Expression of a barley ABA-responsive protein gene in transgenic rice

A cDNA clone encoding an ABA-responsiveprotein HVA1,was isolated by differentialscreening from barley aleurone layers(Hong etal.).Expression of the HVA1 gene is shownto be developmentally regulated,organ-specif-ic,and ABA-and stress-induced(Hong et

Lung-Targeted Transgene Expression of Nanocomplexed Ad5 Enhances Immune Response in the Presence of Preexisting Immunity

Recombinant adenovirus serotype 5(Ad5)vector has been widely applied in vaccine development targeting infectious diseases,such as Ebola virus disease and coronavirus disease 2019(COVID-19).However,the high prevalence of preexisting anti-vector immunity compromises the immunogenicity of Ad5-based vaccines.Thus,there is a substantial unmet need to minimize preexisting immunity while improving the insert-induced immunity of Ad5 vectors.Herein,we address this need by utilizing biocompatible nanoparticles to modulate Ad5–host interactions.We show that positively charged human serum albumin nanoparticles((+)HSAnp),which are capable of forming a complex with Ad5,significantly increase the transgene expression of Ad5 in both coxsackievirus–adenovirus receptor-positive and-negative cells.Furthermore,in charge-and dose-dependent manners,Ad5/(+)HSAnp complexes achieve robust(up to227-fold higher)and long-term(up to 60 days)transgene expression in the lungs of mice following intranasal instillation.Importantly,in the presence of preexisting anti-Ad5 immunity,complexed Ad5-based Ebola and COVID-19 vaccines significantly enhance antigen-specific humoral response and mucosal immunity.These findings suggest that viral aggregation and charge modification could be leveraged to engineer enhanced viral vectors for vaccines and gene therapies.

Differential Expression of Bt Protein in Transgenic Bt Cotton

[Objective] The paper was to study the temporal and spatial dynamics of Bt protein expression in transgenic Bt cotton and to determine the inner relationship of Bt protein expression and transgenic Bt cotton. [Method] With transgenic cotton cultivar（ GK19） as the test material,Bt protein contents in different organs,main stem functional leaves at different growth stages and different positions of main stem leaves at different growth stages were studied by enzyme-linked immunosorbent assay. [Result] There were differences in Bt protein content among different organs of transgenic Bt cotton; the Bt protein content of leaves at seedling stage was the highest,followed by flowers,bubs and bolls,and those of roots and stems were relatively low. The Bt protein content of main stem function leaves gradually decreased with the progressing development. There were great differences in Bt protein content among different positions of main stem leaves at different growth stages; the Bt protein content of the 1^（st）-7^（th） top leaves at seedling stage and full budding stage gradually decreased,while those at full flowering stage and full bolling stage first slowly increased then gradually stabilized. [Conclusion] Bt protein expression was found in all organs of transgenic cotton at all growth stages,and the expression level presented temporal and spatial dynamics.

Improvement of foreign gene expression in transgenic rice(Oryza sativa L.)by matrix attachment regions(MARs)

Matrix attachment regions or matrix associated regions (MARs) were special DNA sequences in chromatin of eukaryotic cells that tightly associated with the nuclear matrix or scaffold in vitro after a combination of nuclease digestion and extraction. They were also called scaffold attachment regions(SARs) . It was found that MARs could improve the expression level and the stability of foreign genes in transgenic plants. The reason might be that a transgene flanked by MARs was transmitted into the plant cells, the MARs would attach the nuclear

Increasing cellulose production and transgenic plant growth in forest tree species

