Cloning of Soybean 24 kDa Oleosin Gene and Its Transient Expression as a Carrier for Foreign Protein

The genomic DNA sequence encoding soybean 24 kDa oleosin and its promoter were cloned andanalyzed for investigation of the potentials of the oleosin acted as a carrier forproduction of recombinant proteins in plant. The -300 box, GA-rich, G-box, SEF-3, SEF-4, RY box, ABA box, CAn and TATA box were found in the upstream region of the soybeanoleosin gene, which shows the functional oleosin promoter available. Homology comparisonreveals that the soybean 24 kDa oleosin shares the highest identity with the soybeanoleosin isoform A (U09118, GenBank), reaching to 98.4% in nucleotide. A soybean oleosin-hirudin fusion gene driven by the oleosin promoter was constructed and inserted intoplant binary expression vector. The intact tobacco plantlets were transformed by meansof vacuum infiltration approach, with the Agrobacterium tumefaciens harboring the abovevector. The transient correct expression of oleosin-hirudin fusion gene was identifiedby SDS/PAGE, western blotting and enterokinase treatment.

Transient Expression of Exogenous Gene into Plant Cell Mediated by PEI Nanovector

This study was carried out to investigate the transfection effect of exogenous gene into plant protoplast cell mediated by polyethylenimine （PEI） nanovector, based on PEI gene delivery system in the field of medical science. PEI/DNA complexes were prepared by using PEI polymer to bind the plant expression plasmid, pCMl205-GFPn. The ability of PEI combining and protecting DNA was investigated by agarose gel electrophoresis retardation assay. The surface characteristics of PEI/DNA complexes were observed with transmission electron microscope. The transfection efficiency of Arabidopsis thaliana protoplasts mediated by PEI/DNA complexes at different N/P ratios was analyzed based on observation of transient expression of green fluorescent protein with confocal laser scanning microscope. PEI could bind and condense DNA, and form stable 100-200 nm PEI/DNA complexes when the proportion of PEl and DNA is in the range of 5：1-1：4. Transfection efficiency of PEI/DNA complexes increased with N/P ratios in range of N/P〈5 and reached the highest at N/P=5, and began to decrease beyond N/P〉5 as higher toxicity to cells. The transfection efficiency of PEI/DNA complexes at N/P=5 was higher than PEG. This study confirmed that PEI nanovector could effectively mediate foreign gene entering into A. thaliana protoplast cell to obtain transient expression, which may be developed as a hopeful and novel transgenic method combined with plant protoplast regeneration.

Efficient gusA Transient Expression in Porphyra yezoensis Protoplasts Mediated by Endogenous Beta-tubulin Flanking Sequences

Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodophyta) are unknown. In this study, the flanking sequences of beta-tubulin gene from P. yezoensis were amplified and two transient expression vectors were con-structed to determine their transcription promoting feasibility for foreign gene gusA. The testing vector pATubGUS was constructed by inserting 5’- and 3’-flanking regions (Tub5’ and Tub3’) up- and down-stream of β-glucuronidase (GUS) gene (gusA), respectively, into pA, a derivative of pCAT?3-enhancer vector. The control construct, pAGUSTub3, contains only gusA and Tub3’. These con-structs were electroporated into P. yezoensis protoplasts and the GUS activities were quantitatively analyzed by spectrometry. The results demonstrated that gusA gene was efficiently expressed in P. yezoensis protoplasts under the regulation of 5’-flanking sequence of the beta-tubulin gene. More interestingly, the pATubGUS produced stronger GUS activity in P. yezoensis protoplasts when com-pared to the result from pBI221, in which the gusA gene was directed by a constitutive CaMV 35 S promoter. The data suggest that the integration of P. yezoensis protoplast and its endogenous beta-tubulin flanking sequences is a potential novel system for foreign gene expression.

