青稞B组醇溶蛋白基因与上游调控区的克隆及基因表达

高等植物种子胚乳贮藏蛋白是种子发芽时的主要氮源，也是人类和动物食用植物蛋白的主要来源。大麦种子胚乳贮藏蛋白主要是醇溶蛋白（hordeins），占大麦胚乳总蛋白的50–60%。根据大麦醇溶蛋白的大小和组成特点，大麦醇溶蛋白被划分为三种类型：富硫蛋白亚类(B，γ-hordeins)、贫硫蛋白亚类（C-hordeins)以及高分子量蛋白亚类(D-hordeins)。B组和C组醇溶蛋白是大麦胚乳的两类主要贮藏蛋白，它们分别占大麦总醇溶蛋白成分的70–80%和10–12%。遗传分析表明，大麦B、C、D和γ-组醇溶蛋白分别是由位于大麦第五染色体1H（5）上的Hor2、Hor1、Hor3和Hor5位点编码。Hor2位点编码大量分子量相同但组成不同的B组醇溶蛋白（B-hordein）。B-hordein的种类、数量和分布是影响大麦酿造、食用及饲养品质的重要因素之一。为深入了解B-hordein基因家族的结构和染色体组织，探明Hor2位点基因表达的发育调控机制，最终达到改良禾谷类作物籽粒品质的目的，本研究以青藏高原青稞为材料，采用同源克隆法，分别克隆B-hordein基因和启动子，通过原核生物表达验证B-hordein基因功能，并利用实时定量PCR探索B-hordein基因表达时空关系，取得如下研究结果： 1. 以具有特殊B组醇溶蛋白亚基组成的9份青藏高原青稞为材料，根据GenBank中三个B-hordein基因序列（GenBank No. X03103, X53690和X53691）设计一对引物，通过PCR扩增，获得23个B-hordein基因克隆并对其进行了序列分析。核苷酸序列分析表明，所有克隆均包含完整的开放阅读框。有11个克隆都存在一个框内终止密码子，推测这11个克隆可能是假基因。推测的氨基酸序列分析表明，所有大麦B-hordein具有相似的蛋白质基本结构，均包括一个高度保守的信号肽、中间重复区以及C-端结构域。不同大麦种重复区内重复基元的数目有较大差异。青稞材料Z07–2和Z26的B-hordeins仅具有12个重复基元结构，更接近于野生大麦。这些重复基元数目的差异导致了重复区序列长度和结构的变异。这种现象极可能是由于醇溶谷蛋白基因在进化过程中染色体的不平衡交换或复制滑动所造成的。对所克隆基因和禾本科代表性醇溶谷蛋白基因进行聚类分析，结果表明所有来自栽培大麦的B-hordeins聚类成一个亚家族，来自野生大麦的B-hordeins以及普通小麦的LMW-GS聚类成另外一个亚家族，表明这两个亚家族的成员存在显著差异。此外，我们发现B-hordein基因推测的C-末端序列具有一些有规律的特征：即具有相同C-末端序列的B-hordein基因在系统发生树中聚类为同一个亚组（除BXQ053，BZ09-1，BZ26-5分别单独聚为一类外）。这个特征将有助于我们对所有B组醇溶蛋白基因家族成员进行分类，避免了在SDS-PAGE电泳图谱上仅依靠大小分类的局限性。 2. 根据上述克隆的青稞B-hordein基因的5’端序列设计三条基因特异的反向引物，以青稞Z09和Z26的基因组DNA为模板，采用SON-PCR和TAIL-PCR技术分离克隆出8个B-hordein基因的上游调控序列（命名为Z09P和Z26P）。序列分析表明，推测的TATA box位于–80 bp，CAAT–like box位于–140 bp处。此外，Z09P和Z26P中有六个序列在–300 bp处均存在一个由高度保守的EM基序和类GCN4基序构成的胚乳盒(Endosperm Box，EB)，在约–560 bp处存在一个胚乳盒类似结构。而Z09P-2和Z26P-3不存在保守的胚乳盒或其类似结构，预示着这两个启动子所调控的基因表达可能受不同类型反式作用因子的调节，推测该启动子对基因的表达调控具有多样性。 3. 将B-hordein基因的开放阅读框定向克隆到表达载体pET-30a中，将其导入大肠杆菌表达菌株BL21中进行外源基因的诱导表达以验证所克隆基因的功能。结果表明仅含重组子pET-BZ07-2和pET-BZ26-5的BL21细菌有目的表达蛋白产生。在诱导3 h时的蛋白表达量最高；3 mM IPTG诱导的蛋白表达量要高于1 mM IPTG诱导的表达量。这为分离纯化B-hordein蛋白以及进一步研究其对大麦籽粒品质的影响奠定基础。 4. 根据从青稞Z09和Z26中分离克隆的B-hordein基因序列设计一对基因特异的引物，同时，选择大麦α-微管蛋白基因（GenBank no. U40042）为看家基因并设计特异引物，利用实时荧光定量PCR检测了青稞籽粒4个胚乳发育时间段的B-hordein基因表达，荧光定量结果显示：两份材料中B-hordein基因的表达量均随发育过程的进行而逐渐升高。Z09中B-hordein基因在开花后7天开始转录，而Z26开花4天后就有低水平B-hordein的表达，这表明Z26中B-hordein基因可能比Z09表达的较早或者Z09中B-hordein基因表达水平较低以致于不能被检测到。此外，在4个不同的胚乳发育时期中，Z26中B-hordein基因的表达量均高于Z09材料。在开花12天到18天的过程中，Z09和Z26中B-hordein基因的表达水平有一个急剧性的升高。这说明在不同胚乳发育时期，Hor2位点的B-hordein等位基因变异体存在mRNA的差异表达。 Seed endosperm storage proteins in higher plants are the main resources of nitrogen for germinating and plant proteins for human and animals. Barley prolamins (also called hordeins) are the major storage proteins in the endosperm and account for 50–60% of total proteins. Hordeins are classically divided into three groups: sulphur-rich (B, γ-hordeins), sulphur-poor (C-hordeins) and high molecular weight (HMW, D-hordeins) hordeins based on the size and composition. B-hordeins and C-hordeins are two major groups and each respectively account for about 70-80% and 10-12% of the total hordein fraction in barley endosperm. Genetic analysis showed that B-, C-, C-, γ-hordeins are encoded by Hor2, Hor1, Hor3 and Hor5 locus on the chromosome 1H (5). Hor2 locus is rich in alleles that encode numerous heterogeneous B-hordein polypeptides. It is reported that B-hordein species, quantity and distribution are significant factors affecting malting, food and feed quality of barley. To understand comprehensively the structure and organization of B-hordein gene family in hull-less barley and explore the developmental control mechanisms of Hor2 locus gene expression and eventually to better exploitation in crop grain quality improvement, we isolated and cloned B-hordein genes and promotors of hull-less barley from Qinghai-Tibet Plateau by PCR, and testified their expression founction in bacteria expression system and explore their spatial and temporal expression pattern by quantitative real time PCR. Our results are as followed, 1. Twenty-three copies of B-hordein gene were cloned from nine hull-less barley cultivars of Qinghai-Tibet Plateau with special B-hordein subunits and molecularly characterized by PCR, based on three B-hordein genes published previously (GenBank No. X03103, X53690 and X53691). DNA sequences analyses confirmed that the