Establishment of a micro-particle bombardment transformation system for Dunaliella saliva

In this study, we chronicle the establishment of a novel transformation system for the unicellular marine green alga, Dunaliella salina. We introduced the CaMV35S promoter-GUS construct into D. saliva with a PDS1000/He micro-particle bombardment system. Forty eight h after transformation, via histochemical staining, we observed the transient expression of GUS in D. salina cells which had been bombarded under rupture-disc pressures of 450 psi and 900 psi. We observed no GUS activity in either the negative or the blank controls. Our findings indicated that the micro-particle bombardment method constituted a feasible approach to the genetic transformation of D. salina. We also conducted tests of the cells' sensitivity to seven antibiotics and one herbicide, and our results suggested that 20 mu g/ ml of Basta could inhibit cell growth completely. The bar gene, which encodes for phosphinothricin acetyltransferase and confers herbicide tolerance, was introduced into the cells via the above established method. The results of PCR and PCR-Southern blot analyses indicated that the gene was successfully integrated into the genome of the transformants.

Expression of the lacZ reporter gene in sporophytes of the seaweed Laminaria japonica (Phaeophyceae) by gametophyte-targeted transformation

The seaweed Laminaria japonica (Phaeophyceae) has a two-generation life cycle consisting of haploid gametophytes and diploid sporophytes. Female and/or male gametophytes were transformed using particle bombardment and the histological LacZ assay was performed on sporophytes generated by either parthenogenesis or inbreeding. Female gametophyte-targeted transformation resulted in similar lower efficiencies in both parthenogenetic and zygotic sporophytes, and only a chimeric expression pattern was observed. Male gametophyte-targeted transformation led to a higher efficiency, with 3.5% of the zygotic sporophytes stained completely blue (all-blue), implying the integration of lacZ at the one-cell stage. Polymerase chain reaction analysis using primers specific for a lacZ-vector juncture fragment and subsequent blotting indicated the presence of the introduced gene in the sporophytes. The method reported here has a potential for seaweed transformation using spore-based bombardment followed by the developmental process.

Three-stage transformation of chlorophyll transient fluorescence pattern under sustained dehydration and the discovery of critical water content in seaweeds

The chlorophyll fluorescence kinetics of marine red alga Grateloupia turutunt Yamada, green alga Ulva pertusa Kjellm and brown alga Laminaria japonica Aresch during natural sustained dehydration were monitored and investigated. The pulse amplified modulation (PAM) system was used to analyze the distinct fluorescence parameters during thallus dehydration. Results proved that the fluorescence kinetics of different seaweed all showed three patterns of transformation with sustained water loss. These were: 1) peak kinetic pattern (at the early stage of dehydration fluorescence enhanced and quenched subsequently, representing a normal physiological state). 2) plateau kinetic pattern (with sustained water loss fluorescence enhanced continuously but quenching became slower, finally reaching its maximum). 3) Platform kinetic pattern (fluorescence fell and the shape of kinetic curve was similar to plateau kinetic pattern). A critical water content (CWC) could be found and defined as the percentage of water content just prior to the fluorescence drop and to be a significant physiological index for evaluation of plant drought tolerance. Once thallus water content became lower than this value the normal peak pattern can not be recovered even through rehydration, indicating an irreversible damage to the thylakoid membrane. The CWC value corresponding to different marine species were varied and negatively correlated with their desiccation tolerance, for example. Laminaria japonica had the highest CWC value (around 90%) and the lowest dehydration tolerance of the three. In addition, a fluorescence "burst" was found only in red algae during rehydration. The different fluorescence parameters F-o, F-v and F-v, F-m were measured and compared during water loss. Both F-o and F-v increased in the first stage of dehydration but F-v/F-m. kept almost constant. So the immediate response of in vivo chlorophyll fluorescence to dehydration was an enhancement. Later with sustained dehydration F-o increased continuously while F-v decreased and tended to become smaller and smaller. The major changes in fluorescence (including fluorescence drop during dehydration and the burst during rehydration) were all attributed to the change in F-o instead of F-v This significance of F-o indicates that it is necessary to do more research on F-o as well as on its relationship with the state of thylakoid membrane.

