Effect of Vector Density and Competence on Macromolecular Vector Transformation Efficiency

To research the effects of vector quantity and competence on the positive cloning rate,with a known gene sequence but in the absence of DNA template,we artificially designed 26 primers to synthesize a target gene of 835 bp in vitro using overlapping PCR technique. The whole experiment design with two factors and six levels( 36 combinations) was applied to study the effects of the vector density and competent cells on the macromolecular vector transformation efficiency. Based on the 1 500 ng target gene,the vector density grades were designed( 50,100,150,200,250,300 ng),and then the recombinant plasmids were transformed into Top10F',DH5,Stbl3,Epi400,JM108,SCSI. Results showed that the positive cloning rates of different vector amount from big to small were in the order of 200,250,300,150,100 and 50 ng.The maximum positive cloning rate of 200 ng reached 75%; and the average value was 28. 5%. The positive cloning rates of different competent cells from big to small were in the order of stbl3,Top10F',DH5,JM108,Epi400 and SCSI. Stbl3 was higher than other competent cells under any vector density,and its average positive cloning rate was 42. 4%. Both the vector density and competent cells had significant effects on the macromolecular vector transformation efficiency. The optimal combination was C4 with 200 ng vector density and Stbl3,the positive cloning rate of which could reach 75%.

Establishment of a transformation system in close relatives of wheat under the assistance of TaWOX5

Species closely related to wheat are important genetic resources for agricultural production,functional genomics studies and wheat improvement.In this study,a wheat gene related to regeneration,TaWOX5,was applied to establish the Agrobacterium-mediated transformation systems of Triticum monococcum,hexaploid triticale,and rye(Secale cereale L.)using their immature embryos.Transgenic plants were efficiently generated.During the transformation process,the Agrobacterium infection efficiency was assessed by histochemical staining forβ-glucuronidase(GUS).Finally,the transgenic nature of regenerated plants was verified by polymerase chain reaction(PCR)-based genotyping for the presence of the GUS and bialaphos resistance(bar)genes,histochemical staining for GUS protein,and the QuickStix strip assay for bar protein.The transformation efficiency of T.monococcum genotype PI428182 was 94.4%;the efficiencies of four hexaploid triticale genotypes Lin456,ZS3297,ZS1257,and ZS3224 were 52.1,41.2,19.4,and 16.0%,respectively;and the transformation efficiency of rye cultivar Lanzhou Heimai was 7.8%.Fluorescence in situ hybridization(FISH)and genomic in situ hybridization(GISH)analyses indicated that the GUS transgenes were integrated into the distal or near centromere(proximal)regions of the chromosomes in transgenic T.monococcum and hexaploid triticale plants.In the transgenic hexaploid triticale plants,the foreign DNA fragment was randomly integrated into the AABB and RR genomes.Furthermore,the transgene was almost stably inherited in the next generation by Mendel’s law.The findings in this study will promote the genetic improvement of the three plant species for grain or forage production and the improvement of cereal species including wheat for functional genomics studies.

Fast,simple,efficient Agrobacterium rhizogenes-mediated transformation system to non-heading Chinese cabbage with transgenic roots

Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However,the immaturity of its stable transformation system and its low transformation efficiency limit gene function research on non-heading Chinese cabbage. Agrobacterium rhizogenes-mediated(ARM) transgenic technology is a rapid and effective transformation method that has not yet been established for non-heading Chinese cabbage plants. Here, we optimized conventional ARM approaches(one-step and two-step transformation methods) suitable for living non-heading Chinese cabbage plants in nonsterile environments. Transgenic roots in composite non-heading Chinese cabbage plants were identified using phenotypic detection, fluorescence observation, and PCR analysis. The transformation efficiency of a two-step method on four five-day-old non-heading Chinese cabbage seedlings(Suzhouqing, Huangmeigui, Wuyueman, and Sijiu Caixin) was 43.33%-51.09%, whereas using the stout hypocotyl resulted in a transformation efficiency of 54.88% for the 30-day-old Sijiu Caixin.The one-step method outperformed the two-step method;the transformation efficiency of different varieties was above 60%, and both methods can be used to obtain transgenic roots for functional studies within one month. Finally, optimized ARM transformation methods can easily,quickly, and effectively produce composite non-heading Chinese cabbage plants with transgenic roots, providing a reliable foundation for gene function research and non-heading Chinese cabbage genetic improvement breeding.