Transient gene expression in western white pine using agroinfiltration

A method for transient gene expression was developed for western white pine(WWP,Pinus monticola Dougl.ex D.Don)using reporter gene uidA encodingβ-glucuronidase(GUS).GUS was transiently expressed in cross sections of primary and secondary needles,cotyledons,and current and second year stems of WWP via vacuum-infiltration with Agrobacterium tumefaciens.Histochemical assays of cross sections of secondary needles showed stronger blue color indicating GUS expression at day 1 and 2 than on other days post agroinfiltration(dpa).GUS activity expressed inside WWP cells was confirmed using light microscopy.In fluorometric assays,GUS expression was high at 1 dpa and lasted until 4 dpa in detached secondary needles,while similarly high expression levels only lasted until 2 dpa in attached secondary needles then dropped significantly.Although the length of GUS-staining zones varied among different WWP organs and between growth and dormant seasons,all tested WWP tissues using the protocol had high levels of transient GUS expression.Thus,heterologous candidate genes or endogenous silencing can be expressed in various WWP tissues or organs using this agroinfiltration approach.The current protocol for efficient transient gene expression will aid functional genomics study of WWP and its pathogens and related conifer species.

Evaluation of parameters affecting Agrobacterium-mediated transient expression in citrus

Agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple,easy to perform,and achieve gene expression rapidly.This study investigated the factors affecting transient gene expression efficiency in citrus by observing the cryo-sectioning of leaf samples under a laser confocal microscope.These factors included the composition of the infiltration buffer,the Agrobacterium cell density,the leaf development stage,the incubation temperature,and plant genotype.The highest transient expression level of yellow fluorescent protein（YFP）was detected in Mexican lime（Citrus aurantifolia）on the third day after the intermediate-aged leaves were infiltrated with the improved infiltration buffer 1（15 mmol L^-1 2-（N-morpholino）ethanesulfonic acid,10 mmol L^-1 MgCl2,and 200μmol L^-1acetosyringone）,which had an optical density of 0.8 and was incubated at 22°C.Additionally,this transient expression assay was applied to other citrus genotypes.Of note,trifoliate orange（Poncirus trifoliata）and kumquat（Fortunella obovate）had higher expression efficiency than other six genotypes of the Citrus genus.Our study provides research basis for the selection of optimization strategies in transient gene expression and improves the method for available genome investigation in citrus.

A Transient Expression System for the Functional Assessment of Early Response Genes on the Powdery Mildew Infected Barley or Wheat Leaves

The principle and the basic steps of the transient assay system for the functional assessment of early response genes on the powdery mildew infected barley or wheat leaves were summarized in brief. The development of this technology and its extensive application were reviewed. Future studies on this approach were recommended in this paper.

Expression Analysis of HMW-GS 1Bx14 and 1By15 in Wheat Varieties and Transgenic Research of 1By15 Gene

High-molecular-weight glutenin subunits （HMW-GSs）, one class of seed storage proteins in wheat, play an important role in determining bread-making quality of flour. More and more proves support that HMW-GS- 1Bx 14 and - 1Bx 15 subunits are strongly positively associated with good bread-making and excellent noodle-making quality. The two subunits are encoded by two genes, Glu-1Bx14 and Glu-1Bx15, which are tightly linked and located on the 1BL. Protein assay by SDS-PAGE indicated that the expression of Glu-1Bx14 gene was always much stronger than that of Glu-1By15 in the same variety. But, variation of expression level for Glu-1By15 gene existed among varieties, such as in Xiaoyan 54, Xiaoyan 6, Yanzhan 1 and Shanyou 225. We also investigated the transcription difference of Glu-1By14 and Glu-1By15 genes in Xiaoyan 54 and Shanyou 225 by semi-quantitative RT-PCR method. The Glu-1By14 always transcripts much more than the Glu-1By15. This was basically consistent with the translation difference between the two genes. Promoters of 1Bx14, 1By15, 1By8, 1Dx2 and 1Dy12 were cloned from Xiaoyan 54, Chinese Spring and Aegilops tauschii. Sequence analysis indicated that the HMW-GS genes had high homology at their promoter regions. However, significant difference existed between sequence of 1Bx14 promoter and those of other HMW-GS genes. The transient expression experiment showed that the promoter of 1By15 has lower activity than that of 1Bx14, which was consistent with their transcription level of the two genes in varieties. In addition, transient expression of the gus driven by the promoter （P2） of HMW-GS 1Dx2 gene was higher than by other HMW-GS promoters. Therefore, we constructed 1By15 gene expression vector driven by the 1Dx2 promoter, and transformed the 1By15 gene into wheat commercial variety, Jimai 20 by pollen tube method. Of 45 independent transgenic lines identified by PCR, 3 were confirmed to contain the HMW-GS 1By15 gene via Southern hybridization. The delivered 1By15 gene expressed the expected HMW-GS protein in the seeds of transgenic plants.