Vertical distribution of bacterial and archaeal communities along discrete layers of a deep-sea cold sediment sample at the East Pacific Rise (similar to 13 degrees N)

The community structure and vertical distribution of prokaryotes in a deep-sea (ca. 3,191 m) cold sediment sample (ca. 43 cm long) collected at the East Pacific Rise (EPR) similar to 13 degrees N were studied with 16SrDNA-based molecular analyses. Total community DNA was extracted from each of four discrete layers EPRDS-1, -2, -3 and -4 (from top to bottom) and 16S rDNA were amplified by PCR. Cluster analysis of DGGE profiles revealed that the bacterial communities shifted sharply between EPRDS-1 and EPRDS-2 in similarity coefficient at merely 49%. Twenty-three sequences retrieved from DGGE bands fell into 11 groups based on BLAST and bootstrap analysis. The dominant groups in the bacterial communities were Chloroflexi, Gamma proteobacteria, Actinobacterium and unidentified bacteria, with their corresponding percentages varying along discrete layers. Pairwise Fst (F-statistics) values between the archaeal clone libraries indicated that the archaeal communities changed distinctly between EPRDS-2 and EPRDS-3. Sequences from the archaeal libraries were divided to eight groups. Crenarchaea Marine Group I (MGI) was prevalent in EPRDS-1 at 83%, while Uncultured Crenarchaea group II B (UCII B) abounded in EPRDS-4 at 61%. Our results revealed that the vertically stratified distribution of prokaryotic communities might be in response to the geochemical settings and suggested that the sampling area was influenced by hydrothermalism. The copresence of members related to hydrothermalism and cold deep-sea environments in the microbial community indicated that the area might be a transitional region from hydrothermal vents to cold deep-sea sediments.

Genetic transformation in Kappaphycus alvarezii using micro-particle bombardment: a potential strategy for germplasm improvement

This study investigated the delivery of a SV40 promoter driving lacZ gene into cells of Kappaphycus alvarezii using particle bombardment. Thallus pieces 0.5-0.8 mm in diameter and 1 cm in length were prepared as gene recipients. Bombardment parameters of 450 psi (rupture pressures) x 6 cm (particle travel distances), 650 psi x 6 cm, 1,100 psi x 6 cm and 1,100 psi x 9 cm were used. A significant increase in transformation efficiency from about 33% under the rupture pressure of 450 psi to 87% at 650 psi was observed in transformed thalli. Most of the positive cells appeared in epidermal cells bombarded at 450 psi, whereas positive signals were seen in both epidermal and medullary cells at 650 psi. No positive transient expression was detected at a bombardment of 1,100 psi, or in negative or blank controls. For the conditions tested, the best parameter was obtained at 650 psi at a distance of 6 cm. Thus, the strategy of taking vegetative thalli as recipients, using particle bombardment, and combining this with micro-propagation, together with developing an in vivo selectable marker, is a viable way to produce stable transformants, to eliminate chimeric expression, and to achieve transgenic breeding in K. alvarezii.

Transformation of Platymonas (Tetraselmis) subcordiformis (Prasinophyceae, Chlorophyta) by agitation with glass beads

A transient transformation system for the unicellular marine green alga, Platymonas subcordiformis, was established in this study. We introduced the pEGFP-N1 vector into P. subcordiformis with a glass bead method. P. subcordiformis was incubated in cell wall lytic enzymes (abalone acetone powder and cellulase solutions) to degrade the cell wall. The applicable conditions for production of viable protoplasts were pH 6.5, 25 degrees C, and 3 h of enzyme treatment. The protoplast yield was 61.2% when P. subcordiformis cells were added to the enzyme solution at a concentration of 10(7) cell ml(-1). The protoplasts were immediately transformed with the pEGFP-N1 vector using glass-bead method. The transformation frequency was about 10(-5), and there was no GFP activity observed in either the negative or the blank controls. This study indicated that GFP was a sensitively transgenic reporter for P. subcordiformis, and the method of cell wall enzymolysis followed by glass bead agitation was applicable for the transformation of P. subcordiformis.

attB site disruption in marine Actinomyces sp M048 via DNA transformation of a site-specific integration vector