Development of powdery mildew resistant derivatives of wheat variety Fielder for use in genetic transformation

Genetic transformation is widely used to improve target traits and to study gene function in wheat.However,transformation efficiency depends on the physiological status of the recipient genotype and that is affected by several factors including powdery mildew(PM)infection.The widely used recipient variety Fielder is very susceptible to PM.Therefore,it would be beneficial to develop PM resistant derivatives with high regeneration ability for use in genetic transformation.In the present study PM resistant lines CB037 and Pm97033 carrying genes Pm21 and PmV,respectively,were backcrossed to Fielder with selection for PM resistance.Five lines,NT89,NT90,NT154,and WT48 with Pm21 and line FL347 with PmV were developed,identified by molecular markers and genomic in situ hybridization(GISH)or fluorescent in situ hybridization(FISH),and further subjected to detailed assessment of agronomic traits and regeneration ability following genetic transformation capacity.Lines FL347,WT48,NT89 and NT154 assessed as being equal to,or superior,to Fielder in regeneration and transformation ability are recommended as suitable materials for the replacement of Fielder for wheat gene transfer and genome editing study.

The Energy Transformation and Efficiency of the May 12,2008 Ms 8.0 Wenchuan Earthquake

The energy transformation and efficiency is now a hot topic among researches of scientific drilling into fault zones （Tanaka et al., 2006; Ma et al., 2006）. This study conducted temperature measurements and fault gouge particle analysis of borehole WFSD-1 from the Wenchuan Earthquake Fault Science Drilling Project （WFSD）, and discussed the earthquake energy budget. The research progress is illuminated as follows.

Economic feasibility and efficiency enhancement approaches for in situ upgrading of low-maturity organic-rich shale from an energy consumption ratio perspective

The technical feasibility of in situ upgrading technology to develop the enormous oil and gas resource potential in low-maturity shale is widely acknowledged.However,because of the large quantities of energy required to heat shale,its economic feasibility is still a matter of debate and has yet to be convincingly demonstrated quantitatively.Based on the energy conservation law,the energy acquisition of oil and gas generation and the energy consumption of organic matter cracking,shale heat-absorption,and surrounding rock heat dissipation during in situ heating were evaluated in this study.The energy consumption ratios for different conditions were determined,and the factors that influence them were analyzed.The results show that the energy consumption ratio increases rapidly with increasing total organic carbon(TOC)content.For oil-prone shales,the TOC content corresponding to an energy consumption ratio of 3 is approximately 4.2%.This indicates that shale with a high TOC content can be expected to reduce the project cost through large-scale operation,making the energy consumption ratio after consideration of the project cost greater than 1.In situ heating and upgrading technology can achieve economic benefits.The main methods for improving the economic feasibility by analyzing factors that influence the energy consumption ratio include the following:(1)exploring technologies that efficiently heat shale but reduce the heat dissipation of surrounding rocks,(2)exploring technologies for efficient transformation of organic matter into oil and gas,i.e.,exploring technologies with catalytic effects,or the capability to reduce in situ heating time,and(3)establishing a horizontal well deployment technology that comprehensively considers the energy consumption ratio,time cost,and engineering cost.