six clones all contained a full-length coding region of the barley B-hordein genes. Eleven clones all contain an in-frame stop codon and they are probably pseudogenes. The analysis of deduced amino acid sequences of the genes shows that they have similar structures including signal peptide domain, central repetitive domain, and C-terminal domain. The number of the repeats was largerly variable and resulted in polypeptides in different sizes or structures among the genes. Twelve such repeated motifs were found in Z07–2 and Z26, and they are close to those of the wild barleys, and it is most probably caused by unequal crossing-over and/or slippage during replication as suggested for the evolution of other prolamins. The relatedness of prolamin genes of barley and wheat was assessed in the phylogenetic tree based on their polypeptides comparison. Our phylogenetic analysis suggested that the predicted B-hordeins of cultivated barley formed a subfamily, while the B-hordeins of wild barleys and the two most similar sequences of LMW-GS of T. aestivum formed another subfamily. This result indicated that the members of the two subfamilys have a distinctive difference. In addition, we found the B-hordeins with identical C-terminal end sequences were clustered into a same subgroup (except BXQ053，BZ09-1 and BZ26-5 as a sole group, respectively), so we believe that B-hordein gene subfamilies possibly can be classified on the basis of the conserved C-terminal end sequences of predicted polypeptide and without the limit of SDS-PAGE protein banding patterns. 2. The specific primers were designed according to the published sequences of barley B-hordein genes from Z09 and Z26. Using total DNA isolated from them as the templates, eight clones (designated Z09Pand Z26P) of upstream sequences of the known B-hordein genes was obtained by TAIL-PCR and SON-PCR. Sequences analysis shows that the putative TATA box was present at position –80 bp and CAAT-like box at position –140 bp. Besides, a putative Endosperm Box including an Endosperm Motif (EM) and a GCN4-Like Motif was found at position –300 bp in six clones, and another Endosperm-like box was found at positon –560 bp. While the Endosperm Box or Endosperm-like box was not found in Z09P-2 and Z26P-3. This may indicate that gene expression drived by the two promtors was probably controlled by different trans-acting factors and the genetic control mechanism of corresponding gene expression may be diverse. 3. The B-hordein genic region coding for the mature peptide was cloned into expression vector pET-30a and transformed into bacterial strain BL21 for identifying gene expression fountion. Protein SDS–PAGE analysis showed that only the transformed lysate with the pET-BZ07-2 and pET-BZ26-5 constructs produced proteins related to B-group hordeins of barley, and the mounts of proteins induced by 3 mM IPTG and 3 h were higher than other conditions. This established a base for isolating and putifying B-hordein and further exploring their effects on barley grain quality. 4. The gene-specific primers of B-hordein genes from Z09 and Z26 were used for the quantification of B-hordein gene expression. The α-tubulin gene from Hordeum vulgare subsp. vulgare (GenBank accession number U40042) was used as a control gene. The result shows the transcription of the B-hordein genes in Z09 was found 7 days after flowering, while the transcription of the B-hordein genes in Z26 was found 4 days after flowering, but at a very low level, and it suggested that the B-hordein genes in Z26 probably expressed earlier than those in Z09, or the B-hordein genes in Z09 expressed at so a lower level than Z26 that it can not detected. In addition, B-hordein genes in Z26 accession showed higher expression levels than those in Z09 in four developing stages. Furthermore, a progressive increase in the expression levels of the B-hordein genes between 12 and 18 days after anthesis was observed in both Z09 and Z26. It implies that the B-hordein allelic variants encoded by Hor2 locus exist the differential expression in mRNA levels of during barley endosperm development.