Cellulose is one of many important polymers in plants. Cellulose is made of repeat units of the monomer glucose. Cellulose is a major industrial biopolymer in the forest products, textile, and chemical industries. It also forms a large portion of the biomass useful in the generation of energy. Moreover, cellulose-based biomass is a renewable energy source that can be used for the generation of ethanol as a fuel. Cellulose is synthesized by a variety of living organisms such as plants and algae. It is the major component of plant cell walls with secondary cell walls having a much higher content of cellulose. The relationship between cellulose and lignin biosynthesis is complicated, but it is confirmed that inhibition of lignin biosynthesis in transgenic trees will increase cellulose biosynthesis and plant growth. Cellulose accumulation may be increased by down-regulating 4-coumarate:coenzyme A ligase (4CL, EC 6.2.1.12) as shown in transgenic aspen. There is no similar reports on down-regulating 4CL in transgenic conifers. Based on our established Agrobacterium tumefaciens-mediated transformation system in loblolly pine, we are able to produce antisense 4-CL transgenic loblolly pine which is predicted to have increas- ing cellulose accumulation. The overall objective of this project is to genetically engineer forest tree species such as loblolly pine with re- duced amount of lignin and increased cellulose content. The research strategy includes: (1) isolate the 4-coumarate:coenzyme A ligase gene from loblolly pine seedlings by reverse transcription-polymerase chain reaction (RT-PCR) and Rapid Amplification of cDNA Ends-Polymerase Chain Reaction (RACE-PCR) techniques from the cDNA library; (2) construct binary expression vectors with antisense 4CL coding sequences and introduce antisense constructs of the 4-coumarate:coenzyme A ligase gene cloned from loblolly pine into the loblolly pine to down regulate the 4-coumarate:coenzyme A ligase gene expression; (3) study the effect of the antisense transgene expres- sion on lignin content, cellulose accumulation, and loblolly pine biomass; and (4) select fast growth and high cellulose accumulation transgenic loblolly pine lines for future commercial application.

Expression profiling of transgenes(Cry1Ac and Cry2A) in cotton genotypes under different genetic backgrounds

Transgenic cotton carrying the CrylAc gene has revolutionized insect pest control since its adoption,although the development of resistance in insect pests has reduced its efficacy.After 10 years of cultivating Bacillus thuringiensis(Bt)cotton with a single Cry1 Ac gene,growers are on the verge of adopting Bt cotton that carries the double gene(Cry1 Ac+Cry2 A)due to its better effectiveness against insect pests.Thus,the current study was designed to evaluate the role of each gene in the effectiveness of Bt cotton carrying the double gene.The expression levels of the Cry1 Ac and Cry2 A genes were evaluated in the leaves of 10 genotypes(2 parents and 8 Fhybrids)at 30 days after sowing(DAS),while samples of leaves,bolls and flowers were taken from the upper and lower canopies at 70 and 110 DAS.The Fhybrids were developed through reciprocal crosses between two Bt(CKC-1,CKC-2)and two non-Bt(MNH-786,FH-942)parents.The differential expression of transgenes was evaluated through Enzyme Linked Immuno-Sorbent Assay(ELISA).The results showed that the MNH786 xCKC-1 hybrid had the highest concentrations of Cry1 Ac gene at30 DAS(3.08μg g^(-1))and 110 DAS(1.01μg g^(-1))in leaves.In contrast,the CKC-2 xMNH-786 hybrid showed the lowest concentrations of Cry1 Ac gene at 30 DAS(2.30μg g^(-1))and 110 DAS(0.86μg g^(-1)).The Fhybrid FH-942×CKC-2 showed the highest concentrations of Cry2 A gene at 30 DAS(8.39μg g^(-1))and 110 DAS(7.74μg g^(-1))in leaves,while the CKC-1 xMNH-786 hybrid expressed the lowest concentrations of Cry2 A gene at 30 DAS(7.10μg g^(-1))and 110 DAS(8.31μg g^(-1)).A comparison between the two stages of plant growth showed that leaves had the highest concentrations at 30 DAS,whereas the lowest concentrations were observed at 110 DAS for both genes in leaves.When the expression pattern was compared between various plant parts in genotype CKC-2,it was found that leaves had higher concentrations of Cry1 Ac(3.12μg g^(-1))and Cry2 A(8.31μg g^(-1))at 70 DAS,followed by bolls(Cry1 Ac(1.66μg g^(-1))and Cry2 A(8.15μg g^(-1)))and flowers(Cry1 Ac(1.07μg g^(-1))and Cry2 A(7.99μg g^(-1))).The genotype CKC-2 had higher concentrations of Cry1 Ac(3.12μg g^(-1))and Cry2 A(8.31μg g^(-1))in the upper canopy but less accumulation(2.66μg g^(-1)of Cry1 Ac,8.09μg g^(-1)of Cry2 A)in the lower canopy at 70 DAS.Similarly,at 110 DAS,the expression levels of Cry1 Ac and Cry2 A in upper and lower canopy leaves were 1.52 and 7.92μg 9,and 0.99 and 7.54μg 9,respectively.Hence,the current study demonstrates that different genotypes showed variable expression for both of the Cry1 Ac and Cry2 A genes during plant growth due to different genetic backgrounds.The Cry2 A gene had three-fold higher expression than Cry1 Ac with significant differences in expression in different plant parts.The findings of this study will be helpful for breeding insect-resistant double-gene genotypes with better gene expression levels of Cry1 Ac and Cry2 A for sustainable cotton production worldwide.

