chloroplast rbcL genes. It is not present in MC but is abundant in adjacent BSC of green leaves. As reported previously, the separate regions of rbcS-m3, which are required for stimulating transcription of the gene in BSC and for suppressing expression of reporter genes in MC, were identified by an in situ expression assay; expression was not suppressed in MC until after leaves of dark-grown seedlings had been illuminated for 24 h. Now we have found that transient expression of rbcS-m3 reporter genes is stimulated in BSC via a red/far-red reversible phytochrome photoperception and signal transduction system but that blue light is required for suppressing rbcS-m3 reporter gene expression in MC. Blue light is also required for the suppression system to develop in MC. Thus, the maize gene rbcS-m3 contains certain sequences that are responsive to a phytochrome photoperception and signal transduction system and other regions that respond to a UVA/blue light photoperception and signal transduction system. Various models of "coaction" of plant photoreceptors have been advanced; these observations show the basis for one type of coaction.

Plasma ATP concentration and venous oxygen content in the forearm during dynamic handgrip exercise

Background: It has been proposed that adenosine triphosphate (ATP) released from red blood cells (RBCs) may contribute to the tight coupling between blood flow and oxygen demand in contracting skeletal muscle. To determine whether ATP may contribute to the vasodilatory response to exercise in the forearm, we measured arterialised and venous plasma ATP concentration and venous oxygen content in 10 healthy young males at rest, and at 30 and 180 seconds during dynamic handgrip exercise at 45% of maximum voluntary contraction (MVC). Results: Venous plasma ATP concentration was elevated above rest after 30 seconds of exercise (P < 0.05), and remained at this higher level 180 seconds into exercise (P < 0.05 versus rest). The increase in ATP was mirrored by a decrease in venous oxygen content. While there was no significant relationship between ATP concentration and venous oxygen content at 30 seconds of exercise, they were moderately and inversely correlated at 180 seconds of exercise (r = -0.651, P = 0.021). Arterial ATP concentration remained unchanged throughout exercise, resulting in an increase in the venous-arterial ATP difference. Conclusions: Collectively these results indicate that ATP in the plasma originated from the muscle microcirculation, and are consistent with the notion that deoxygenation of the blood perfusing the muscle acts as a stimulus for ATP release. That ATP concentration was elevated just 30 seconds after the onset of exercise also suggests that ATP may be a contributing factor to the blood flow response in the transition from rest to steady state exercise.

The transformation of banana with potential virus resistance genes

One approach to reducing the yield losses caused by banana viral diseases is the use of genetic engineering and pathogen-derived resistance strategies to generate resistant cultivars. The development of transgenic virus resistance requires an efficient banana transformation method, particularly for commercially important 'Cavendish' type cultivars such as 'Grand Nain'. Prior to this study, only two examples of the stable transformation of banana had been reported, both of which demonstrated the principle of transformation but did not characterise transgenic plants in terms of the efficiency at which individual transgenic lines were generated, relative activities of promoters in stably transformed plants, and the stability of transgene expression. The aim of this study was to develop more efficient transformation methods for banana, assess the activity of some commonly used and also novel promoters in stably transformed plants, and transform banana with genes that could potentially confer resistance to banana bunchy top nanovirus (BBTV) and banana bract mosaic potyvirus (BBrMV). A regeneration system using immature male flowers as the explant was established. The frequency of somatic embryogenesis in male flower explants was influenced by the season in which the inflorescences were harvested. Further, the media requirements of various banana cultivars in respect to the 2,4-D concentration in the initiation media also differed. Following the optimisation of these and other parameters, embryogenic cell suspensions of several banana (Musa spp.) cultivars including 'Grand Nain' (AAA), 'Williams' (AAA), 'SH-3362' (AA), 'Goldfinger' (AAAB) and 'Bluggoe' (ABB) were successfully generated. Highly efficient transformation methods were developed for both 'Bluggoe' and 'Grand Nain'; this is the first report of microprojectile bombardment transformation of the commercially important 'Grand Nain' cultivar. Following bombardment of embryogenic suspension cells, regeneration was monitored from single transfom1ed cells to whole plants using a reporter gene encoding the green fluorescent protein (gfp). Selection with kanamycin enabled the regeneration of a greater number of plants than with geneticin, while still preventing the regeneration of non-transformed plants. Southern hybridisation confirmed the neomycin phosphotransferase gene (npt II) was stably integrated into the banana genome and that multiple transgenic lines were derived from single bombardments. The activity, stability and tissue specificity of the cauliflower mosaic virus 358 (CaMV 35S) and maize polyubiquitin-1 (Ubi-1) promoters were examined. In stably transformed banana, the Ubi-1 promoter provided approximately six-fold higher p-glucuronidase (GUS) activity than the CaMV 35S promoter, and both promoters remained active in glasshouse grown plants for the six months they were observed. The intergenic regions ofBBTV DNA-I to -6 were isolated and fused to either the uidA (GUS) or gfjJ reporter genes to assess their promoter activities. BBTV promoter activity was detected in banana embryogenic cells using the gfp reporter gene. Promoters derived from BBTV DNA-4 and -5 generated the highest levels of transient activity, which were greater than that generated by the maize Ubi-1 promoter. In transgenic banana plants, the activity of the BBTV DNA-6 promoter (BT6.1) was restricted to the phloem of leaves and roots, stomata and root meristems. The activity of the BT6.1 promoter was enhanced by the inclusion of intron-containing fragments derived from the maize Ubi-1, rice Act-1, and sugarcane rbcS 5' untranslated regions in GUS reporter gene constructs. In transient assays in banana, the rice Act-1 and maize Ubi-1 introns provided the most significant enhancement, increasing expression levels 300-fold and 100-fold, respectively. The sugarcane rbcS intron increased expression about 10-fold. In stably transformed banana plants, the maize Ubi-1 intron enhanced BT6.1 promoter activity to levels similar to that of the CaMV 35S promoter, but did not appear to alter the tissue specificity of the promoter. Both 'Grand Nain' and 'Bluggoe' were transformed with constructs that could potentially confer resistance to BBTV and BBrMV, including constructs containing BBTV DNA-1 major and internal genes, BBTV DNA-5 gene, and the BBrMV coat protein-coding region all under the control of the Ubi-1 promoter, while the BT6 promoter was used to drive the npt II selectable marker gene. At least 30 transgenic lines containing each construct were identified and replicates of each line are currently being generated by micropropagation in preparation for virus challenge.