An efficient conjugation method has been developed for the marine Actinomyces sp. isolate M048 to facilitate the genetic manipulation of the chandrananimycin biosynthesis gene cluster. A phi C31-derived integration vector pIJ8600 containing oriT and attP fragments was introduced into strain M048 by bi-parental conjugation from Escherichia coli ET12567 to strain M048. Transformation efficiency was (6.38 +/- 0.41) x 10(-5) exconjugants per recipient spore. Analysis of eight exconjugants showed that the plasmid pIJ8600 was stably integrated at a single chromosomal site (attB) of the Actinomyces genome. The DNA sequence of the attB was cloned and shown to be conserved. The results of antimicrobial activity analysis indicated that the insertion of plasmid pIJ8600 seemed to affect the biosynthesis of antibiotics that could strongly inhibit the growth of E. coli and Mucor miehei (Tu284). HPLC-MS analysis of the extracts indicated that disruption of the attB site resulted in the complete abolition of chandrananimycin A-C production, proving the identity of the gene cluster. Instead of chandrananimycins, two bafilomycins were produced through disruption of the attB site from the chromosomal DNA of marine Actinomyces sp. M048.

Transformation of accommodation space of the Cretaceous Qingshankou Formation, the Songliao Basin, NE China

Analysis of accommodation space variation during deposition of the Cretaceous Qingshankou Formation in the Songliao Basin, NE China, indicates that accommodation space changed both through time and across the basin as a seesaw movement. The mid-upper Qingshankou Formation is divided into three units. In each unit, changes of accommodation space differ in the southern and northern part of the basin. Increasing accommodation in the southern part is accompanied by a decrease in the northern part, and vice versa. Between the northern and southern basin, there was a neutral belt that is like a fulcrum, called the transformation belt here, where the accommodation did not change to any significant degree. We call this response 'accommodation transformation', whose characteristics are defined by tectonic subsidence analysis, palaeontological and sedimentary analyses. The accommodation increasing belt, decreasing belt, transformation belt and accommodation transformation boundary together constitute the accommodation transformation system. The recognition of accommodation transformation in the Songliao Basin provides a new insight into sequence stratigraphy and might be widely applicable.

Transformation field method for calculating the effective properties of isotropic graded composites having arbitrary shapes

A method of transformation field is developed to estimate the effective properties of graded composites whose inclusions have arbitrary shapes and gradient profiles by means of a periodic cell model. The boundary-value problem of graded composites having arbitrary inclusion shapes is solved by introducing the transformation field into the inclusion region. As an example, the effective dielectric response of isotropic graded composites having arbitrary shapes and gradient profiles is handled by the transformation field method (TFM). Moreover, TFM results are validated by the exact solutions of isotropic graded spherical inclusions having a power-law profile and good agreement is obtained in the dilute limit. Furthermore, it is found that the inclusion shapes and the parameters of the gradient profiles can have profound effect on the effective properties of composite systems at high concentration of inclusions.

Possible suppression of exogenous beta-1,3-glucanase gene gluc78 on rice transformation and growth

Three genes encoding for fungal cell wall degrading enzymes (CWDE), ech42, nag7O and gluc78 from the biocontrol fungus Trichoderma atroviride were transformed into rice mediated by Agrobacterium tumefaciens singly and in all possible combinations. A total of more than 1800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. Our data indicated that gluc78 gene had negative effects on transformation frequency and plant growth. Some regenerated plants with gluc78 gene were stunted; spontaneously produced brown specks; could not tassel. The combination with either one of the two other genes (ech42, nag70) present in the same T-DNA region reduced the negative effect of gluc78 on plant growth. These results indicated that expression of several genes in one T-DNA region interfered with each other and expression of exogenous gene in recipient plant was a complex behavior. (c) 2007 Published by Elsevier Ireland Ltd.