TRANSIENT SOLUTION FOR QUEUE-LENGTH DISTRIBUTION OF Geometry/G/1 QUEUEING MODEL

In this paper, the Geometry/G/1 queueing model with inter-arrival times generated by a geometric（parameter p） distribution according to a late arrival system with delayed access and service times independently distributed with distribution {gj }, j≥ 1 is studied. By a simple method （techniques of probability decomposition, renewal process theory） that is different from the techniques used by Hunter（1983）, the transient property of the queue with initial state i（i ≥ 0） is discussed. The recursion expression for u -transform of transient queue-length distribution at any time point n^＋ is obtained, and the recursion expression of the limiting queue length distribution is also obtained.

OsPIN1a Gene Participates in Regulating Negative Phototropism of Rice Roots

The complete open reading frame of OsPINla was amplified through reverse transcriptase- polymerase chain reaction （RT-PCR） based on the sequence deposited in GenBank to explore the relationship between the auxin efflux protein OsPINla and the negative phototropism of rice roots. Sequencing results showed that the GC content of OsPINla was 65.49%. The fusion expression vector pCAMBIA-1301-OsP/N1a：：GFP containing the OsPINla gene and a coding green fluorescent protein （gfp） gene was constructed. The fusion vector was transferred into onion epidermal cells by Agrobacterium tumefaciens transformation. The transient expression of OsPINla-GFP was mainly located in the nucleus and cell membrane. Moreover, the transgenic plants were obtained by Agrobacterium-mediated genetic transformation. Molecular detection performed by using PCR and β-glucuronidase staining showed that the target construct was integrated into the genome of rice. The negative phototropic curvatures of the transgenic rice roots were higher than those of the wild type. Similarly, the expression levels of OsPINla in the transgenic plants were considerably higher than those in the wild-type plants. These results suggest that OsPINla is crucial in the negative phototropic curvature of rice roots.

Transient expression of a TaGRF4-TaGIF1 complex stimulates wheat regeneration and improves genome editing

Genome editing is an unprecedented technological breakthrough but low plant regeneration frequencies and genotype dependence hinder its implementation for crop improvement. Here, we found that transient expression of a complex of the growth regulators TaGRF4 and TaGIF1(TaGRF4-TaGIF1) increased regeneration and genome editing frequency in wheat. When we introduced synonymous mutation in the miR396 target site of TaGRF4, the resulting complex(mTaGRF4-TaGIF1) performed better than original TaGRF4-TaGIF1. Use of m TaGRF4-TaGIF1 together with a cytosine base editor targeting TaALS resulted in 2-9-fold increases in regeneration and transgene-free genome editing in 11 elite common wheat cultivars. Therefore, m TaGRF4-TaGIF1 will undoubtedly be of great value in crop improvement and especially in commercial applications, since it greatly increased the range of cultivars available for transformation.

Optimization of Electroporation Parameters for Immature Embryos of indica Rice (Oryza sativa)

To obtain a suitable condition for electroporation transformation in indica rice, the 10-day-old immature embryos were selected for optimization experiments. The results showed that one pulse at 850 V/cm, 950μF capacitance, 200 μL electroporation buffer with 70 mmol/L sodium glutamate, 100 μg/mL plasmid, 50μg/mL carrier DNA, 20 embryos per cuvette, 0℃ treatment and CC medium were the best parameters, which not only improved the transformation efficiency to 30.89%, but also ameliorated the embryo survival ratio to 95.92%. To further verify the practicability of this condition, the embryos from another indica rice variety and a rice type Ⅱ metallothionein-like gene （OsMT2bL） promotec：mgfp5：：gusA construct were tested, and specific GUS expression on the embryos was visualized by histochemical staining. The results showed that the GUS expression on the embryos activated by the OsMT2bL promoter was mainly concentrated on the apical point of the plumule whereas the expression driven by CaMV35S promoter was distributed on nearly all areas of the electroporated tissues. These results indicated that the optimized embryo electroporation conditions could be used not only in genetic transformation of indica rice but also in assay of gene regulation on embryos.