Numerical simulation of red blood cells’ motion : a review

The micro-circulation of blood plays an important role in human body by providing oxygen and nutrients to the cells and removing carbon dioxide and wastes from the cells. This process is greatly affected by the rheological properties of the Red Blood Cells (RBCs). Changes in the rheological properties of the RBCs are caused by certain human diseases such as malaria and sickle cell diseases. Therefore it is important to understand the motion and deformation mechanism of RBCs in order to diagnose and treat this kind of diseases. Although, many methods have been developed to explore the behavior of the RBCs in micro-channels, they could not explain the deformation mechanism of the RBCs properly. Recently developed Particle Methods are employed to explain the RBCs’ behavior in micro-channels more comprehensively. The main objective of this study is to critically analyze the present methods, used to model the RBC behavior in micro-channels, in order to develop a computationally efficient particle based model to describe the complete behavior of the RBCs in micro-channels accurately and comprehensively

Preferential invasion by Plasmodium merozoites and the self-regulation of parasite burden

The preferential invasion of particular red blood cell (RBC) age classes may offer a mechanism by which certain species of Plasmodia regulate their population growth. Asexual reproduction of the parasite within RBCs exponentially increases the number of circulating parasites; limiting this explosion in parasite density may be key to providing sufficient time for the parasite to reproduce, and for the host to develop a specific immune response. It is critical that the role of preferential invasion in infection is properly understood to model the within-host dynamics of different Plasmodia species. We develop a simulation model to show that limiting the range of RBC age classes available for invasion is a credible mechanism for restricting parasite density, one which is equally as important as the maximum parasite replication rate and the duration of the erythrocytic cycle. Different species of Plasmodia that regularly infect humans exhibit different preferences for RBC invasion, with all species except P. falciparum appearing to exhibit a combination of characteristics which are able to selfregulate parasite density.

Deformation properties of single red blood cell in a stenosed microchannel

Red Blood Cells (RBCs) exhibit different types of motions and different deformed shapes, when they move through capillaries. RBCs can travel through capillaries having smaller diameters than RBCs’ diameter, due to the capacity of high deformability of the viscoelastic RBC membrane. The motion and the steady state shape of the RBCs depend on many factors, such as the geometrical parameters of the microvessel through which blood flows, the RBC membrane bending stiffness and the flow velocity. In this study, the effect of the RBC’s membrane stiffness on the deformation of a single RBC in a stenosed capillary is comprehensively examined. Smoothed Particle Hydrodynamics (SPH) in combination with the two-dimensional spring network membrane model is used to investigate the motion and the deformation property of the RBC. The simulation results demonstrate that the membrane bending stiffness of the RBC has a significant impact on the RBCs’ deformability.