基于状态构形矢量和状态构形矩阵的可重构模块星球机器人构形水

可重构模块星球机器人系统由母体和多个子机器人模块组成,单个模块可以独立运动和操作,多个模块可以重构组合成不同构形,模块采用非对称式轮手一体机构,具有姿态方位性和运动方向性,重构目的是组成在某种环境下更好地完成有向性运动的构形。基于此,提出矢量构形概念,将运动趋势方向和方位性融合到构形拓扑结构中。在模块矢量分析模型基础上,提出并构建状态构形矢量(State configuration vector,SCV)和状态构形矩阵(State configuration matrix,SCM),对非对称式单模块和整体构形的状态信息进行描述,同时支持预定义的数学变换操作,可以表达并触发模块的基础动作、构形重构。提出离散模块组合重构成目标构形的优化分析算法,通过实例仿真计算获得优化分析的选择结果。

jλ变换与离散振荡指标Md在海洋机器人控制系统动态综合中的应用

本文给出由连续系统的S域,到离散领域?的转换的通式。并给出了离散传递函数W(?)的基本属性、离散振荡指标Md的基本概念、?变换的稳定判据、二阶无静差数字随动系统的动态综合、Md综合在海洋机器人航向控制中的应用。

滑坡型泥石流转化机理研究—以四川省达县青宁乡滑坡为例

Slide-debris flow is debris flow which is transformed from landslide consecutively in a short time, it comprises of two phases: First, Landslide starts to slide; Second, landslide changes to debris flow. Slide-debris flow which brings great property and life loss happens frequently at home and abroad. In order to forecast the happening possibility and scope of slide-debris flow, transfromation mechanism of Slide-debris flow must be studied. Research on transformation mechanism of slide-debris flow is intersectant science of landslide kinetics and debris flow starting theory, It is a fringe problem as well as front problem of geological hazard. This paper takes Qingning slide-debris flow in Da County, Sichuan Province for example and has studied the mechanism of its instability and transfromation into debris flow through indoor test (including usual soil test and ring shear test) and digital modeling method.The research gets the following conclusions. Qingning Landslide took place mainly because of confined water head arising from rainfall infiltration. Before Landslide occurring, it rained continuously for 22 days, accumulated precipitation arrived at 521.6mm.Investigation shows that strata of Qingning Landslide contains quaternary loose accumulation, slip soil and highly weathered bedrock, which is a good condition for formation of confined water in the slope. Further more, groundwater seepage in the slope body and corresponding slope safety factor before landslide occurring have been computed through finite element method. The result shows that because of infiltration of rainfall, confined water head in the slope arose sharply, accordingly, the safety factor of the slope declined quickly. The result also shows that force put on the slide body by the rock mass detached from Dazhaiyan mountain was the direct factor for landslide occurring. Qingning slide-debris transformation mode has been summarized, the process the landslide changed into debris flow is divided into three phases in the prospective of macroscopic geological condition: landslide occurring, transformation and debris flow. Landslide occurring phase is from slope’ local creeping slide to Landslide occurring; transformation phase contains slide body sliding on the slide bed after slide occurring and sliding on the slope after shearing opening; debris flow phase is that slide body breaks up completely and flows downward into the ditches. The transformation mechanism of Qingning slide-debris flow has been studied through indoor ring shear test of slip soil. The result shows that transformation mechanism contains two points: first, during slide body sliding on the slide bed and slope after shearing opening, shearing shrinkage, grain crushing and grain layering brought about declining of its volume and produced excess pore water pressure, and because producing velocity of excess pore water pressure is much greater than its dissipating velocity, shear strength of slide body decreased sharply because of accumulated pore water pressure. Second, grains crushing and grains layering during slide body sliding brought about thick liquefied layer at the bottom of the slidebody, liquefied layer contained high water content and its shear strength was very low, its thickness increased as the sliding displacement increasing. Liquefied layer makes slide body sliding fast and easily break down to debris flow. Excess pore water pressure and liquefied layer made shear strength of slidebody became very low, furthermore, water in the pit of slope joining in the slidebody was also a facter that made slidebody accelerate the transformation. Influence of slide body thickness and fine grains content to transformation of slide-debris flow has been studied through ring shear test. The result reaches two conclusions. First, thickness of slide body affects transformation of slide-debris flow by two ways, porewater pressure and effect of “soft base” increases as thickness of slide body increasing.so the thicker slide body is ,the easier transformation is. Second, actual dissipating velocity of porewater pressure should be considered when studying the influence of fine grains content to tranformation of slide-debris flow. There should be a critical content of fine grains which makes the difference of producing and dissipating velocity of water pore pressre greatest, this value is the best for slide-debris transformation. The whole process of slide-debris flow transformation is reproduced through discrete element method. Transformation mechanism of slide-debris flow is studied through monitoring various parameters including pore water pressure, grain crushing and grain layering in the slide body during the transformation. The result confirms and supplements the transformation mechanism of slide-debris flow got from ring shear test well.