A Highly Efficient Agrobacterium-Mediated Method for Transient Gene Expression and Functional Studies in Multiple Plant Species

Although the use of stable transformation technology has led to great insight into gene function,its application in high-throughput studies remains arduous.Agro-infiltration have been widely used in species such as Nicotiana benthamiana for the rapid detection of gene expression and protein interaction analysis,but this technique does not work efficiently in other plant species,including Arabidopsis thaliana.As an efficient high-throughput transient expression system is currently lacking in the model plant species A.thaliana,we developed a method that is characterized by high efficiency,reproducibility,and suitability for transient expression of a variety of functional proteins in A.thaliana and 7 other plant species,including Brassica oleracea,Capsella rubella,Thellungiella salsuginea,Thellungiella halophila,Solanum tuberosum,Capsicum annuum,and N.benthamiana.Efficiency of this method was independently verified in three independent research facilities,pointing to the robustness of this technique.Furthermore,in addition to demonstrating the utility of this technique in a range of species,we also present a case study employing this method to assess protein–protein interactions in the sucrose biosynthesis pathway in Arabidopsis.

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.

Optimization of the uidA Gene Transfer of Rosa hybrida via Agrobacterium tumefaciens: an Assessment of Factors Influencing the Efficiency of Gene Transfer

To develop a transformation protocol of Rosa hybrida Samantha via Agrobacterium tumefaciens, the authors examined the effect of different factors on T-DNA transfer by measuring transient expression levels of an intron-containing -glucuronidase gene. The results indicate that explant, light condition, salt concentration and acetosyringone (AS) concentration in co-culture medium are the most important factors, and factors like co-culture temperature, co-culture period and bacteria density have a strong effect on the growth of bacteria and then T-DNA transfer. Optimized co-cultivation was performed by inoculation of embryogenic callus with bacteria at a density of OD600= 0.50.8 for 20 min and co-culture in darkness under 23 C on medium with 1/2 MS salts and 300 mol稬1 AS for 3 d.

Assessment of Lipid Transfer Protein (LTP1) Gene in Wheat Powdery Mildew Resistance

This study is to investigate the role of lipid transfer protein （LTP1） gene of wheat （Triticum aestivum L.） in powdery mildew （Blumeria graminis f.sp. tritici, Bgt） resistance. A pair of primers based on the full length cDNA of wheat LTP1 was used for amplifying the coding regions of LTP in hexaploid （AABBDD） wheat and its diploid donors T. urartu （AA）, Ae. speltoides ssp speltoide （SS） and Ae. tauchii ssp strangulate （DD）. LTP1 and LTP2 of wheat were isolated from the tested two hexaploid （ABD） materials： powdery mildew resistance near isogenic line （NIL） Mardler/7 × Bainong 3217 and its susceptible parent Bainong 3217 at the same time, while only one kind ofLTP gene was found in the tested three diploid materials respectively by using the above PCR primer pairs. Two peaks of the expression of LTP1 and LTP2 induced by powdery mildew were observed [one occurred at 3 h after inoculation （hai）; the other occurred at 10 hai] in resistant NIL Mardler/7 × Bainong3217 in comparison with a steady transcript level of LTP1 and LTP2 in susceptible Bainong3217. Transient over-expression result showed that LTP1 reduced the penetration efficiency （PE） of powdery mildew in susceptible cultivar by about 28.3%. This result indicated an obvious effectiveness of LTP1 in powdery mildew resistance. Expression analysis also showed that LTP1 and LTP2 of wheat are generally involved in salt/drought, but not in low temperature stress early responses.

An in vivo Transient Expression System Can Be Applied for Rapid and Effective Selection of Artificial MicroRNA Constructs for Plant Stable Genetic Transformation

The utility of artificial microRNAs（amiRNAs） to induce loss of gene function has been reported for many plant species,but expression efficiency of the different amiRNA constructs in different transgenic plants was less predictable,In this study,expressions of amiRNAs through the gene backbone of Arabidopsis miR168a were examined by both Agrobacterium-mediated transient expression and stable plant genetic transformation.A corresponding trend in expression of amiRNAs by the same amiRNA constructs between the transient and the stable expression systems was observed in the experiments.Plant genetic transformation of the constructs that were highly expressible in amiRNAs in the transient agro-infiltration assays resulted in generation of transgenic lines with high level of amiRNAs.This provides a simple method for rapid and effective selection of amiRNA constructs used for a time-consuming genetic transformation in plants.