Deformation of a three-dimensional red blood cell in a stenosed micro-capillary

Red blood cells (RBCs) exhibit different types of motions and deformations when the blood flows through capillaries. Interestingly, due to the complex three-dimensional structure of the RBC membrane, RBCs show three-dimensional motions and deformations in the blood flow. These motions and deformations of the RBCs highly depend on the stiffness of the RBC membrane and on the geometrical parameters of the capillary through which blood flows. However, capillaries always do not have uniform cross sections and some capillaries have stenosed segments, where cross sectional area suddenly reduces. Further, some diseases can alter the stiffness of the RBC membrane drastically. In this study, the deformation behaviour of a single three-dimensional RBC is examined, when it moves through a stenosed capillary. A three-dimensional spring network is used to model the RBC membrane. The RBC’s inside and outside fluids are discretized into a finite number of mass points and treated by smoothed particle hydrodynamics (SPH) method. The capillary is considered as a rigid tube with a stenosed section. The deformation index, mean velocity and total energy of the RBC are analysed when it flows through the stenosed capillary. Further, motion and deformation of the RBCs with different membrane stiffness (KB) are compared when they flow through the stenosed segment of the capillary. The simulation results demonstrate the RBCs are subjected to a larger deformation when they move through the stenosed part of the capillary and the RBCs with lower KBvalues easily pass through the stenosed segment of the capillary. Further, RBCs having higher KBvalues have a lower mean velocity and it leads to slow down the overall blood flow rate

Deformation of a single red blood cell in a microvessel

Red blood cells (RBCs) are the most common type of cells in human blood and they exhibit different types of motions and deformed shapes in capillary flows. The behaviour of the RBCs should be studied in order to explain the RBC motion and deformation mechanism. This article presents a numerical simulation method for RBC deformation in microvessels. A two dimensional spring network model is used to represent the RBC membrane, where the elastic stretch/compression energy and the bending energy are considered with the constraint of constant RBC surface area. The forces acting on the RBC membrane are obtained from the principle of virtual work. The whole fluid domain is discretized into a finite number of particles using smoothed particle hydrodynamics concepts and the motions of all the particles are solved using Navier--Stokes equations. Minimum energy concepts are used to simulate the deformed shape of the RBC model. To verify the model, the motion of a single RBC is simulated in a Poiseuille flow and the characteristic parachute shape of the RBC is observed. Further simulations reveal that the RBC shows a tank treading motion when it flows in a linear shear flow.

Formation of the three-dimensional geometry of the red blood cell membrane

Red blood cells (RBCs) are nonnucleated liquid capsules, enclosed in deformable viscoelastic membranes with complex three dimensional geometrical structures. Generally, RBC membranes are highly incompressible and resistant to areal changes. However, RBC membranes show a planar shear deformation and out of plane bending deformation. The behaviour of RBCs in blood vessels is investigated using numerical models. All the characteristics of RBC membranes should be addressed to develop a more accurate and stable model. This article presents an effective methodology to model the three dimensional geometry of the RBC membrane with the aid of commercial software COMSOL Multiphysics 4.2a and Fortran programming. Initially, a mesh is generated for a sphere using the COMSOL Multiphysics software to represent the RBC membrane. The elastic energy of the membrane is considered to determine a stable membrane shape. Then, the actual biconcave shape of the membrane is obtained based on the principle of virtual work, when the total energy is minimised. The geometry of the RBC membrane could be used with meshfree particle methods to simulate motion and deformation of RBCs in micro-capillaries

Numerical investigation of motion and deformation of a single red blood cell in a stenosed capillary

It is generally assumed that influence of the red blood cells (RBCs) is predominant in blood rheology. The healthy RBCs are highly deformable and can thus easily squeeze through the smallest capillaries having internal diameter less than their characteristic size. On the other hand, RBCs infected by malaria or other diseases are stiffer and so less deformable. Thus it is harder for them to flow through the smallest capillaries. Therefore, it is very important to critically and realistically investigate the mechanical behavior of both healthy and infected RBCs which is a current gap in knowledge. The motion and the steady state deformed shape of the RBCs depend on many factors, such as the geometrical parameters of the capillary through which blood flows, the membrane bending stiffness and the mean velocity of the blood flow. In this study, motion and deformation of a single two-dimensional RBC in a stenosed capillary is explored by using smoothed particle hydrodynamics (SPH) method. An elastic spring network is used to model the RBC membrane, while the RBC's inside fluid and outside fluid are treated as SPH particles. The effect of RBC's membrane stiffness (kb), inlet pressure (P) and geometrical parameters of the capillary on the motion and deformation of the RBC is studied. The deformation index, RBC's mean velocity and the cell membrane energy are analyzed when the cell passes through the stenosed capillary. The simulation results demonstrate that the kb, P and the geometrical parameters of the capillary have a significant impact on the RBCs' motion and deformation in the stenosed section.