地震波场模拟及叠前深度偏移的李群方法

The processes of seismic wave propagation in phase space and one way wave extrapolation in frequency-space domain, if without dissipation, are essentially transformation under the action of one parameter Lie groups. Consequently, the numerical calculation methods of the propagation ought to be Lie group transformation too, which is known as Lie group method. After a fruitful study on the fast methods in matrix inversion, some of the Lie group methods in seismic numerical modeling and depth migration are presented here. Firstly the Lie group description and method of seismic wave propagation in phase space is proposed, which is, in other words, symplectic group description and method for seismic wave propagation, since symplectic group is a Lie subgroup and symplectic method is a special Lie group method. Under the frame of Hamiltonian, the propagation of seismic wave is a symplectic group transformation with one parameter and consequently, the numerical calculation methods of the propagation ought to be symplectic method. After discrete the wave field in time and phase space, many explicit, implicit and leap-frog symplectic schemes are deduced for numerical modeling. Compared to symplectic schemes, Finite difference (FD) method is an approximate of symplectic method. Consequently, explicit, implicit and leap-frog symplectic schemes and FD method are applied in the same conditions to get a wave field in constant velocity model, a synthetic model and Marmousi model. The result illustrates the potential power of the symplectic methods. As an application, symplectic method is employed to give synthetic seismic record of Qinghai foothills model. Another application is the development of Ray+symplectic reverse-time migration method. To make a reasonable balance between the computational efficiency and accuracy, we combine the multi-valued wave field & Green function algorithm with symplectic reverse time migration and thus develop a new ray+wave equation prestack depth migration method. Marmousi model data and Qinghai foothills model data are processed here. The result shows that our method is a better alternative to ray migration for complex structure imaging. Similarly, the extrapolation of one way wave in frequency-space domain is a Lie group transformation with one parameter Z and consequently, the numerical calculation methods of the extrapolation ought to be Lie group methods. After discrete the wave field in depth and space, the Lie group transformation has the form of matrix exponential and each approximation of it gives a Lie group algorithm. Though Pade symmetrical series approximation of matrix exponential gives a extrapolation method which is traditionally regarded as implicit FD migration, it benefits the theoretic and applying study of seismic imaging for it represent the depth extrapolation and migration method in a entirely different way. While, the technique of coordinates of second kind for the approximation of the matrix exponential begins a new way to develop migration operator. The inversion of matrix plays a vital role in the numerical migration method given by Pade symmetrical series approximation. The matrix has a Toepelitz structure with a helical boundary condition and is easy to inverse with LU decomposition. A efficient LU decomposition method is spectral factorization. That is, after the minimum phase correlative function of each array of matrix had be given by a spectral factorization method, all of the functions are arranged in a position according to its former location to get a lower triangular matrix. The major merit of LU decomposition with spectral factorization (SF Decomposition) is its efficiency in dealing with a large number of matrixes. After the setup of a table of the spectral factorization results of each array of matrix, the SF decomposition can give the lower triangular matrix by reading the table. However, the relationship among arrays is ignored in this method, which brings errors in decomposition method. Especially for numerical calculation in complex model, the errors is fatal. Direct elimination method can give the exact LU decomposition But even it is simplified in our case, the large number of decomposition cost unendurable computer time. A hybrid method is proposed here, which combines spectral factorization with direct elimination. Its decomposition errors is 10 times little than that of spectral factorization, and its decomposition speed is quite faster than that of direct elimination, especially in dealing with a large number of matrix. With the hybrid method, the 3D implicit migration can be expected to apply on real seismic data. Finally, the impulse response of 3D implicit migration operator is presented.