蛇形机器人的翻滚运动及其越障研究

提出了一种新型蛇形机器人机构。该机构既可以实现平面运动又可以实现空间运动。建立了其空间运动学模型并实现了空间翻滚运动。实验结果表明 :翻滚运动不需依靠模块与地面之间动摩擦力方向差来实现驱动 ,而是通过模块与地面之间静摩擦力和互相垂直的两个运动平面内异相波的相互作用产生驱动力。翻滚的方向和姿态由两个波形曲线的相差和传播方向决定。利用翻滚运动 ,实现了少模块数蛇形机器人的越障运动

Dust-Cloud Structures behind a Shock Wave Moving Over a Deposited Layer of Fine Particles

The present paper investigates dispersed-phase flow structures of a dust cloud induced by a normal shock wave moving at a constant speed over a flat surface deposited with fine particles. In the shock-fitted coordinates, the general equations of dusty-gas

Effect of ambient pressure and radiation reabsorption of atmosphere on the flame spreading over thermally thin combustibles in microgravity

For the flame spread over thermally thin combustibles in an atmosphere, if the atmosphere cannot emit and absorb the thermal radiation (e.g. for atmosphere Of O-2-N-2), the conductive heat transfer from the flame to the fuel surface dominates the flame spread at lower ambient atmosphere. As the ambient pressure increases, the flame spread rate increases, and the radiant heat transfer from the flame to the fuel surface gradually becomes the dominant driving force for the flame spread. In contrast, if the atmosphere is able to emit and absorb the thermal radiation (e.g. for atmosphere Of O-2-CO2), at lower pressure, the heat transfer from flame to the fuel surface is enhanced by the radiation reabsorption of the atmosphere at the leading edge of the flame, and both conduction and thermal radiation play important roles in the mechanism of flame spread. With the increase in ambient pressure, the oxygen diffuses more quickly from ambient atmosphere into the flame, the chemical reaction in the flame is enhanced, and the flame spread rate increases. When the ambient pressure is greater than a critical value, the thermal radiation from the flame to the solid surface is hampered by the radiation reabsorption of ambient atmosphere with the further increase in ambient pressure. As a result, with the increase in ambient pressure, the flame spread rate decreases and the heat conduction gradually dominates the flame spread over the fuel surface.