Mutation in the Agrobacterium his I gene enhances transient expression in pepper

Agrobacterium-mediated plant transformation is widely used in plant genetic engineering.However,its efficiency is limited by plant immunity against Agrobacterium.Chili pepper(Capsicum annuum L.)is an important vegetable that is recalcitrant to Agrobacterium-mediated transformation.In this work,Agrobacterium was found to induce a strong immune response in pepper,which might be the reason for T-DNA being difficult to express in pepper.An Agrobacterium mutant screen was conducted and a point mutation in the hisI gene was identified due to a weak immune response and enhanced transient expression mediated by this Agrobacterium mutant in pepper leaves.Further genetic analysis revealed that histidine biosynthesis deficiency caused by mutations in many genes of this pathway led to reduced pepper cell death,presumably due to reduced bacterial growth.However,mutation analysis of threonine and tryptophan biosynthesis genes showed that the biosynthesis of different amino acids may play different roles in Agrobacterium growth and stimulating the pepper immune response.The possible application of Agrobacterium amino acid biosynthesis mutations in plant biology was discussed.

Optimization of protoplast isolation,transformation and its application in sugarcane (Saccharum spontaneum L.)

Sugarcane is a prominent source of sugar and ethanol production.Genetic analysis for trait improvement of sugarcane is greatly hindered by its complex genome,long breeding cycle,and recalcitrance to genetic transformation.The protoplast-based transient transformation system is a versatile and convenient tool for in vivo functional gene analysis;however,quick and effective transformation systems are still lacking for sugarcane.Here,we developed an efficient protoplast-based transformation system by optimizing conditions of protoplasts isolation and PEG-mediated transformation in S.spontaneum.The yield of viable protoplasts was approximately 1.26×107 per gram of leaf material,and the transformation efficiency of 80.19%could be achieved under the optimized condition.Furthermore,using this approach,the nuclear localization of an ABI5-like bZIPs transcription factor was validated,and the promoter activity of several putative DNase I hypersensitive sites(DHSs)was assessed.The results indicated this system can be conveniently applied to protein subcellular localization and promoter activity assays.A highly efficient S.spontaneum mesophyll cell protoplast isolation and transient transformation method was developed,and it shall be suitable for in vivo functional gene analysis in sugarcane.

Study on the influence of armature on the efficiency of reluctance accelerator

The armature is an important part affecting the energy conversion efficiency of a reluctance accelerator.In this paper,six kinds of soft magnetic materials are chosen and four structures are designed for the armature.At first,the circuit and magnetic force are theoretically analyzed.Then the armatures with different materials and structures are used in the simulation,and the performances are compared and analyzed.At last,the experiment verifies the theory analysis and simulation design.It is concluded that the saturation flux density and conductivity of the material are the key factors affecting the armature force,and the optimization of armature structure can effectively restrain the eddy current,reduce negative force and improve efficiency.Compared with cutting slits in solid armatures,laminating the sheets radially can reduce the eddy current more efficiently.Although slitting can prevent the eddy current to a certain extent,meanwhile,it will decrease the magnetic force because of the losing of magnetized volume and the surface area.Hence,choosing the high saturation flux density material and making out the armature with radially_laminated sheets will improve the efficiency of the reluctance accelerator.In this paper,the silicon steel radially_laminated armature is a better choice for the armature design of the reluctance accelerator.

The Mandatory Energy Efficiency Standard for Distribution Transformer soon Publicized in China

Owning to the rapid economy development in China and sharp increase of energy consumption in recent years, energy shortage is increasingly apparent and becoming an important obstacle to the sustainable development of our economy. With the aim to relieve the problem of energy shortage, the State Development and Reform Commission places the great emphasis of energy-saving work on improving energy efficiency of industrial energy-consuming products in energy-saving work. The General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China(AQSIQ) has issued and implemented two energy efficiency standards for three-phase induction motors and displacement air compressor, and will soon issue another two energy efficiency standards for clean- water centrifugal pumps and ventilation fans. The transformer is a kind of electric equipment widely used in many industries in national economy, with features of great amount of use, long period of operation and tremendous potential of energy-saving. So, it is necessary to develop the standard of energy efficiency for distribution transformers.It has been an issue of common concern for all countries in the world to reduce the consumption of transformers and upgrade their distribution efficiency.