Preparation, Characterization, and Application of Antiharpin_(xoo) Antibody

Polyclonal antiharpinxoo rabbit antibody has been prepared successfully using purified harpinxoo protein as an immunogen. The ELISA titer of the antiserum against harpinxoo was about 1：2 000. Western blot analysis showed that the antiserum could bind to the expression harpinxoo protein in particular, hrfl, encoding harpinxoo, is an expression in transgenic rice, detected by antiharpinxoo rabbit antibody. The rabbit antibody against harpinxoo can be used to study further about the biological function, harpinxoo localization, and hrfl gene expression in other plants.

A preliminary analysis of antineoplastic activity ofparvovirus MVMp NS-1 proteins

Human gastric cancer MKN-45 cells were transfectedwith pULB 3238, a plasmid carrying MVMp NS-1 genewith its original P4 promoter replaced by the glucocorticoid inducible promoter MMTV-LTR. After the integration and expression of NS-1 gene, some of the transfectantsdied, while others remained alive, but the growth featuresof survived cells were changed. For further study on theantineoplastic function of parvoviral NS-1 protein in vivo,transgenic mice carrying NS-1 genes were established byconventional method. Among 4 founders, one of them wasfound to be able to transmit the transgene to around 50%of their offsprings. RT-PCR was performed to indicate theexpression of NS-1 gene in transgenic mice and its mRNAappeared in a variety of tissues. The expression of integrated NS-1 gene may correlate with the decreased incidence of tumor induced in vivo by chemical carcinogens.

Two Lycopene β-Cyclases Genes from Sweet Orange (Citrus sinensis L. Osbeck) Encode Enzymes With Different Functional Efficiency During the Conversion of Lycopene-to-Provitamin A

Citrus fruits are rich in carotenoids.In the carotenoid biosynthetic pathway,lycopene β-cyclase（LCYb,EC：1.14.-.-） is a key regulatory enzyme in the catalysis of lycopene to β-carotene,an important dietary precursor of vitamin A for human nutrition.Two closely related lycopene β-cyclase cDNAs,designated CsLCYb1 and CsLCYb2,were isolated from the pulp of orange fruits（Citrus sinensis）.The expression level of CsLCYb genes is lower in the flavedo and juice sacs of a lycopeneaccumulating genotype Cara Cara than that in common genotype Washington,and this might be correlated with lycopene accumulation in Cara Cara fruit.The CsLCYb1 efficiently converted lycopene into the bicyclic β-carotene in an Escherichia coli expression system,but the CsLCYb2 exhibited a lower enzyme activity and converted lycopene into the β-carotene and the monocyclic γ-carotene.In tomato transformation studies,expression of CsLCYb1 under the control of the cauliflower mosaic virus（CaMV） 35S constitutive promoter resulted in a virtually complete conversion of lycopene into β-carotene,and the ripe fruits displayed a bright orange colour.However,the CsLCYb2 transgenic tomato plants did not show an altered fruit colour during development and maturation.In fruits of the CsLCYb1 transgenic plants,most of the lycopene was converted into β-carotene with provitamin A levels reaching about 700 μg g-1DW.Unexpectedly,most transgenic tomatoes showed a reduction in total carotenoid accumulation,and this is consistent with the decrease in expression of endogenous carotenogenic genes in transgenic fruits.Collectively,these results suggested that the cloned CsLCYb1 and CsLCYb2 genes encoded two functional lycopene β-cyclases with different catalytic efficiency,and they may have potential for metabolite engineering toward altering pigmentation and enhancing nutritional value of food crops.