The relationship between the expression of transient receptor potential vanilloid 1 and the airway remodeling in elderly patients with chronic obstructive pulmonary disease

Objective To investigate the relationship between the expression of trannsient receptor potential vanilloid(TRPV1)and the severity of airway remodeling in elderly patients with chronic obstructive pulmonary disease(COPD).Methods According to airflow obstruction severity,totally 100 cases of elderly patients with

Immunosuppression induced by expression of a viral RNase enhances susceptibility of Plutella xylostella to microbial pesticides

Polydnaviruses are a group of insect DNA viruses and are characterized in their segmented genome that is located in the chromosome（s） of host wasps. A polydnavirus, Cotesiaplutellae bracovirus （CpBV）, encodes a viral ribonuclease （RNase） T2 in a specific segment #3 （CpBV-S3）. This study tested its effect on gene expression associated with host immune responses in the diamondback moth, Plutella xylostella. Micro-inj ection of CpBV- $3 into nonparasitized larvae induced expression of its two encoded genes, CpBV-ORF301 （= CpBV-RNase T2） and CpBV-ORF302. In response to a bacterial challenge, four antimi- crobial peptide genes （hemolin, gloverin, cecropin and lysozyme） and six phenoloxidase （PO）-associated genes （proPO-activating proteinase, PO, serine proteinase homolog and serpins 1-3） were up-regulated in their expressions. However, the transient expression of CpBV-S3 suppressed the expressions of cecropin, PO and serpin 1. Double-stranded RNA specific to the viral RNase T2 could specifically knockdown the viral gene expression and restored the three gene expressions suppressed in the larvae injected with CpBV-S3. The inhibitory activity of the viral RNase T2 on the target genes was further proven by the suppression of PO activation in response to bacterial challenge in the larvae injected with CpBV-S3. This immunosuppression by the expression of the viral RNase T2 resulted in significant increase of pathogen susceptibility ofP. xylostella against Bacillus thuringiensis or baculovirus infection.

Optimization of Agrobacterium tumefaciens-Mediated Transformation Systems in Tea Plant(Camellia sinensis)

In this study, an efficient plant regeneration protocol in vitro and transformation by Agrobacterium-mediated method of Camellia sinensis was achieved, which would lay the foundation for genetic improvement of tea plant by genetic engineering technology. The cotyledon callus of C.sinensis were used as the receptors for transformation by Agrobacterium tumefaciens EHA105 containing PS1aG-3. Some factors which affected the result of Agrobacterium-mediated transformation of C. sinensis were studied on the basis of GUS transient expression system. The optimum system of Agrobacterium-mediated transformation was that the cotyledon callus were pre-cultured for 3 d, and then infected by EHA105 for 15 min followed by 3 d co-culture in the dark on the YEB medium containing 150 μmol·L^(-1) acetosyringone(AS). The transient expression rate of GUS gene was 62.6%. After being delayed selective culture for 3 d, infected callus were transferred into the differentiation medium and the root induction medium both of which were supplemented with 100 mg·L^(-1) spectinomycin, and then resistant seedlings of C. sinensis were obtained. The conversion rate was 3.6%.

Environmentally applied nucleic acids and proteins for purposes of engineering changes to genes and other genetic material

In this article we summarize the development of vehicles for penetrating living cells,tissue and organisms with nucleic acids(DNA and RNA)and proteins that damage or repair DNA.The purpose in doing so is to provide an assessment of the potential for these technologies to unintentionally cause harm to human health or the environment or to be retasked with an intention to cause harm.Two new types of biological-molecule-based products are being developed for use in medicine,agriculture and food production or preservation.The first type are genetically modified organisms,such as those that express bio-pesticides.They produce molecules and that are difficult to alter at scale after release.Products of this type are usually evaluated by both food and environmental regulators.The second type comprises topical chemical or physical agents.Most of these are in pre-commercial testing phase.Topically applied products use nucleic acids and/or proteins wherein the active biological is transferred by contact,ingestion or inhalation.From a survey of the research and patent literature we suggest that chemical formulations and physical manipulations that can be used to ferry nucleic acid and protein cargo into cells,tissues or organisms could be assembled de novo or repurposed from existing commercial products and loaded with proteins and/or nucleic acids designed using publicly available genome sequences.Biological actives may evade risk assessment and regulatory review because they are often excluded from the category of hazardous chemicals and are actively being excluded as agents of genetic modifi-cation.This emerging gap in oversight could lead to either dual use appropriation or unintended harm to human health or the environment.