Shock-Induced Near-Wall Two-Phase Flow Structure Over a Micron-Sized Particles Bed

The present paper studies numerical modelling of near-wall two-phase flows induced by a normal shock wave moving at a constant speed, over a micronsized particles bed. In this two-fluid model, the possibility of particle trajectory intersection is considered and a full Lagrangian formulation of the dispersed phase is introduced. The finiteness of the Reynolds and Mach numbers of the flow around a particle as well as the fineness of the particle sizes are taken into account in describing the interactions between the carrier- and dispersed- phases. For the small mass-loading ratio case, the numerical simulation of flow structure of the two phases is implemented and the profiles of the particle number density are obtained under the constant-flux condition on the wall. The effects of the shock Mach number and the particle size and material density on particle entrainment motion are discussed in detail.The obtained results indicate that interphase non-equilibrium in the velocity and temperature is a common feature for this type of flows and a local particle accumulation zone may form near the envelope of the particle trajectory family.

Ocean Waves Propagating over a Porous Seabed: Residual and Oscillatory Mechanisms

Two mechanisms for the wave-induced pore pressures in a porous seabed, i.e. oscillatory and residual excess pore pressures, have been observed in laboratory experiments and field measurements. Most previous investigations have focused on one of the mechanisms individually. In this paper, an analytical solution for the wave-induced residual pore pressure, which is not available yet, is derived, and compared with the existing experimental data. With the new solution, a parametric analysis is performed to clarify the applicable ranges of two mechanisms. Then, a simplified approximation for the prediction of wave-induced liquefaction potential is proposed for engineering practice.

A Numerical Study for Turbulent Flow and Thermal Influence Over Inhomogenous Canopy of Roughness Elements

A large-eddy simulation with transitional structure function(TSF) subgrid model we previously proposed was performed to investigate the turbulent flow with thermal influence over an inhomogeneous canopy, which was represented as alternative large and small roughness elements. The aerodynamic and thermodynamic effects of the presence of a layer of large roughness elements were modelled by adding a drag term to the three-dimensional Navier-Stokes equations and a heat source/sink term to the scalar equation, respectively. The layer of small roughness elements was simply treated using the method as described in paper (Moeng 1984, J. Atmos Sci. 41, 2052-2062) for homogeneous rough surface. The horizontally averaged statistics such as mean vertical profiles of wind velocity, air temperature, et al., are in reasonable agreement with Gao et al.(1989, Boundary layer meteorol. 47, 349-377) field observation (homogeneous canopy). Not surprisingly, the calculated instantaneous velocity and temperature fields show that the roughness elements considerably changed the turbulent structure within the canopy. The adjustment of the mean vertical profiles of velocity and temperature was studied, which was found qualitatively comparable with Belcher et al. (2003, J Fluid Mech. 488, 369-398)'s theoretical results. The urban heat island(UHI) was investigated imposing heat source in the region of large roughness elements. An elevated inversion layer, a phenomenon often observed in the urban area (Sang et al., J Wind Eng. Ind. Aesodyn. 87, 243-258)'s was successfully simulated above the canopy. The cool island(CI) was also investigated imposing heat sink to simply model the evaporation of plant canopy. An inversion layer was found very stable and robust within the canopy.

DNS of Compressible Turbulent Boundary Layer Over a Blunt Wedge

Direct numerical simulation of spatially evolving compressible boundary layer over a blunt wedge is performed in this paper. The free-stream Mach number is 6 and the disturbance source produced by wall blowing and suction is located downstream of the sound-speed point. Statistics are studied and compared with the results in incompressible flat-plate boundary layer. The mean pressure gradient effects on the vortex structure are studied.

Turbulent Modeling of ABL over Wavy Water Surface

A new model accounting for both turbulence and sea state effects has been proposed in this paper to describe momentum, heat and moisture exchanges through air-sea interface. While long wave components mainly change air flow profile, short wave components

On the design of the piecewise linear vibration absorber

This paper explores the potential of the piecewise linear vibration absorber in a system subject to narrow band harmonic loading. Such a spring is chosen because the design of linear springs is common knowledge among engineers. The two-degrees-of-freedom system is solved by using the Incremental Harmonic Balance method, and response aspects such as stiffness crossing frequency and jump behaviour are discussed. The effects of mass, stiffness, natural frequency ratios, and stiffness crossing positions on the suppression zone are probed. It is shown that a hardening absorber can deliver a wider bandwidth than a linear one over a range of frequencies. The absorber parameters needed to produce good designs have been determined and the quality of the realized suppression zone is discussed. Design guidelines are formulated to aid the parameter selection process.