Sustained high level transgene expression in mammalian cells mediated by the optimized piggyBac transposon system

Sustained,high level transgene expression in mammalian cells is desired in many cases for studying gene functions.Traditionally,stable transgene expression has been accomplished by using retroviral or lentiviral vectors.However,such viral vector-mediated transgene expression is often at low levels and can be reduced over time due to low copy numbers and/or chromatin remodeling repression.The piggyBac transposon has emerged as a promising nonviral vector system for efficient gene transfer into mammalian cells.Despite its inherent advantages over lentiviral and retroviral systems,piggyBac system has not been widely used,at least in part due to their limited manipulation flexibilities.Here,we seek to optimize piggyBac-mediated transgene expression and generate a more efficient,user-friendly piggyBac system.By engineering a panel of versatile piggyBac vectors and constructing recombinant adenoviruses expressing piggyBac transposase(PBase),we demonstrate that adenovirusmediated PBase expression significantly enhances the integration efficiency and expression level of transgenes in mesenchymal stem cells and osteosarcoma cells,compared to that obtained from co-transfection of the CMV-PBase plasmid.We further determine the drug selection timeline to achieve optimal stable transgene expression.Moreover,we demonstrate that the transgene copy number of piggyBac-mediated integration is approximately 10 times higher than that mediated by retroviral vectors.Using the engineered tandem expression vector,we show that three transgenes can be simultaneously expressed in a single vector with high efficiency.Thus,these results strongly suggest that the optimized piggyBac system is a valuable tool for making stable cell lines with sustained,high transgene expression.

Costs of resistance to fungal pathogens in genetically modified wheat

Aims Many resistance genes against fungal pathogens show costs of resistance.Genetically modified(GM)plants that differ in only one or a few resistance genes from control plants present ideal systems for measuring these costs in the absence of pathogens.Methods To assess the ecological relevance of costs of pathogen resistance,we grew individual plants of four transgenic spring wheat lines in a field trial with three pathogen levels and varied the genetic diversity of the crop.Important Findings We found that two lines with a Pm3b transgene were more resistant to powdery mildew than their sister lines of the variety Bobwhite,whereas lines with chitinase(A9)or chitinase and glucanase(A13)transgenes were not more resistant than their mother variety Frisal.Nevertheless,in the absence of the pathogen,both the GM lines of Bobwhite as well as those of Frisal performed significantly worse than their controls,i.e.Pm3b#1 and Pm3b#2 had 39%or 53%and A9 and A13 had 14%or 23%lower yields.In the presence of the pathogen,all GM lines except Pm3b#2 could increase their yields and other fitness-related traits,reaching the performance levels of the control lines.Line Pm3b#2 seemed to have lost its phenotypic plasticity and had low performance in all environments.This may have been caused by very high transgene expression.No synergistic effects of mixing different GM lines with each other were detected.This might have been due to high transgene expression or the similarity between the lines regarding their resistance genes.We conclude that costs of resistance can be high for transgenic plants with constitutive transgene expression and that this can occur even in cases where the non-transgenic control lines are already relatively resistant,such as in our variety Frisal.Transgenic plants could only compete with conventional varieties in environments with high pathogen pressure.Furthermore,the large variability among the GM lines,which may be due to unpredictable transgene expression,suggests that case-by-case assessments are necessary to evaluate costs of resistance.

An Open-Source System for In Planta Gene Stacking by Bxbl and Cre Recombinases

The rapid development of crops with multiple transgenic traits arouses the need for an efficient system for creating stacked cultivars. Most major crops rely on classical breeding to introgress the transgene from a laboratory variety to the numerous cultivars adapted to different growing regions. Even with vegetative propagated crops, genetic crosses are conducted during varietal improvement prior to vegetative cloning. The probability to assort the ＇x＇ number of transgenic loci into a single genome may seem trivial, （~）x for a diploid species, but given the ＇y＇ number of other nontransgenic traits that breeders also need to assemble into the same genome, the （~）~＊y probability for a ＇breeding stack＇ could quickly make the line conversion process unmanageable. Adding new transgenes onto existing transgenic varieties without creating a new segregating locus would require site-specific integration of new DNA at the existing transgenic locus. Here, we tested a recombinase-mediated gene-stacking scheme in tobacco. Sequential site-specific inte- gration was mediated by the mycobacteriophage Bxbl integrase-catalyzed recombination between attP and attB sites. Transgenic DNA no longer needed after integration was excised by Cre recombinase-mediated recombination of Iox sites. Site-specific integration occurred in -10% of the integration events, with half of those events usable as substrates for a next round of gene stacking. Among the site-specific integrants, however, a third experienced gene silencing. Overall, precise structure and reproducible expression of the sequentially added triple traits were obtained at an overall rate of -3% of the transformed clones--a workable frequency for the development of commercial cultivars. Moreover, since nei- ther the Bxbl-att nor the Cre-lox system is under patent, there is freedom to operate,