Direct Numerical Simulation Of Hypersonic Boundary-Layer Transition Over A Blunt Cone

Direct numerical simulation of transition How over a blunt cone with a freestream Mach number of 6, Reynolds number of 10,000 based on the nose radius, and a 1-deg angle of attack is performed by using a seventh-order weighted essentially nonoscillatory scheme for the convection terms of the Navier-Stokes equations, together with an eighth-order central finite difference scheme for the viscous terms. The wall blow-and-suction perturbations, including random perturbation and multifrequency perturbation, are used to trigger the transition. The maximum amplitude of the wall-normal velocity disturbance is set to 1% of the freestream velocity. The obtained transition locations on the cone surface agree well with each other far both cases. Transition onset is located at about 500 times the nose radius in the leeward section and 750 times the nose radius in the windward section. The frequency spectrum of velocity and pressure fluctuations at different streamwise locations are analyzed and compared with the linear stability theory. The second-mode disturbance wave is deemed to be the dominating disturbance because the growth rate of the second mode is much higher than the first mode. The reason why transition in the leeward section occurs earlier than that in the windward section is analyzed. It is not because of higher local growth rate of disturbance waves in the leeward section, but because the growth start location of the dominating second-mode wave in the leeward section is much earlier than that in the windward section.

Receptivity To Free-Stream Disturbance Waves For Hypersonic Flow Over A Blunt Cone

A high-order shock-fitting finite difference scheme is studied and used to do direction numerical simulation (DNS) of hypersonic unsteady flow over a blunt cone with fast acoustic waves in the free stream, and the receptivity problem in the blunt cone hypersonic boundary layers is studied. The results show that the acoustic waves are the strongest disturbance in the blunt cone hypersonic boundary layers. The wave modes of disturbance in the blunt cone boundary layers are first, second, and third modes which are generated and propagated downstream along the wall. The results also show that as the frequency decreases, the amplitudes of wave modes of disturbance increase, but there is a critical value. When frequency is over the critial value, the amplitudes decrease. Because of the discontinuity of curvature along the blunt cone body, the maximum amplitudes as a function of frequencies are not monotone.

Experimental study of gaseous detonation propagation over acoustically absorbing walls

The paper presents results front an experimental investigation of the propagation of gaseous detonation waves over tube sections lined with acoustically absorbent materials. The measurements were compared with results from control tests in a smooth wall section. The results show the increasing effectiveness of a perforated steel plate, wire mesh and steel wool in attenuating detonation.

Air flow over a mountain and the convective boundary-layer

In this paper, the analytical model coupling the convective boundary layer (CBL) with the free atmosphere developed by Qi and Fu (1992) is improved. And by this improved model, the interaction between airflow over a mountain and the CBL is further discussed. The conclusions demonstrate: (1) The perturbation potential temperatures in the free atmosphere can counteract the effect of orographic thermal forcing through entraining and mixing in the CBL. If u(M)BAR > u(F)BAR, the feedback of the perturbation potential temperatures in the free atmosphere is more important than orographic thermal forcing, which promotes the effect of interfacial waves. If u(M)BAR < u(F)BAR, orographic thermal forcing is more important, which makes the interfacial height and the topographic height identical in phase, and the horizontal speeds are a maximum at the top of the mountain. (2) The internal gravity waves propagating vertically in the free atmosphere cause a strong downslope wind to become established above the lee slope in the CBL and result in the hydraulic jump at the top of the CBL. (3) With the CBL deepening, the interfacial gravity waves induced by the potential temperature jump at the top of the CBL cause the airflow in the CBL to be subcritical.

Character of simplified Navier-Stokes (SNS) equations near separation point for two-dimensional laminar flow over a flat plate

It is proved that the simplified Navier-Stokes (SNS) equations presented by Gao Zhi[1], Davis and Golowachof-Kuzbmin-Popof (GKP)[3] are respectively regular and singular near a separation point for a two-dimensional laminar flow over a flat plate. The order of the algebraic singularity of Davis and GKP equation[2,3] near the separation point is indicated. A comparison among the classical boundary layer (CBL) equations, Davis and GKP equations, Gao Zhi equations and the complete Navier-Stokes (NS) equations near the separation point is given.